U.S. patent application number 15/587636 was filed with the patent office on 2017-08-24 for identification of t cell target antigens.
The applicant listed for this patent is Ludwig-Maximilians-Universitat Munchen. Invention is credited to Klaus Dornmair, Reinhard Hohlfeld, Song-Min Kim, Jorg Prinz, Katherina Siewert.
Application Number | 20170240884 15/587636 |
Document ID | / |
Family ID | 44023005 |
Filed Date | 2017-08-24 |
United States Patent
Application |
20170240884 |
Kind Code |
A1 |
Dornmair; Klaus ; et
al. |
August 24, 2017 |
IDENTIFICATION OF T CELL TARGET ANTIGENS
Abstract
The present invention relates to a method of identifying a
target antigen of T cells comprising (a) contacting (aa) cells
expressing (i) a functional T cell receptor complex comprising
predefined matching T cell receptor .alpha. and .beta. chains; and
(ii) a read-out system for T cell activation; with (ab)
antigen-presenting cells carrying (iii) peptide libraries encoded
by randomised nucleic acid sequences; and (iv) WIC molecules
recognised by the T cell receptor of (i); (b) assessing T cell
activation using said read-out system; (c) isolating
antigen-presenting cells that are in contact with the cells in
which the read-out system indicates T cell activation; (d)
identifying the target antigen or the nucleic acid molecule
encoding said target antigen.
Inventors: |
Dornmair; Klaus; (Olching,
DE) ; Hohlfeld; Reinhard; (Martinsried, DE) ;
Prinz; Jorg; (Munchen, DE) ; Siewert; Katherina;
(Munchen, DE) ; Kim; Song-Min; (Munchen,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ludwig-Maximilians-Universitat Munchen |
Munchen |
|
DE |
|
|
Family ID: |
44023005 |
Appl. No.: |
15/587636 |
Filed: |
May 5, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13814263 |
Apr 19, 2013 |
9678061 |
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PCT/EP2011/063538 |
Aug 5, 2011 |
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15587636 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6883 20130101;
G01N 2333/70539 20130101; A61P 37/06 20180101; C12Q 2600/16
20130101; G01N 33/56977 20130101; G01N 33/505 20130101; A61P 35/00
20180101; C12N 15/1037 20130101; A61K 39/00 20130101; A61P 31/00
20180101; G01N 2333/7051 20130101 |
International
Class: |
C12N 15/10 20060101
C12N015/10; G01N 33/569 20060101 G01N033/569 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 6, 2010 |
EP |
10008233.8 |
Claims
1. A peptide library, wherein the peptide library comprises a
plurality of vectors comprising nucleic acid sequences encoding
peptides, wherein the peptides of the peptide library are encoded
by plasmid vectors, wherein the peptides are potential target
antigens of T cells and wherein the nucleic acid sequences are
randomized nucleic acid sequences.
2. The peptide library of claim 1, wherein the peptides have a
length of between 4 to 20 amino acids.
3. The peptide library of claim 2, wherein the peptides have a
length of between 8 to 10 amino acids.
4. The peptide library of claim 1, wherein the peptides, as the
potential target antigens of T cells bind and are represented by
MHC class I molecules.
5. A method of preparing antigen-presenting cells that express
potential target antigens of T cells, comprising transfecting or
transforming cells with a peptide library according to claim 1.
6. The method of claim 5, wherein the antigen-presenting cells are
cells capable of amplifying the peptide libraries.
7. The method of claim 6, wherein the antigen presenting cells are
selected from the group consisting of COS-7, HEK, Hela, H9, Jurkat,
NIH3T3, C127, COS-1, CV1, QC1-3, mouse L cells, mouse C2C12 cells
and Chinese hamster ovary (CHO), Wi-38, MRC-5, insect cells like
Sf9, and Hi-5 cells.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional Application of U.S. patent
application Ser. No. 13/814,263 filed on Apr. 19, 2013, which is a
35 U.S.C. .sctn.371 U.S. National Stage Entry of International
Application No. PCT/EP2011/063538 filed Aug. 5, 2011, which claims
the benefit of priority of European Application No. 10008233.8
filed Aug. 6, 2010, the contents of which are each incorporated
herein by reference in its entirety.
REFERENCE TO SEQUENCE LISTING
[0002] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled SEQLST20011003USDIV.txt created on May 5, 2017, which
is 33,283 bytes in size. The information in electronic format of
the sequence listing is incorporated herein by reference in its
entirety.
DETAILED DESCRIPTION
[0003] The present invention relates to a method of identifying a
target antigen of T cells comprising (a) contacting (aa) cells
expressing (i) a functional T cell receptor complex comprising
predefined matching T cell receptor .alpha. and .beta. chains; and
(ii) a read-out system for T cell activation; with (ab)
antigen-presenting cells carrying (iii) peptide libraries encoded
by randomised nucleic acid sequences; and (iv) MHC molecules
recognised by the T cell receptor of (i); (b) assessing T cell
activation using said read-out system; (c) isolating
antigen-presenting cells that are in contact with the cells in
which the read-out system indicates T cell activation; (d)
identifying the target antigen or the nucleic acid molecule
encoding said target antigen. The present invention further relates
to a method of identifying nucleic acid molecules encoding
variable, hypervariable and/or joining regions of T cell receptor
.beta. chains as well as to this method of the invention further
comprising identifying the nucleic acid molecules encoding
variable, hypervariable and/or joining regions of the matching T
cell receptor .alpha. chains. The present invention also relates to
a method of identifying patient-specific T cell antigens comprising
(A) isolating T cells from a sample obtained from said patient; (B)
identifying matching T cell receptor a and chains from the T cells
isolated in (A); and (C) identifying T cell antigens in accordance
with the method of the invention, wherein the cell comprising a
functional T cell receptor and a read-out system for T cell
activation expresses matching T cell receptor .alpha. and .beta.
chains from the T cells identified in (B). The present invention
further relates to a composition comprising a T cell antigen
identified by a method of the invention, a peptide library as well
as a method of preparing antigen-presenting cells and a primer or a
set of primers.
[0004] In this specification, a number of documents including
patent applications and manufacturer's manuals are cited. The
disclosure of these documents, while not considered relevant for
the patentability of this invention, is herewith incorporated by
reference in its entirety. More specifically, all referenced
documents are incorporated by reference to the same extent as if
each individual document was specifically and individually
indicated to be incorporated by reference.
[0005] T cells play crucial roles in many infectious, tumor, and
autoimmune diseases, but apart from very few exceptions, the target
antigens of pathogenic human T cells have remained unknown. The
specificity of T cells towards their target antigens is determined
by their heterodimeric, hyper-variable T cell receptor (TCR)
molecules, which recognize antigenic peptides that are presented by
MHC molecules. In immune defense situations, the MHC molecules are
of "self"-origin, whereas the antigenic peptides are "non-self",
i.e. they are derived from viral or microbial peptides. Typically,
class-I MHC molecules present peptides of intracellular (viral)
origin to CD8+ T cells, whereas class-II MHC molecules present
phagocytosed (microbial) peptides to CD4+ T cells. In addition,
"self" MHC molecules also present "self" peptides, but these are
normally ignored because of T cell tolerance. It is assumed that in
autoimmune diseases the tolerance is broken and recognition of
"self" peptides results in chronic inflammation, disturbed organ
function or tissue destruction. Another important role of T cells
is during tumor defense where T cells may mount anti-tumor
responses. In this case, however, they recognize tumor-associated
antigens.
[0006] Despite tremendous efforts by many groups of investigators
who applied very different techniques, there is so far no simple,
reliable, and unbiased method to determine T cell antigens. One
reasons is that straightforward biochemical techniques such as
immunoprecipitation or affinity chromatography cannot be used,
because the affinities of TCRs to MHC/peptide complexes are several
orders of magnitude too low. Such techniques work well if the
dissociation constant is in the nanomolar range or below, as it is
typical for antibodies or conventional receptor-ligand
interactions, but for TCR-MHC/peptide interactions, the
dissociation constants are usually greater than 10.sup.-6 M
(Rudolph et al., 2006; J. D. Stone et al (2009).
[0007] In the past, some antigens were identified by generating T
cell lines in vitro against known or bona fide candidate autoimmune
or tumor antigens, and the precise epitopes were mapped later using
synthetic peptides.
[0008] In other approaches, antigenic peptides were eluted from MHC
molecules of tumor or autoimmune tissue and analyzed by mass
spectrometry (Cox et al., 1994; Fissolo et al., 2009).
[0009] A third method uses randomized synthetic peptide libraries
(Nino-Vasquez et al., 2005). With this approach the idea is that
TCR molecules recognize patterns rather than defined sequences or
structures. In other words, the recognition of target structures is
poly-specific (also termed "promiscuous" or "degenerate")
(Wucherpfennig et al., 2007). Such libraries contain random amino
acids in all but one position and may allow identification of
"recognition patterns", i.e. of mimotopes of the natural peptides,
which then may be identified by database searches. These approaches
are limited to some TCRs which show an appropriate balance between
specificity and poly-specificity. If the specificity is too high,
the few activating peptides are too dilute in the library, if the
specificity is too low, the pattern can not be recognized any more.
These approaches are well suited to determine the degree of
polyspecificity of TCR, but have so far in most cases failed to
identify new, yet unknown antigens.
[0010] Fourth, cDNA libraries have been used in particular to
investigate tumor T cell antigens. They were either transfected
into COS cells via plasmids bearing the SV40 origin (Van der
Bruggen et al., 1991; Wong et al., 1999; Boon et al., 2006), or via
retroviral constructs into appropriate recipient cells (Smith et
al., 2001). In both cases the plasmids or viral particles may be
recovered from the transfected cells and amplified in bacteria.
Usually many pools of plasmids are used, and positive pools are
subjected to further rounds of transfections until a single
antigen-bearing plasmid may be isolated and characterized. Such
amplification strategies are of course advantageous as compared to
biochemical or combinatorial synthetic peptide libraries. Although
several tumor antigens were detected, these methods have not
reached general applicability. The reasons are diverse: the
libraries usually come from diseased tissue (which is often not
available) and a tremendous number of clones must be screened by
laborious and expensive cytotoxicity or cytokine assays,
which--another limitation--require large numbers of T cells. Most
importantly, however, the proteins expressed from cDNA libraries
require extensive and correct processing. Hence, the processing
pattern of the APCs used in vitro must be identical to the
processing pattern of the original APCs. This may often not be the
case.
[0011] A fifth strategy is reminiscent of phage display libraries.
Here baculovirus infected insect cells display randomized peptide
libraries in the binding groove of recombinant MHC molecules
(Crawford et al., 2004; Wang et al., 2005; Crawford et al., 2006).
These libraries are screened with fluorescent oligomerized soluble
TCR molecules. Although this technology could reveal mimotopes of
peptides known to activate class-I and class-II restricted TCRs, it
has several intricacies. The most important drawback is, as
discussed above, that TCRs have notoriously low affinities to their
MHC/peptide ligands. This impedes detection of positive insect cell
clones. TCR-oligomerization facilitates recognition by increasing
avidity, but may presumably not completely overcome this
limitation. Further, library cloning is possible only by homologous
recombination which yields libraries of limited size, or by cloning
directly into baculovirus DNA which is difficult, owing to its
large size.
[0012] WO2003068800 describes isolated peptides that bind to HLA
molecules and stimulate cytolytic T cells specific for complexes of
the peptide and the HLA molecule. A transfection of antigen
presenting cells with recombinant combinatorial peptide libraries
is not envisaged in WO2003068800.
[0013] U.S. Pat. No. 6,037,135 discloses methods of making HLA
binding peptides, but does not disclose the loading of MHC
molecules with and presentation of antigens by antigen presenting
cells via a peptide library encoding such peptides.
[0014] In summary, even though some of the different techniques
used in the past occasionally allowed the detection of mimotopes of
previously known antigens, an a priori unknown antigen could only
in rare cases be discovered. Thus, despite the above described
advances in the field of T cell antigen identification, there
remains a need to provide improved methods for an unbiased
identification of novel T cell antigens.
[0015] This need is addressed by the provision of the embodiments
characterised in the claims.
[0016] Accordingly, the present invention relates to a method of
identifying a target antigen of T cells comprising (a) contacting
(aa) cells expressing (i) a functional T cell receptor complex
comprising predefined matching T cell receptor .alpha. and .beta.
chains; and (ii) a read-out system for T cell activation; with (ab)
antigen-presenting cells carrying (iii) peptide libraries encoded
by randomised nucleic acid sequences; and (iv) MHC molecules
recognised by the T cell receptor of (i); (b) assessing T cell
activation using said read-out system; (c) isolating
antigen-presenting cells that are in contact with the cells in
which the read-out system indicates T cell activation; (d)
identifying the target antigen or the nucleic acid molecule
encoding said target antigen.
[0017] The term "target antigen of T cells", as used in accordance
with the present invention, relates to an antigen that is
recognised and bound by T cells. The binding of the antigen to T
cells subsequently results in the activation of said T cells. T
cell target antigens are recognised by T cells via a functional T
cell receptor complex consisting of T cell receptors (TCR) and the
CD3 complex. T cell target antigens are presented by major
histocompatibility complex (MHC) molecules on the surface of
antigen-presenting cells (APCs). In accordance with the present
invention, the term "target antigen of T cells" refers to the
peptide epitope. When the peptide epitope is complexed with an MHC
molecule, reference is made to the "antigen-MHC complex" herein.
Target antigens presented by MHC class I molecules are recognized
by CD8+ T cells and are typically of intracellular origin, such as
for example viral target antigens. Target antigens presented by MHC
class II molecules are recognized by CD4+ T cells. Typically they
are peptides, which originate from extracellular sources that were
phagocytosed, such as for example peptides derived from microbes.
Target antigens of T cells can be of an origin that is foreign to
the host organism, such as for example viral or microbial peptides.
Target antigens may also be of self-origin, such as for example
tumor-associated antigens or self-antigens that trigger an
autoimmune response in the host organism.
[0018] In a preferred embodiment, the target antigen of T cells is
a target antigen of CD8+ T cells.
[0019] As used herein, the term "functional T cell receptor
complex" refers to a complex capable of eliciting activation of the
T cell in which the complex is expressed. The T cell receptor
complex is composed of six subunits. The T cell receptor is made up
of two subunits (also referred to herein as chains), TCR .alpha.0
and .beta., which form a disulfide-linked heterodimer, which
comprises the variable, hypervariable and joining region of the TCR
receptor complex that interacts with the antigen/WIC-complex, thus
forming one single antigen-binding site. In a subgroup of T cells
the T cell receptor is made up of TCR .gamma. and .delta. subunits.
In addition, TCR .alpha. and .beta.-chains comprise conserved
(constant) regions which interact with the proteins of the
CD3-complex and fix the TCR in the membrane. The T-cell receptor
complex further contains four CD3 subunits. Each CD3 complex
contains one CD3.gamma. subunit, one CD3.delta. subunit, and two
CD3.epsilon. subunit. One of the CD3.epsilon. subunits forms a
heterodimer with the CD3.gamma. subunit, while the other
CD3.epsilon. subunit forms a heterodimer with the CD3.delta.
subunit. Antigen binding leads to the cross-linking and activation
of the TCR hexamers. The signal is then conducted by the
.zeta.-chains to further downstream intracellular compounds.
Functionality of a T cell receptor complex can be analysed using
methods well known in the art, such as for example measurements of
phosphorylation and de-phosphorylation of proteins and other
intracellular molecules (such as for example IP3), Ca.sup.2+-influx
into T-cells, production of cytokines (such as for example
Interferon-.gamma., interleukins (such as IL-2, -4, -6, -17),
TNF-.alpha.), secretion of cytotoxic granules containing perforin
and granzymes or killing of target cells (Smith-Garvin et al.
(2009); Murphy et al. "Janeway's Immunobiology" 2008, 7th
Edition).
[0020] The term "predefined matching T cell receptor .alpha. and
.beta. chains", according to the invention, relates to .alpha. and
.beta. chains derived from one molecular type of T cell receptor,
i.e. paired .alpha. and .beta. chains. In other words, the chains
are matched to represent functional T cell receptor heterodimers.
The term "predefined" as used in this context refers to the
purposive selection of the T cell receptor chains to represent a T
cell receptor of interest, for which corresponding antigens are to
be identified by the method of the invention. Preferably, the T
cell receptor is a vertebrate T cell receptor, more preferably it
is a mammalian T cell receptor. Even more preferably, the T cell
receptor is a T cell receptor from horse, bovine, swine, canine,
feline or primate. Most preferably, the T cell receptor is a human
T cell receptor.
[0021] T cell receptor chains may be obtained by any method known
in the art, such as for example the method described in Seitz et
al. 2006, where TCR chains are cloned from morphologically
characterised single cells. Such cells may be obtained from a
sample obtained from a patient, for example, by laser
micro-dissection of biopsy tissue. Furthermore, T cell receptors of
known sequence may be employed, in which case the chains may be
obtained by transfecting the cell of (aa) with a nucleic acid
molecule encoding said T cell receptor chains.
[0022] In accordance with the present invention, the term "read-out
system for T cell activation" relates to a system that provides a
measurable signal upon activation of the T cell receptor. T cell
receptor activation normally results in the activation of the
respective T cell expressing said T cell receptor. Thus, the signal
provided by the read-out system is representative of a successful T
cell activation via T cell receptor binding of the antigen
presented by the antigen-presenting cell. Said activation of the T
cell receptor expressed in the cells of (aa) results in a signal
such as for example the expression of a reporter protein.
Non-limiting examples of reporter genes suitable to provide a
measurable signal upon T cell activation include enzymes such as
for example .beta.-galactosidase, CAT, .beta.-glucuronidase,
peroxidase, .beta.-xylosidase, catechol dioxygenase (XylE),
trehalase (TreA), alkaline phosphatase or secreted alkaline
phosphatase as well as fluorescent compounds, bioluminescent
compounds and chemiluminescent compounds. Such read-out systems are
well known in the art and are described, for example, in
Suter-Crazzolara et al. (1995) or Shaner, et al. (2005).
[0023] Thus, the cells of (aa) are characterised by the expression
of a functional T cell receptor complex and a read-out system,
which indicates T cell activation. Such cells may be any cells
comprising a T cell receptor complex, such as for example cells
naturally expressing a T cell receptor complex or cells transformed
or transfected with a T cell receptor or a T cell receptor complex.
Such cells are also referred to as T cell receptor transfectants.
Non-limiting examples of such cells are T cells, T cell hybridomas,
lymphomas and artificially immortalized T cells (Katakura et al.
(1998)). Preferably, the cells of (aa) are T cell hybridomas. T
cell hybridomas are well known in the art and may be obtained as
described in Ozaki et al. (1988); Shirahata et al. (1998); Chen et
al. (2007) or as described in the examples below.
[0024] It is preferred that the cells of (aa) express only one
molecular type of T cell receptor. Such cells may be, for example,
naturally occurring T cells or T cell hybridomas devoid of an
endogenous T cell receptor and being transfected or transformed
with a T cell receptor of interest.
[0025] In an alternative embodiment, cells already expressing an
endogenous T cell receptor may be transfected or transformed with a
further T cell receptor of interest. In that case, the T cell
receptor of interest is modified such as to prevent
hetero-dimerisation between chains of the endogenous T cell
receptor and chains of the T cell receptor of interest. Such
modifications are well known in the art and include, without being
limiting, the introduction of mutated amino acids at the interface
of the constant regions of the T cell receptor chains of interest,
or choosing T cells of very limited heterogeneity, such as oligo-
or monoclonal T cell populations, as recipients (Voss et al.
(2008); Cohen et al. (2007); Kuball et al. (2007); Weinhold et al.
(2007); van Loenen et al. (2010); Bendle et al. (2010)). It is
preferable that if cells expressing different molecular types of T
cell receptor are employed in the method of the invention, further
rounds of screening are carried out using different cells, such as
for example cells expressing the T cell receptor of interest with
only one endogenous T cell receptor, wherein said endogenous T cell
receptor differs from the T cell receptor(s) present in the cells
of the first or previous rounds of screening. Preferably, the cells
employed in such further rounds of screening only express the T
cell receptor of interest, in order to confirm that the antigen
identified is an antigen of the T cell receptor of interest and not
an antigen of the endogenous T cell receptor.
[0026] The term "antigen-presenting cells", as used herein, relates
to cells that display antigen in complex with the major
histocompatibility complex (MHC) on its surface. Any cell is
suitable as an antigen-presenting cell in accordance with the
present invention, as long as it expresses an MHC and presents an
antigen. Cells that have in vivo the potential to act as antigen
presenting cells include for example dendritic cells, macrophages,
B-cells or activated epithelial cells but also fibroblasts, glial
cells, pancreatic beta cells or vascular endothelial cells. Such
cells may be employed in accordance with the present invention
after transfection or transformation with a peptide library as
defined below. Also cells not endogenously expressing MHC may be
employed, in which case suitable MHC are to be transformed or
transfected into said cells.
[0027] The term "peptide libraries encoded by randomised nucleic
acid sequences", according to the present invention, relates to a
collection of peptides, i.e. a library, wherein the peptides are
encoded by nucleic acid sequences of arbitrary sequences. In other
words, the nucleic acid sequences represent a variety of possible
sequence variations randomly chosen. That is, if the peptide
library represents peptides having, for example, a length of 9
amino acids, then the encoding nucleic acid sequences would have to
be 27 nucleotides long, which means a total of
4.sup.27=1.8.times.10.sup.16 different nucleic acid sequences would
represent each possible nucleotide combination. Preferably, the
peptide library is encoded by a set of nucleic acid sequences
representing at least 10.sup.3 different nucleic acid sequences,
such as at least 10.sup.4, at least 10.sup.5, at least 10.sup.6, at
least 10.sup.7, at least 10.sup.8, at least 10.sup.9, at least
10.sup.10, at least 10.sup.11, at least 10.sup.12, at least
10.sup.13, at least 10.sup.14, at least 10.sup.15 or at least
10.sup.16 different nucleic acid sequences. Most preferably, the
peptide library is encoded by a set of nucleic acid sequences
representing all possible permutations for a given peptide length.
Furthermore, peptide libraries encompassing peptides of different
lengths are also envisaged herein and the above defined amounts of
nucleic acid sequence representation within said library apply
mutatis mutandis.
[0028] In addition, for a number of target antigens of MHC
complexes so-called anchor positions are known to be required for
binding between the antigen and the MHC. Where these anchor
positions are known, the randomised nucleic acid sequences may also
be generated by introducing in these positions fixed amino acids
and randomising solely the remaining positions. Anchor positions
for various MHCs are known, such as for example in HLA-A2, as
discussed in the examples, where anchor positions are isoleucine in
position 2, valine in position 6 and leucine in position 9
(Rammensee et al., 1999). Further anchors are known in the art and
are described, for example in (Rammensee et al., 1999).
[0029] The term "peptide" as used herein describes a group of
molecules consisting of up to 50 amino acids. Peptides may further
form dimers, trimers and higher oligomers, i.e. consisting of more
than one molecule which may be identical or non-identical. The
corresponding higher order structures are, consequently, termed
homo- or heterodimers, homo- or heterotrimers etc. The term
"peptide" (wherein "polypeptide" is interchangeably used with
"protein") also refers to naturally modified peptides wherein the
modification is effected e.g. by glycosylation, acetylation,
phosphorylation and the like. Such modifications are well-known in
the art. Preferably, the peptides have a minimum length of at least
4 amino acids, such as for example at least 5, at least 6, at least
7, at least 8, at least 9 or at least 10 amino acids. Also
preferred is that the peptides have a length of at the most 50
amino acids, such as for example at most 45, such as at most 40, at
most 35, at most 30, at most 25, at most 20 amino acids. Any of the
intermediate numbers not explicitly mentioned are also envisaged
herein. More preferably, peptides represented by MHC class I
molecules have a length of between 4 and 20 amino acids. Thus, said
peptides may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19 or 20 amino acids in length. Also preferred is that peptides
represented by MHC class II molecules have a length of between 4
and 50 amino acids. The class II peptides may in principle be
infinitely long, because they may reach out from the MHC binding
groove at both sides. The epitope itself is normally 8 to 10 amino
acids long. Thus, said peptides may be 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49 or 50 amino acids in length. Even more preferably,
the peptides of the present invention have a length of between 8 to
10 amino acids. Most preferably, the peptides have a length of 9
amino acids.
[0030] "Nucleic acid sequences", in accordance with the present
invention, include DNA, such as cDNA or genomic DNA, and RNA. It is
understood that the term "RNA" as used herein comprises all forms
of RNA and preferably refers to mRNA. Further included are nucleic
acid mimicking sequences known in the art such as synthetic or
semisynthetic derivatives of DNA or RNA and mixed polymers, both
sense and antisense strands. Such nucleic acid mimicking molecules
or nucleic acid derivatives according to the invention include
phosphorothioate nucleic acid, phosphoramidate nucleic acid,
2'-O-methoxyethyl ribonucleic acid, morpholino nucleic acid,
hexitol nucleic acid (HNA) and locked nucleic acid (LNA) (see
Braasch and Corey (2001) Chem Biol. 8, 1). LNA is an RNA derivative
in which the ribose ring is constrained by a methylene linkage
between the 2'-oxygen and the 4'-carbon. They may contain
additional non-natural or derivatized nucleotide bases, as will be
readily appreciated by those skilled in the art.
[0031] Due to the randomised nature of the nucleic acid sequences,
considerations based on a known target sequence are not required.
Thus, the peptide libraries employed in the present invention
differ from the prior art in that in the prior art, one would have
to design the nucleic acid sequences according to a known sequence
of the gene, whereas from the present library one can screen
through a fully random panel of different peptides (without the
need of prior knowledge of their sequences) to look for a target
antigen of T cells. In a preferred embodiment, the randomised
nucleic acid sequences encoding the peptide library/libraries do
not encode a naturally occurring gene. In other words, the term
"randomised nucleic acid sequences" does not include full length
genes encoding a particular protein nor cDNAs, such as for example
cDNAs derived from mRNA.
[0032] Preferably, the nucleic acid sequences encoding the peptides
of the peptide library are incorporated into a vector.
[0033] Preferably, the vector is a plasmid, cosmid, virus,
bacteriophage or another vector used e.g. conventionally in genetic
engineering. The nucleic acid sequences of the present invention
may be inserted into several commercially available vectors.
Non-limiting examples include vectors compatible with an expression
in mammalian cells like pREP (Invitrogen), pcDNA3 (Invitrogen),
pCEP4 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5
(Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo,
pSV2-dhfr, pIZD35, pLXIN, pSIR (Clontech), pIRES-EGFP (Clontech),
pEAK-10 (Edge Biosystems) pTriEx-Hygro (Novagen) and pCINeo
(Promega).
[0034] For vector modification techniques, see Sambrook and Russel
(2001), loc. cit. Generally, vectors can contain one or more origin
of replication (ori) and inheritance systems for cloning or
expression, one or more markers for selection in the host, e. g.,
antibiotic resistance, and one or more expression cassettes.
Suitable origins of replication (ori) include, for example, the Col
E1, the SV40 viral and the M 13 origins of replication.
[0035] The coding nucleic acid sequences inserted in the vector can
e.g. be synthesized by standard methods, or isolated from natural
sources. Ligation of the coding sequences to transcriptional
regulatory elements and/or to other amino acid encoding sequences
can be carried out using established methods. Transcriptional
regulatory elements (parts of an expression cassette) ensuring
expression in eukaryotic cells are well known to those skilled in
the art. These elements comprise regulatory sequences ensuring the
initiation of the transcription (e. g., translation initiation
codon, promoters, enhancers, and/or insulators), internal ribosomal
entry sites (IRES) (Owens, Proc. Natl. Acad. Sci. USA 98 (2001),
1471-1476) and optionally poly-A signals ensuring termination of
transcription and stabilization of the transcript. Additional
regulatory elements may include transcriptional as well as
translational enhancers, and/or naturally-associated or
heterologous promoter regions. Preferably, the nucleic acid
sequences of the invention are operatively linked to such
expression control sequences allowing expression in eukaryotic
cells. The vector may further comprise nucleotide sequences
encoding secretion signals as further regulatory elements. Such
sequences are well known to the person skilled in the art.
Furthermore, depending on the expression system used, leader
sequences capable of directing the expressed polypeptide to a
cellular compartment may be added to the coding sequence of the
polynucleotide of the invention. Such leader sequences are well
known in the art.
[0036] Possible examples for regulatory elements ensuring the
initiation of transcription comprise the cytomegalovirus (CMV)
promoter, SV40-promoter, RSV-promoter (Rous sarcome virus), the
lacZ promoter, the gai10 promoter, human elongation factor
1.alpha.-promoter, CMV enhancer, CaM-kinase promoter, the
Autographa californica multiple nuclear polyhedrosis virus (AcMNPV)
polyhedral promoter or the SV40-enhancer. Examples for further
regulatory elements in eukaryotic cells comprise transcription
termination signals, such as SV40-poly-A site or the tk-poly-A site
or the SV40, lacZ and AcMNPV polyhedral polyadenylation signals,
downstream of the nucleic acid sequences.
[0037] Furthermore, it is preferred that the vector of the
invention comprises a selectable marker. Examples of selectable
markers include neomycin, ampicillin, and hygromycin resistance and
the like. Specifically-designed vectors allow the shuttling of DNA
between different hosts, such as bacteria-fungal cells or
bacteria-animal cells.
[0038] An expression vector according to this invention is capable
of directing the replication, and the expression, of the nucleic
acid sequences and encoded peptides of this invention. Suitable
expression vectors which comprise the described regulatory elements
are known in the art such as Okayama-Berg cDNA expression vector
pcDV1 (Pharmacia), pRc/CMV, pcDNA1, pcDNA3 (Invitrogene, as used,
inter alia in the appended examples), pSPORT1 (GIBCO BRL), Gateway
plasmids (Invitrogen) or pGEMHE (Promega).
[0039] The nucleic acid sequences of the invention as described
herein above may be designed for direct introduction or for
introduction via electroporation (using for example Multiporator
(Eppendorf) or Genepulser (BioRad)), PEI (Polysciences Inc.
Warrington, Eppelheim), Ca.sup.2+-mediated transfection or via
liposomes (example: "Lipofectamine" (Invitrogen)), non-liposomal
compounds (example: "Fugene" (Roche)), liposomes, phage vectors or
viral vectors (e.g. adenoviral, retroviral, lentiviral) into the
cell. Additionally, baculoviral systems or systems based on
Vaccinia Virus or Semliki Forest Virus can be used as eukaryotic
expression system for the nucleic acid sequences of the
invention.
[0040] The term "MHC" is used interchangeably with HLA herein. In
accordance with the present invention it is a prerequisite that
"MHC molecules recognised by the T cell receptor" are employed.
Thus, when screening for a CD8+ T cell target antigen, the MHC
expressed by the cell of (ab) has to be a class I MHC while when
screening for CD4+ T cell target antigen, the MHC has to be a class
II MHC. Furthermore, the specific MHC recognised by the T cell
receptor expressed by the cell of (aa) needs to be employed. When
the specific HLA gene encoding the MHC recognised by the T cell
receptor under investigation is not know, then a preceding
experiment may be performed to screen the complete set of all MHC
(HLA) molecules expressed from all alleles of the subject, e.g. of
a patient, using methods well established in the art, such as for
example as discussed in Robinson J, et al. (2003) (see:
http://www.ebi.ac.uk/imgt/h1a/citations.html); Bettinotti et al.
(2003); Marsh et al (2010).
[0041] Accordingly, the cells of (aa) that express the T cell
receptor of interest will be activated when in contact with those
cells (ab) that present an antigen of said T cell receptor. T cell
activation is observable due to the read-out system and allows for
convenient isolation of the cell complex of (ab) with (aa) or of
the activating cells of (ab) only. Methods of isolation of cells
are well known in the art and include, without being limiting,
manual picking of cells or automated picking by use of a robot,
laser-capture microdissection or FACS (fluorescence activated cell
sorting). Such methods are described for example in Murray (2007)
or Tung et al. (2007) as well as in the examples below.
[0042] After isolation of the antigen presenting cell of (ab), the
nucleic acid molecule encoding the peptide(s) of the peptide
library present in said individual cell are isolated. Methods of
isolation of a nucleic acid molecule are well known in the art and
exemplary methods are also described in the examples below. Prior
to identification of the target antigen or the nucleic acid
molecule encoding it, said nucleic acid molecule may be amplified.
Amplification can include, for example, amplification of the
nucleic acid molecule via PCR technology as further detailed below
and/or transfection of the nucleic acid molecule into bacterial
cells and subsequent culture of the bacteria.
[0043] The amplified nucleic acid molecules or the peptides encoded
by these nucleic acid molecules are then identified. The term
"identifying", as used in accordance with the present invention,
refers to determining the amino acid or nucleic acid sequence of
the target antigen(s). Methods of identification of nucleic acid
molecules include, without being limiting, nucleic acid
sequencing.
[0044] Subsequently, the target antigen or the nucleic acid
molecule encoding said target antigen is identified.
[0045] In accordance with the present invention, an unbiased and
straightforward technique for determining the target antigens of T
cells is provided. As is shown in the examples below, the
intricacies of protein processing are circumvented by
co-transfecting a recombinant combinatorial library encoding short
peptides and the appropriate MHC-I molecules into cells, such as
for example COS cells. The library contains plasmids comprising
random nucleotide sequences encoding short peptides. These plasmids
can be easily recovered. Transfected COS cells are, as for example
shown in the examples below, screened with cells which carry
functional CD3 and downstream signalling molecules and are
co-transfected with TCR .alpha.- and .beta.-chains (T-cell receptor
transfectants), human CD8.alpha.- and .beta.-chains, and sGFP under
the control of the response element of the nuclear factor of
activated T cells (NFAT) in cells that carry NFAT (Fiering et al.,
1990; Karttunen and Shastri, 1991). Preferably, these cells are
TCR-transfected T-hybridoma cells, as this provides unlimited
numbers of "revived" T cells and avoids establishing "real" T cell
clones, which is often more problematic.
[0046] A confluent layer of library-presenting COS cells is
overlaid with the TCR-transfectants. Whenever a particular single
TCR-transfectant is activated by a peptide from the library, it
lights up green, and the underlying COS cell can be picked with a
micromanipulator under a fluorescent microscope. By this
high-throughput approach, several millions of library-peptides can
be examined within a few hours. This approach is more efficient
than previously described methods, which measured cytotoxicity or
secreted cytokines in pools of plasmid transfected APCs or by
several rounds of FACSort (Van der Bruggen et al., 1991; Smith et
al., 2001; Nino-Vasquez et al., 2005; Crawford et al., 2006).
[0047] The library-plasmids from the recovered COS-cell may then be
isolated, subjected to a new round of screening, and are finally
sequenced. Then the T cell receptor activating motif can be
analyzed and compared to existing proteins in the database. The
feasibility of this technique is demonstrated in the appended
examples using the well investigated TCR JM22 (Gotch et al., 1987;
Stewart-Jones et al., 2003; Ishizuka et al., 2008) that is specific
for HLA-A2 and the influenza matrix protein peptide flu(58-66).
[0048] A major advantage of the inventive technology is that it
does not depend on the antigen processing machinery of the
antigen-presenting cells by providing plasmid vectors encoding
short peptides. As is shown in the examples, a high number of
antigen-presenting cells efficiently present the encoded peptides
(e.g. flu(58-66)) after transfection with plasmid pcDNA (FIG.
2c,2e). The examples also provide evidence that the peptides, such
as flu(58-66), are efficiently transported after expression into
the lumen of the endoplasmic reticulum where they are loaded onto
HLA-A*0201. Strikingly, the full-length protein flu(1-252) was not
recognized (FIG. 2g). This suggests that the full length protein is
not correctly processed by COS-7 cells and illustrates a general
problem of cDNA libraries, namely that they rely on the processing
machinery of the chosen APC. A situation that exactly resembles
antigen presentation in a lesion of a patient will presumably never
be available in vitro, for several reasons: First, in many cases it
is not even known which cells present the (auto)antigen in situ.
Second, even if this were known, it would be almost impossible or
at least very difficult to obtain and propagate the original APCs.
Third, antigen processing in inflamed tissue may be altered as
compared to healthy tissue (Martinon et al., 2009). This may lead
to a very different peptide spectrum from identical parent
proteins. Fourth, the antigens, i.e. the cDNA libraries, must be
introduced into primary cells, and it is known that the
transfection and transformation efficacies are very low. The method
presented herein circumvents all these problems since it does not
depend on antigen processing at all. Further, COS cells can be
transfected with high efficacy and they amplify the plasmid vector
in their cytosol, which allows for it to be recovered and
analyzed.
[0049] The results provided herein demonstrate that it is possible
to identify mimotopes that allow unequivocal identification of the
parent, naturally occurring peptide by a simple database search.
The list of identified mimotopes generated by the present
technology may not be complete, because some peptides may be
digested further by endo- or exopeptidases in the cytosol of the
COS cells, or some may be lost due to their chemical properties,
i.e. they may be hydrophobic and insert into membranes, or they may
bind to proteins or other cellular components. There are indeed
technologies which are based on chemically synthesized peptide
libraries, that provide a comprehensive overview on the
polyspecificity of a particular TCR (Nino-Vasquez et al., 2005).
However, reports on identification of a priori unknown antigens are
scarce. This is presumably due to the fact that chemically
synthesized peptides may only be analyzed in pools of a tremendous
multitude of other peptides: The "correct" peptide may therefore be
present in the library, but only at a dilution that is too low to
be detected. In contrast, the high-throughout technology of the
present invention allows to pick a single activating cell from
millions of negative cells, and then to amplify the positive
plasmid. Thus, the present approach is oriented pragmatically: even
if the list of mimotopes may be incomplete, it is still sufficient
to find enough mimotopes for detecting the parent peptide in a
database.
[0050] Although the present technology may be reminiscent of
methods for screening phage libraries or approaches that use
oligomerised soluble T cell receptors as detection tools (Crawford
et al., 2006), there is a fundamental difference, because the
present method does not dependent on high affinities of
TCR-MHC/peptide interactions. Screening of phage libraries involves
several panning steps, where reasonable affinities are a
prerequisite. For usual applications of phage libraries, i.e. for
investigating interactions of ligands with antibodies or receptors,
the affinities are surely high enough. T cell receptors, by
contrast, have notoriously low affinities: They are typically
several orders of magnitude lower than those observed for
antibodies. Even TCR-oligomerisation may not be able to overcome
this disadvantage, because the readout of such an assay is still a
binding assay with moderate sensitivity. This is presumably the
reason why, as discussed above, a priori unknown T cell antigens
have not yet been discovered by this technique. By contrast, the
present invention makes use of an extremely sensitive bioassay: the
activation of a cell, such as a T hybridoma cell, that just needs
to be loosely attached to its antigen-presenting cell, is measured.
The tremendous sensitivity of this assay compensates for the low
binding affinity of the T cell receptor to its MHC/peptide
complex.
[0051] The technology of the present invention may have several
implications not only for scientific purposes, but also for medical
applications. First, it is a generally applicable method, i.e., it
may be applied to any T cell of interest. It is not restricted to
human T cells, but the antigen recognition properties of any TCR
from any species may be investigated. This is of course highly
interesting for many scientific questions, where T cells are
involved. Technically, the only proviso is that the specific
class-I MHC molecule is known, or that the specific class-II MHC
molecule is known. If this is not the case, then the complete set
of all candidate alleles has to be identified in a preceding
experiment, using methods well established in the art (Robinson J,
et al. (2003); http://www.ebi.ac.uk/imgt/h1a/citations.html;
Bettinotti et al. (2003); Marsh et al (2010)). Then all candidate
HLA alleles can be tested by the method provided here. Further,
there is no need to generate primary cells, neither T cells, nor
antigen presenting cells, which may often be highly problematic, in
particular if human samples are investigated.
[0052] The most important perspective, however, is in the medical
field: T cells are involved in many diseases. They defeat
infections and tumours, and they attack "self" tissue in
autoimmunity. In most cases their precise antigens are not known.
Knowing these epitopes will enable scientists to utilise this
knowledge in diagnostics and therapy. Thus, the antigens may serve
as biomarkers to detect infections and tumours and to improve
diagnosis of many autoimmune diseases. Further, such biomarkers may
be of great prognostic value for the progression of diseases.
Knowing the antigens may also be helpful in therapeutic approaches:
They could be used in vaccinations against infections or tumours
(Boon et al., 2006), and they may allow to selectively delete or
modulate auto-aggressive T cell clones in many autoimmune diseases,
where e.g. CD8+ T cells are known to play prominent roles, i.e. in
multiple sclerosis, psoriasis, inflammatory myopathies and many
others more (Friese and Fugger, 2009; Dalakas, 2006; Walter and
Santamaria, 2005).
[0053] Further, knowledge of the antigens may allow the rational
design and development of new, improved T cell receptors that are
engineered for higher affinities to their target antigens. Such
improved T cell receptors may be produced by replacement or
introduction of amino acids in the complementarity determining
regions by site-specific mutagenesis. The improved T cell receptors
may be used for detection of their antigens in vitro and in vivo
for scientific, diagnostic and therapeutic purposes. They may be
used to detect, block, or delete (by coupling toxic substances)
cells that present certain antigens. If required, T cell receptors
may be oligomerized to increase avidity. Thus, in an alternative
embodiment, the present invention also refers to engineered T cell
receptors, wherein amino acids are altered as compared to a natural
T cell receptor and wherein said alteration results in an increased
affinity to the target antigen as compared to the unaltered T cell
receptor.
[0054] In addition, the knowledge of specific antigens may also
allow the design of altered peptide ligands for a particular T cell
receptor of interest. Such altered antigens may be of importance
for inducing T cell tolerance or improving the immunogenicity of a
particular antigen vaccine. Methods for the rational design of
altered and/or improved T cell antigens are well known in the art
(Fontoura et al. (2005); Bielekova and Martin (2001)).
[0055] In a preferred embodiment, the method of the invention
comprises repeating steps (a) to (c) of the above defined method
until a single molecular type of nucleic acid molecule encoding a
single type of antigen is isolated. Thus, steps (a) to (c) may be
repeated at least one more time, such as for example two more
times, three more times, four more times, five more times, six more
times, seven more times, eight more time, nine more times or ten
more times. A higher number of repeats of steps (a) to (c) is also
envisaged herein.
[0056] Employing standard methods of introducing the peptide
library into the antigen-presenting cells of (ab) may result in the
presence of more than one copy of the encoding nucleic acid
molecule within said cell. Furthermore, nucleic acid molecules
present in the culture medium may be transferred together with the
antigen-presenting cell when isolating said cell in step (c). Thus,
performing the method of the invention with only one round of steps
(a) to (c) may result in the isolation of an antigen-presenting
cell that carries multiple diverse copies of nucleic acid molecules
encoding different antigen peptides or to which multiple diverse
copies of nucleic acid molecules encoding different antigen
peptides are attached. By repeating steps (a) to (c), the correct
target antigen may be identified in the subsequent step (d). It is
understood that when repeating step (a), the antigen-presenting
cell is a cell carrying the nucleic acid molecules isolated from
the antigen-presenting cell isolated in step (c) in the previous
round of screening.
[0057] FIG. 6 below provides an overview over a preferred way of
carrying out these repeated method steps in order to obtain a
single molecular type of nucleic acid molecule encoding a single
type of antigen. In brief, in a first step an activated, green
fluorescent TCR-transfectant is picked together with the subjacent
antigen presenting cell that carries the activating plasmid. The
inserts of the plasmids are cloned, and transfected into bacteria.
To count the numbers of bacterial clones, a fraction of the
bacterial clones is plated onto agar plates. Then 30 or more
sub-pools of bacteria are created that contain 500 independent
bacterial clones, each. They are transfected into COS-7 cells and
tested again. From a positive sub-pool, further sub-pools are
generated, which contain less clones per sub-pool. Finally, single
bacterial clones are analyzed. Positive clones are sequenced and
reveal the antigenic mimotope.
[0058] In another preferred embodiment, the cells of (aa) further
express CD8 chains, more preferably CD8.alpha. and .beta.
chains.
[0059] The additional expression of CD8 on the cells of (aa) serves
as a co-receptor for the T cell receptor (TCR). The extracellular
IgV-like domain of CD8-.alpha. interacts with the .alpha..sub.3
portion of Class I MHC molecules. This interaction keeps the cell
expressing the T cell receptor closely together with the target
cell bound during antigen-specific activation. Thus, additional
expression of CD8 on the cells of (aa) further enhances the
interaction between the two cells in accordance with the
invention.
[0060] In another preferred embodiment of the method of the
invention, the identification of the target antigen in (d)
comprises sequencing of the nucleic acid molecule encoding said
target antigen.
[0061] Methods for sequencing comprise, without being limiting,
approaches of sequence analysis by direct sequencing, fluorescent
SSCP in an automated DNA sequencer and pyro-sequencing. These
methods are well known in the art, see e.g. Adams et al. (Ed.),
"Automated DNA Sequencing and Analysis", Academic Press, 1994;
Alphey, "DNA Sequencing: From Experimental Methods to
Bioinformatics", Springer Verlag Publishing, 1997; Ramon et al., J.
Transl. Med. 1 (2003) 9; Meng et al., J. Clin. Endocrinol. Metab.
90 (2005) 3419-3422.
[0062] In a more preferred embodiment, the nucleic acid molecule
encoding said target antigen is amplified prior to sequencing.
[0063] Techniques for amplifying nucleic acid molecules include,
but are not limited to, PCR and its various modifications such as
RT-PCR (also referred to as reverse transcriptase-PCR). PCR is well
known in the art and is employed to make large numbers of copies of
a target sequence. This is done on an automated cycler device,
which can heat and cool containers with the reaction mixture in a
very short time. The PCR, generally, consists of many repetitions
of a cycle which consists of: (a) a denaturing step, which melts
both strands of a DNA molecule and terminates all previous
enzymatic reactions; (b) an annealing step, which is aimed at
allowing the primers to anneal specifically to the melted strands
of the DNA molecule; and (c) an extension step, which elongates the
annealed primers by using the information provided by the template
strand. Generally, PCR can be performed for example in a 50 .mu.l
reaction mixture containing 5 .mu.l of 10.times. PCR buffer with
1.5 mM MgCl2, 200 .mu.M of each deoxynucleoside triphosphate, 0.5
.mu.l of each primer (10 .mu.M), about 10 to 100 ng of template DNA
and 1 to 2.5 units of Taq Polymerase. The primers for the
amplification may be labelled or be unlabelled. DNA amplification
can be performed, e.g., with a model 2400 thermal cycler (Applied
Biosystems, Foster City, Calif.): 2 min at 94.degree. C., followed
by 30 to 40 cycles consisting of annealing (e. g. 30 s at
50.degree. C.), extension (e. g. 1 min at 72.degree. C., depending
on the length of DNA template and the enzyme used), denaturing (e.
g. 10 s at 94.degree. C.) and a final annealing step at 55.degree.
C. for 1 min as well as a final extension step at 72.degree. C. for
5 min. Suitable polymerases for use with a DNA template include,
for example, E. coli DNA polymerase I or its Klenow fragment, T4
DNA polymerase, Tth polymerase, Taq polymerase, a heat-stable DNA
polymerase isolated from Thermus aquaticus Vent, Amplitaq, iProof,
Pfu and KOD, some of which may exhibit proof-reading function
and/or different temperature optima. However, the person skilled in
the art knows how to optimize PCR conditions for the amplification
of specific nucleic acid molecules with primers of different length
and/or composition or to scale down or increase the volume of the
reaction mix. The "reverse transcriptase polymerase chain reaction"
(RT-PCR) is used when the nucleic acid to be amplified consists of
RNA. The term "reverse transcriptase" refers to an enzyme that
catalyzes the polymerization of deoxyribonucleoside triphosphates
to form primer extension products that are complementary to a
ribonucleic acid template. RT-PCR is particularly suitable when RNA
viruses are employed in order to encode the library plasmids for
use in the method of the invention. The enzyme initiates synthesis
at the 3'-end of the primer and proceeds toward the 5'-end of the
template until synthesis terminates. Examples of suitable
polymerizing agents that convert the RNA target sequence into a
complementary, copy-DNA (cDNA) sequence are avian myeloblastosis
virus reverse transcriptase and Thermus thermophilus DNA
polymerase, a thermostable DNA polymerase with reverse
transcriptase activity marketed by Perkin Elmer. Typically, the
genomic RNA/cDNA duplex template is heat denatured during the first
denaturation step after the initial reverse transcription step
leaving the DNA strand available as an amplification template.
High-temperature RT provides greater primer specificity and
improved efficiency. U.S. patent application Ser. No. 07/746,121,
filed Aug. 15, 1991, describes a "homogeneous RT-PCR" in which the
same primers and polymerase suffice for both the reverse
transcription and the PCR amplification steps, and the reaction
conditions are optimized so that both reactions occur without a
change of reagents. Thermus thermophilus DNA polymerase, a
thermostable DNA polymerase that can function as a reverse
transcriptase, can be used for all primer extension steps,
regardless of template. Both processes can be done without having
to open the tube to change or add reagents; only the temperature
profile is adjusted between the first cycle (RNA template) and the
rest of the amplification cycles (DNA template). The RT Reaction
can be performed, for example, in a 20 .mu.l reaction mix
containing: 4 .mu.l of 5.times.AMV-RT buffer, 2 .mu.l of Oligo dT
(100 .mu.g/ml), 2 .mu.l of 10 mM dNTPs, 1 .mu.l total RNA, 10 Units
of AMV reverse transcriptase, and H.sub.2O to 20 .mu.l final
volume. The reaction may be, for example, performed by using the
following conditions: The reaction is held at 70 C..degree. for 15
minutes to allow for reverse transcription. The reaction
temperature is then raised to 95 C..degree. for 1 minute to
denature the RNA-cDNA duplex. Next, the reaction temperature
undergoes two cycles of 95.degree. C. for 15 seconds and 60
C..degree. for 20 seconds followed by 38 cycles of 90 C..degree.
for 15 seconds and 60 C..degree. for 20 seconds. Finally, the
reaction temperature is held at 60 C..degree. for 4 minutes for the
final extension step, cooled to 15 C.degree., and held at that
temperature until further processing of the amplified sample. Any
of the above mentioned reaction conditions may be scaled up
according to the needs of the particular case.
[0064] Suitable primers for both the sequencing as well as the
amplification of nucleic acid molecules can be derived by the
skilled person using well-established methods. For example, primer
sequences may be derived based on the knowledge of up-stream and
down-stream flanking regions surrounding the randomised nucleic
acid sequences encoding the peptide libraries, such as for example
the plasmid backbone into which said nucleic acid sequences are
inserted to, the promoter sequence employed to express the peptides
or other regulatory sequences, such as enhancer sequences.
[0065] In a further preferred embodiment of the method of the
invention, the identification of the target antigen in (d)
comprises the identification of at least one mimotope of the
antigen.
[0066] The term "mimotope", in accordance with the present
invention, relates to a molecule, which mimics the structure of an
antigen epitope. Said molecule may, for example, be a peptide. Due
to the mimicking property the mimotope causes a T cell receptor
response identical to the one elicited by the naturally occurring
antigen epitope. A T cell receptor recognizing a particular antigen
epitope will thus also recognize a mimotope which mimics that
antigen epitope. Preferably, a mimotope differs from the natural
target antigen epitope of a particular T cell receptor in its amino
acid structure, e.g. the amino acid sequence and composition, such
as for example by a difference of less than 40 amino acids in amino
acids structure, such as for example less than 30 amino acids, less
than 20 amino acids, less than 15 amino acids less than 10 amino
acids, less than 5 amino acids, less than 4 amino acids, less than
3 amino acids and most preferably by one amino acid.
[0067] "At least one mimotope", as used herein, refers to any
number of possible mimotopes for a given antigen. It includes, for
example, at least two mimotopes, such as for example at least
three, at least four, at least five, at least six, at least seven,
at least eight, at least nine or at least 10 mimotopes. Also
included is at least 15, such as at least 20, at least 30, at least
40, at least 50, at least 75, at least 100, at least 150, at least
200, at least 300, at least 400 or at least 500 mimotopes.
[0068] This embodiment is based on the finding that it is possible
to identify mimotopes by the method of the present invention, which
may then be employed in a database search to identify the naturally
occurring antigen. Thus, even if the actual antigen is not directly
identified by the method of the invention, it can indirectly be
identified via said mimotopes, as described in the examples
below.
[0069] In another preferred embodiment of the method of the
invention, the antigen-presenting cells are cells capable of
amplifying the peptide libraries.
[0070] The term "cells capable of amplifying the peptide
libraries", as used herein, refers to cells capable to replicate
the nucleic acid sequences or the vector. Such cells are well known
in the art. Non-limiting examples include cells comprising the
large T antigen when employing plasmids having a SV40 origin as
described above; cells comprising the EBNA system as described in
Durocher et al. (2002) or cells that are suited to be transformed
by a virus, such as for example a retrovirus, adenovirus or
lentivirus, which may host and amplify such retroviral vectors
(Smith et al. (2001). Also cells not endogenously expressing said
systems may be employed, in which case the cells may simply be
transfected or transformed with the required components of the
respective system, for example with the large T antigen.
[0071] A suitable degree of amplification, in accordance with the
present invention, is achieved when more copies of the nucleic acid
sequences are present in the cell than were originally transfected
or transformed into said cell. Thus, amplification is achieved when
the amount of nucleic acid sequence copies is at least duplicated,
such as for example when at least 5% more copies of one molecular
species of nucleic acid sequence is present in the cell, such as
for example at least 10% more copies, such as at least 20% more
copies, at least 30% more copies, at least 50% more copies, at
least 100% more copies, at least 200% more copies, at least 500%
more copies, at least 1.000% more copies, at least 2.000% more
copies, at least 3.000% more copies, at least 4.000% more copies,
at least 5.000% more copies, at least 10.000% more copies, at least
15.000% more copies, at least 50.000% more copies, at least
100.000% more copies, at least 250.000% more copies or at least
500.000% more copies.
[0072] In order to verify whether a particular cell is capable of
amplifying the peptide libraries, some of said cells may be
analysed immediately after transfection/transformation for the
amount of copies of one molecular type of the nucleic acid
sequence. In addition, the same analysis may be repeated with cells
obtained at (a) later time point(s) and the copy numbers thus
determined may be compared. Methods for the quantitative or
semi-quantitative determination of copy numbers of any given
nucleic acid sequence are well known in the art and include,
without being limiting, PCR methods as described elsewhere
herein.
[0073] In a more preferred embodiment, the antigen-presenting cells
are selected from the group consisting of COS-7, HEK, Hela, H9,
Jurkat, NIH3T3, C127, COS-1, CV1, QC1-3, mouse L cells, mouse C2C12
cells and Chinese hamster ovary (CHO), Wi-38, MRC-5, insect cells
like Sf9, Hi-5 cells.
[0074] In another preferred embodiment, the read-out system
comprises the activation of a reporter protein.
[0075] The term "reporter protein", as used herein, refers to a
protein that is expressed in response to a certain stimulus to be
investigated and that can easily be detected. Thus, in accordance
with the present invention, it is preferred that the reporter
protein is expressed upon T cell activation. A reporter protein
preferably is a protein that is easy to detect and, more
preferably, it is a protein that is not present normally in the
cells investigated. Non-limiting examples of reporter proteins
include: .beta.-galactosidase (encoded by the bacterial gene lacZ),
luciferase, such as for example bacterial luciferase (luxAB),
Photinus luciferase and Renilla luciferase, chloramphenicol
acetyltransferase (CAT; from bacteria), GUS .beta.-glucuronidase;
commonly used in plants) as well as green fluorescent protein (GFP;
from jelly fish) and variants thereof, such as CFP, YFP, EGFP,
GFP+. Further non-limiting examples include alkaline phosphatase or
secreted alkaline phosphatase, peroxidase, .beta.-xylosidase, XylE
(catechol dioxygenase), TreA (trehalase) as well as coral-derived
photoproteins including DSRed, HcRed, AmCyan, ZsGreen, ZsYellow,
AsRed.
[0076] Furthermore, the reporter protein may be a protein that
confers resistance to an antibiotic. In this case, it is preferred
that the cells be cultivated in the presence of an antibiotic so
that only clones expressing the reporter protein are capable of
propagating. Generally, the protein can mediate resistance to an
antibiotic such as hygromycin, geneticin (G418), puromycin,
blasticidine, zeocin, histidinol, methotrexate, media with
xanthine/hypoxynthin-aminopterin-mycophenolic (Gpt selection), HAT
selection, indole media without tryptopha, or phleomycin. (R.Vile
(1991) Meth Mol. Biol. 8:49-60)
[0077] In a more preferred embodiment, the reporter protein is
selected from the group consisting of fluorescent compounds,
bioluminescent compounds and chemiluminescent compounds.
[0078] More preferably, the reporter protein is selected from the
group consisting of GFP and variants of GFP such as CFP, YFP, EGFP,
GFP+, sGFP, bacterial luciferase (luxAB), Photinus luciferase,
Renilla luciferase and coral-derived photoproteins including DSRed,
HcRed, AmCyan, ZsGreen, ZsYellow, AsRed.
[0079] The present invention further relates to a method of
identifying nucleic acid molecules encoding variable, hypervariable
and/or joining regions of T cell receptor .beta. chains, comprising
amplifying said nucleic acid molecules obtained from T cells
expressing .alpha..beta. T cell receptors using a primer or a set
of primers selected from the primers represented by SEQ ID NOs: 1
to 9.
[0080] The definitions as well as the preferred embodiments
provided herein above with regard to the other methods of the
invention apply mutatis mutandis also to this method of identifying
nucleic acid molecules encoding T cell receptor chains as well as
to the preferred embodiments thereof described herein below.
[0081] Preferably, the T cells are single T cells, i.e. individual
T cells or clonal expansions of individual T cells. In other words,
the T cells represent one molecular type of cell only.
[0082] In accordance with this method of the invention, nucleic
acid molecules are obtained from T cells using methods well known
in the art, e.g. as described elsewhere herein.
[0083] The term "set", as used herein, relates to a combination of
primers. The set of primers of the present invention requires that
more than one molecular species of primer is present. Thus, at
least two different primers, such as at least three, at least four,
at least five, at least six, at least seven, at least eight or at
least nine different primers selected from the primers represented
by SEQ ID NOs: 1 to 9 are comprised in the set of primers.
[0084] In accordance with the present invention, T cell receptor
V.beta.-primers were identified based on sequence homologies of the
various functional V.beta.-gene families. The primer positions are
such that direct sequencing can identify the respective TCR
V.beta.-gene. In addition, these primers for the V.beta. repertoire
are adjusted to each other as well as to a set of 24
V.alpha.-primers in order to minimize potential interactions during
PCR amplification. The set of nine V.beta. primers (Vp1-9, Table 1)
covers all functional VP genes. Except Vp1, which is located in the
leader segment, all primers are positioned in the V.beta.-gene
segment. In combination with a C.beta.(out)-primer (SEQ ID NO: 11)
each of these primers efficiently amplifies the corresponding
V.beta.-gene rearrangements, as shown by agarose
gel-electrophoresis and ethidium-bromide staining (FIG. 8a).
TABLE-US-00001 TABLE 1 V.beta. Primers and TRBV subfamilies covered
by V.sub.p primers. SEQ Vp ID primer Sequence NO: TRBV family
Basepairs Vp1 5'-TSY TTT GTC TCC TGG GAG CA- 1 5, 9, 13, 14, 19
29-48 3' Vp2 5'-CCT GAA GTC GCC CAG ACT CC- 2 2, 16 4-23 3' Vp3
5'-GTC ATS CAG AAC CCA AGA 3 15, 18 10-31 YAC C-3' Vp4 5'-GGW TAT
CTG TMA GMG TGG 4 20, 29 30-53 AAC CTC-3' Vp5 5'-ATG TAC TGG TAT
CGA CAA 5 6, 10, 24, 25, 94-115 GAY C-3' 27, 28 Vp6 5'-CAC TGT GGA
AGG AAC ATC 6 30 69-91 AAA CC-3' Vp7 5'-TCT CCA CTC TSA AGA TCC 7
7, 11, 12 221-241 AGC-3' Vp8 5'-CAG RAT GTA RAT YTC AGG 8 7 50-75
TGT GAT CC-3' Vp9 5'-CCA GAC WCC AAR AYA CCT 9 3, 4 15-37 GGT CA-3'
The nomenclature of degenerate basepairs relates to the IUPAC
nomenclature of mixed bases. Examples: S = C or G; Y = T or C; W =
T or A; M = C or A; R = A or G; K = G or T; V = A or C or G; H = A
or T or C; D = A or T or G; B = T or C or G; N = A or T or C or G
(Cornish-Bowden A: IUPAC-IUB symbols for nucleotide nomenclature.
Nucleic Acids Res 1985, 13: 3021-3030.).
[0085] In a preferred embodiment of the method of identifying
nucleic acid molecules encoding T cell receptor .beta. chains, the
amplification further comprises the use of a C.beta.(out)-primer
(SEQ ID NO: 11).
TABLE-US-00002 TABLE 2 C primer and universal primer (UP) SEQ C
primer Sequence ID NO: C.alpha. out 5'-GCA GAC AGA CTT GTC ACT
GG-3' 10 C.beta. out 5'-TGG TCG GGG AAG AAG CCT GTG-3' 11 C.alpha.
in 5'-AG TCT CTC AGC TGG TAC ACG-3' 12 UP 5'-ACA GCA CGA CTT CCA
AGA CTC A-3' 13 C.beta. in 5'-TCT GAT GGC TCA AAC ACA GC-3' 14
[0086] In a further preferred embodiment of the method of
identifying nucleic acid molecules encoding T cell receptor .beta.
chains, the method further comprises identifying the nucleic acid
molecules encoding the variable, hypervariable and/or joining
regions of matching T cell receptor .alpha. chains by amplifying
said nucleic acid molecules using a primer or a set of primers
selected from the primers represented by SEQ ID NOs: 15 to 38.
[0087] The 24 T cell receptor V.alpha.-primers represented by SEQ
ID NOs: 15 to 38 have been recently described for the amplification
of the TCR V.alpha. repertoire (Seitz, Schneider et al. 2006) and
are shown in Table 3.
TABLE-US-00003 TABLE 3 V.alpha. primers. SEQ ID Vp primer Sequence
NO: V.alpha.-1.sup.14-for-out 5'-AGS AGC CTC ACT GGA GTT G- 15 3'
V.alpha.-1.sup.235-for-out 5'-CTG AGG TGC AAC TAC TCA 16 TC-3'
V.alpha.-2-for-out 5'-CAR TGT TCC AGA GGG AGC C- 17 3'
V.alpha.-3,25-for-out 5'-GAA RAT GYC WCC ATG AAC 18 TGC-3'
V.alpha.-4,20-for-out 5'-WTG CTA AGA CCA CCC AGC C- 19 3'
V.alpha.-5-for-out 5'-AGA TAG AAC AGA ATT CCG 20 AGG-3'
V.alpha.-6,14-for-out 5'-RYT GCA CAT ATG ACA CCA 21 GTG-3'
V.alpha.-7-for-out 5'-CAC GTA CCA GAC ATC TGG G- 22 3'
V.alpha.-8,21-for-out 5'-CCT GAG YGT CCA GGA RGG- 23 3'
V.alpha.-9-for-out 5'-GTG CAA CTA TTC CTA TTC 24 TGG-3'
V.alpha.-10,24-for-out 5'-AST GGA GCA GAG YCC TCA G- 25 3'
V.alpha.-11-for-out 5'-TCT TCA GAG GGA GCT GTG G- 26 3'
V.alpha.-12-for-out 5'-GGT GGA GAA GGA GGA TGT G- 27 3'
V.alpha.-13,19,26-for- 5'-SAA STG GAG CAG AGT CCT C- 28 out 3'
V.alpha.-15-for-out 5'-CCT GAG TGT CCG AGA GGG- 29 3'
V.alpha.-16-for-out 5'-ATG CAC CTA TTC AGT CTC 30 TGG-3'
V.alpha.-17-for-out 5'-TGA TAG TCC AGA AAG GAG 31 GG-3'
V.alpha.-18-for-out 5'-GTC ACT GCA TGT TCA GGA 32 GG-3'
V.alpha.-22,31-for-out 5'-CCC TWC CCT TTT CTG GTA TG- 33 3'
V.alpha.-23,30-for-out 5'-GGC ARG AYC CTG GGA AAG G- 34 3'
V.alpha.-27-for-out 5'-CTG TTC CTG AGC ATG CAG G- 35 3'
V.alpha.-28-for-out 5'-AGA CAA GGT GGT ACA AAG 36 CC-3'
V.alpha.-29-for-out 5'-CAA CCA GTG CAG AGT CCT C- 37 3'
V.alpha.-32-for-out 5'-GCA TGT ACA AGA AGG AGA 38 GG-3' The
nomenclature of degenerate basepairs relates to the IUPAC
nomenclature of mixed bases. Examples: S = C or G; Y = T or C; W =
T or A; M = C or A; R = A or G; K = G or T; V = A or C or G; H = A
or T or C; D = A or T or G; B = T or C or G; N = A or T or C or G
(Cornish-Bowden A: IUPAC-IUB symbols for nucleotide nomenclature.
Nucleic Acids Res 1985, 13: 3021-3030.).
[0088] In a preferred embodiment, the amplification further
comprises the use of a C.alpha. out-primer (SEQ ID NO: 10).
[0089] In a more preferred embodiment of the method of identifying
nucleic acid molecules encoding T cell receptor .beta. chains, the
method comprises the steps: (i) amplifying nucleic acid molecules
using a set of primers comprising (a) the primers represented by
SEQ ID NOs: 1 to 9; and/or (b) the primers represented by SEQ ID
NOs: 15 to 38; (ii) amplifying the reaction product of (i)(a) using
a set of primers comprising the primers represented by SEQ ID NOs:
39 to 47; and/or (iii-a) amplifying the reaction product of (ii)
using (a) the primer of SEQ ID NO: 13; and (b) the primer of SEQ ID
NO: 14 and/or (iii-b) amplifying the reaction product of (i)(b)
using (a) a set of primers represented by SEQ ID NOs: 48 to 83 and
(b) the primer of SEQ ID NO: 12.
TABLE-US-00004 TABLE 4 Vp+ primers. SEQ Vp+ ID primer Sequence NO:
Vp1+ 5'-ACAGCACGACTTCCAAGACTCA 39 CYTTTGTCTCCTGGGAGCA-3' Vp2+
5'-ACAGCACGACTTCCAAGACTCA 40 CCTGATGTCGCCCAGACTCC-3' Vp3+
5'-ACAGCACGACTTCCAAGACTCA 41 GTCATSCAGAACCCAAGAYACC-3' Vp4+
5'-ACAGCACGACTTCCAAGACTCA 42 GGWTATCTGTMAGMGTGGAACCTC-3' Vp5+
5'-ACAGCACGACTTCCAAGACTCA 43 ATGTACTGGTATCGACAAGAYC-3' Vp6+
5'-ACAGCACGACTTCCAAGACTCA 44 CACTGTGGAAGGAACATCAAACC-3' Vp7+
5'-ACAGCACGACTTCCAAGACTCA 45 TCTCCACTCTSAAGATCCAGC-3' Vp8+
5'-ACAGCACGACTTCCAAGACTCA 46 CAGRATGTARATYTCAGGTGTGATCC-3' Vp9+
5'-ACAGCACGACTTCCAAGACTCA 47 TCAGACWCCAARAYACCTGGTCA-3' The
nomenclature of degenerate basepairs relates to the IUPAC
nomenclature of mixed bases. Examples: S = C or G; Y = T or C; W =
T or A; M = C or A; R = A or G; K = G or T; V = A or C or G; H = A
or T or C; D = A or T or G; B = T or C or G; N = A or T or C or G
(Cornish-Bowden A: IUPAC-IUB symbols for nucleotide nomenclature.
Nucleic Acids Res 1985, 13: 3021-3030.).
TABLE-US-00005 TABLE 5 V.alpha. nested primers and preferred sets
thereof. SEQ ID Vp+ primer Sequence NO: Set 1 V.alpha.-4/1-for-in
5'-ACA GAA GAC AGA AAG TCC AGC-3' 48 V.alpha.-4/2-for-in 5'-GTC CAG
TAC CTT GAT CCT GC-3' 49 V.alpha.-6-for-in 5'-GCA AAA TGC AAC AGA
AGG TCG-3' 50 V.alpha.-8/1-for-in 5'-CAG TGC CTC AAA CTA CTT CC-3'
51 V.alpha.-8/2-for-in 5'-GCC TCA GAC TAC TTC ATT TGG-3' 52
V.alpha.-14-for-in 5'-ACA GAA TGC AAC GGA GAA TCG-3' 53
V.alpha.-24-for-in 5'-CCT TCA GCA ACT TAA GGT GG-3' 54
V.alpha.-28-for-in 5'-TCT CTG GTT GTC CAC GAG G-3' 55 Set 2
V.alpha.-2/1-for-in 5'-TGG AAG GTT TAC AGC ACA GC-3' 56
V.alpha.-2/2-for-in 5'-TGG AAG GTT TAC AGC ACA GG-3' 57
V.alpha.-5-for-in 5'-CAG CAT ACT TAC AGT GGT ACC-3' 58
V.alpha.-10-for-in 5'-TCA CTG TGT ACT GCA ACT CC-3' 59
V.alpha.-12-for-in 5'-TAC AAG CAA CCA CCA AGT GG-3' 60
V.alpha.-22-for-in 5'-AGG CTG ATG ACA AGG GAA GC-3' 61
V.alpha.-31-for-in 5'-GTG GAA TAC CCC AGC AAA CC-3' 62 Set 3
V.alpha.-7-for-in 5'-CTC CAG ATG AAA GAC TCT GC-3' 63
V.alpha.-13-for-in 5'-TTA AGC GCC ACG ACT GTC G-3' 64
V.alpha.-17-for-in 5'-CTG TGC TTA TGA GAA CAC TGC-3' 65
V.alpha.-18-for-in 5'-CC TTA CAC TGG TAC AGA TGG-3' 66
V.alpha.-21-for-in 5'-TGC TGA AGG TCC TAC ATT CC-3' 67
V.alpha.-23-for-in 5'-GTG GAA GAC TTA ATG CCT CG-3' 68
V.alpha.-32-for-in 5'-TCA CCA CGT ACT GCA ATT CC-3' 69 Set 4
V.alpha.-3-for-in 5'-TTC AGG TAG AGG CCT TGT CC-3' 70
V.alpha.-11-for-in 5'-AGG GAC GAT ACA ACA TGA CC-3' 71
V.alpha.-15-for-in 5'-CCT CCA CCT ACT TAT ACT GG-3' 72
V.alpha.-19-for-in 5'-CCT GCA CAT CAC AGC CTC C-3' 73
V.alpha.-25-for-in 5'-AGA CTG ACT GCT CAG TTT GG-3' 74
V.alpha.-26-for-in 5'-CCT GCA TAT CAC AGC CTC C-3' 75
V.alpha.-29-for-in 5'-ACT GCA GTT CCT CCA AGG C-3' 76 Set 5
V.alpha.-1/235-for-in 5'-AAG GCA TCA ACG GTT TTG AGG-3' 77
V.alpha.-1/14-for-in 5'-CTG AGG AAA CCC TCT GTG C-3' 78
V.alpha.-9-for-in 5'-ATC TTT CCA CCT GAA GAA ACC-3' 79
V.alpha.-16-for-in 5'-TCC TTC CAC CTG AAG AAA CC-3' 80
V.alpha.-20-for-in 5'-ACG TGG TAC CAA CAG TTT CC-3' 81
V.alpha.-27-for-in 5'-ACT TCA GAC AGA CTG TAT TGG-3' 82
V.alpha.-30-for-in 5'-CTC TTC ACC CTG TAT TCA GC-3' 83
[0090] The above described method allows in a first step (step (i))
to simultaneously amplify all V.alpha.- and/or .beta.-chains from
cDNA isolated from cells of interest by using a pool of primers
comprising 24 V.alpha.-specific and 9 V.beta.-specific primers
(Vp1-Vp9), which cover all functional .alpha. and .beta. TCR
variable region genes. In a second step, i.e. step (ii), the
respective TCR V.beta.-chain rearrangements are amplified from the
pre-amplification product. Because the multitude of 23 different
V.beta.-gene subfamilies prohibits a V.beta.-specific nested PCR in
one sample, a universal primer sequence is introduced at the 5' end
of the TCR .beta.-chain PCR-products in step (ii). For this purpose
a unique 21-nucleotide sequence was designed lacking primer
interactions or homologies with human genes and appended to the 5'
end of nine different Vp (Vp1-9) primers (referred to herein as Vp+
primers). Parts of the pre-amplification product is subjected to a
run-off PCR using these elongated primers, followed by a third
semi-nested PCR, which amplifies the respective single cell
TCR-V.beta. rearrangement independent from the TCR V.beta.-gene
family using the universal primer together with a nested
C.beta.(2)-primer (SEQ ID NO: 14) (step (iii-a)). This PCR strategy
is shown in FIG. 9. In parallel or subsequently, the corresponding
TCR V.alpha.-rearrangements are amplified from the
pre-amplification (i.e. step (i)) multiplex PCR in different nested
PCRs (i.e. step (iii-b)). Preferably, only five different nested
PCRs are carried out using five V.alpha.-primer pools as shown in
Table 5 instead of individual reactions, described by Seitz et al.
The amplified TCR .alpha.- and .beta.-chain rearrangements can then
be characterized by direct sequencing.
[0091] In a preferred embodiment, the method described above is
preceded by a step of reverse transcription of mRNA of cells,
preferably a single cell or a clonally expanded single type of
cell, into cDNA. Such methods of reverse transcription are well
known and may be carried out, e.g. by using a one step RT-PCR kit
(such as provided by QIAGEN) and gene specific C.alpha.- and
.beta.-primers.
[0092] Molecular analysis of the paired .alpha..beta.-TCR
rearrangements of single T cells has to encompass the complete
spectrum of approx. 70 TCR V.alpha.- and approx. 50 TCR
V.beta.-region genes. In accordance with the present invention, a
PCR strategy was established that can amplify all different
V.beta.-gene families of the TCR .beta.-chain repertoire together
with a set of 24 TCR V.alpha.-primers recently described for the
simultaneous amplification of the TCR V.alpha. repertoire (Seitz,
Schneider et al. 2006). The complexity of this approach results
from potential interactions between the multitude of primers, which
may interfere with specific amplification. In accordance with the
present invention, a PCR protocol was established which starts with
a multiplex RT-PCR capable of pre-amplifying all TCR V.alpha.- and
V.beta.-genes in a single reaction. Subsequently, the TCR V.alpha.-
and V.beta.-PCR products are handled separately.
[0093] The present invention further relates to a method of
identifying patient-specific T cell antigens comprising (A)
isolating T cells from a sample obtained from said patient; (B)
identifying matching T cell receptor .alpha. and .beta. chains from
the T cells isolated in (A); and (C) identifying T cell antigens in
accordance with the method of the invention, wherein the cell
comprising a functional T cell receptor and a read-out system for T
cell activation expresses the matching T cell receptor .alpha. and
.beta. chains identified in (B).
[0094] The definitions as well as the preferred embodiments
provided herein above with regard to the method of the invention
apply mutatis mutandis also to this patient-specific method of
identifying T cell antigens.
[0095] In accordance with this patient-specific method of
identifying T cell antigens, T cells are isolated from a patient.
Methods for isolating T cells from patients are well known in the
art and include, without being limiting the isolation of T cells by
laser-micro-dissection from frozen tissue biopsy samples, by
picking of living or dead cells from fresh biopsy specimens
harbouring living cells by hand or by using a micromanipulator, by
isolating cells using a FACSort apparatus, by ex vivo cloning by
limiting dilution, with or without previous fusion of the cell with
a suitable tumor cell line to generate immortalized hybridoma cells
(Burgemeister R. (2005) J Histochem Cytochem. 53:409-12; Erickson H
S et al. (2008) Methods Mol Biol. 424:433-48; Dainiak M B et al.
(2007) Adv Biochem Eng Biotechnol. 106:1-18; Tung J W et al. (2007)
Clin Lab Med. 27:453-68).
[0096] In a subsequent step, the heterodimeric T cell receptors
expressed in said cells are identified. Methods for identifying T
cell receptors from T cells are known in the art and include,
without being limiting, amplification of T cell receptor .alpha.-
and .beta.-chains by PCR using clone-specific primers, e.g. in
cases where the sequence of one of the chains is known, for example
by CDR3-spectratyping (Pannetier et al. (1995)) or by
immunohistochemistry employing antibodies that recognize epitopes
on the variable regions of the TCR, or amplification of T cell
receptor .alpha.- and .beta.-chains by PCR using one or more sets
of PCR primers that allow the amplification of many different, e.g.
unknown, TCR chains. Such methods include, without being limiting,
the above described method of the invention for identifying nucleic
acid molecules encoding variable, hypervariable and/or joining
regions of T cell receptor .alpha. and .beta. chains (for example
employing the primers disclosed in Seitz et al. (2006) for
.alpha.-chains, and the V.beta. primers disclosed herein for the
variable, hypervariable and/or joining regions of .beta.-chains).
Alternative sets of primers suitable for the identification of T
cell receptors from T cells include, without being limiting, those
disclosed in Genevee (1992); Monteiro et al. (1996); Roers et al.
(2000); Gagne et al. (2000) or Zhou et al. (2006). The T cell
receptor thus identified is then employed in the cell according to
(aa) of the method of identifying a target antigen of T cells
according to the invention and the inventive method is carried out
as described above.
[0097] In a preferred embodiment, the T cells isolated from the
patient represent one single molecular type of T cells.
Furthermore, it is preferred that the T cell receptor isolated from
these cells represents one single molecular type of T cell
receptor. Furthermore, the embodiment may also relate to two
different T-cell receptors expressed in a single cell, i.e. T-cell
receptors with two different V.alpha.-chains and one V.beta.-chain
and vice versa.
[0098] The main advantage of this inventive patient-specific
antigen search technology is that it allows to transfer a highly
complex in vivo situation to a straightforward in vitro condition.
This is of outstanding interest, because many different cell types
and many different T cells are present in an inflammatory lesion,
but only few of them are relevant to disease pathogenesis or
progression. A method to analyze individual T cells of particular
interest in inflammatory autoimmune lesions has recently been
described (Seitz et al., 2006). This allows to distinguish relevant
T cells from irrelevant T cells in various in vivo settings: For
example, relevant T cells may be auto-aggressive T cells in
autoimmune diseases, tumor-infiltrating T cells in neoplastic
diseases, or anti-viral T cells in infectious diseases. Irrelevant
cells may be bystander cells that were non-specifically attracted
into the inflammatory milieu, cells in blood vessels, or patrolling
T cells during immune surveillance. Individual T cells of choice
can, for example, be isolated by laser-micro-dissection from frozen
tissue biopsy samples or other methods for single cell isolation
such as described above, their matching TCR .alpha.- and
.beta.-chains are cloned and expressed in
58.alpha..sup.-.beta..sup.- T hybridoma cells. Using this detection
method in combination with the antigen search technology described
herein, it will be possible to characterise the antigens of
putatively pathogenic or beneficial T cells in a straightforward,
quick, and cheap high-throughput assay. Using such T cell receptor
transfected hybridoma cells provides the advantage that unlike
other strategies where "real" T cells or T cell lines are used,
there are virtually no limitations in cell numbers, because the
hybridoma cells grow fast and reliable.
[0099] In a preferred embodiment of the patient-specific method of
identifying T cell antigens, the matching T cell receptor .alpha.
and .beta. chains are identified in step (B) by identifying the
nucleic acid molecules encoding variable, hypervariable and/or
joining regions of said T cell receptor .alpha. and .beta. chains
according to the method of the invention described herein
above.
[0100] As detailed in example 4 below, the method of the present
invention employing the primers or sets of primers disclosed herein
enables the identification of variable, hypervariable and/or
joining regions of T cell receptor .beta. chains in combination
with matching a chains.
[0101] The present invention also relates to a composition
comprising a T cell antigen identified by the method of the
invention and/or the identified by the method of identifying a
patient-specific T cell antigen of the invention.
[0102] The term "composition", as used in accordance with the
present invention, relates to a composition which comprises at
least one T cell antigen identified in accordance with the present
invention. It may, optionally, comprise further molecules capable
of altering the characteristics of the T cell antigen of the
invention thereby, for example, stabilizing the T cell antigen. The
composition may be in solid, liquid or gaseous form and may be,
inter alia, in the form of (a) powder(s), (a) tablet(s), (a)
solution(s) or (an) aerosol(s).
[0103] As mentioned herein above, the T cell antigens identified by
the methods of the present invention may serve as biomarkers to
detect infections and tumours and to improve diagnosis of
autoimmune diseases and are of great prognostic value for the
progression of diseases. Furthermore, said T cell antigens can be
helpful in therapeutic approaches: They can be used in vaccinations
against infections or tumours, and they may allow to selectively
delete or modulate auto-aggressive T cell clones in autoimmune
diseases, such as for example in multiple sclerosis, psoriasis,
inflammatory myopathies and many others more. Thus, the composition
of the invention may have a T cell activating activity, e.g. for
use in vaccines, or it may have a T cell neutralising activity,
e.g. for use in deletion of T cell clones.
[0104] In a preferred embodiment, the composition is a
pharmaceutical composition.
[0105] In accordance with the present invention, the term
"pharmaceutical composition" relates to a composition for
administration to a patient, preferably a human patient. The
pharmaceutical composition of the invention comprises the
compounds, i.e. T cell antigens, recited above. The pharmaceutical
composition of the present invention may, optionally and
additionally, comprise a pharmaceutically acceptable carrier and/or
excipient. By "pharmaceutically acceptable carrier and/or
excipient" is meant a non-toxic solid, semisolid or liquid filler,
diluent, encapsulating material or formulation auxiliary of any
type. Examples of suitable pharmaceutical carriers and/or
excipients are well known in the art and include sodium chloride
solutions, phosphate buffered sodium chloride solutions, water,
emulsions, such as oil/water emulsions, various types of wetting
agents, sterile solutions, organic solvents including DMSO etc.
Preferably the carrier/excipient is a parenteral carrier/excipient,
more preferably a solution that is isotonic with the blood of the
recipient. The carrier/excipient suitably contains minor amounts of
additives such as substances that enhance isotonicity and chemical
stability. Such materials are non-toxic to recipients at the
dosages and concentrations employed, and include buffers such as
phosphate, citrate, succinate, acetic acid, and other organic acids
or their salts; antioxidants such as ascorbic acid; low molecular
weight (less than about ten residues) (poly)peptides, e.g.,
polyarginine or tripeptides; proteins, such as serum albumin,
gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,
aspartic acid, or arginine; monosaccharides, disaccharides, and
other carbohydrates including cellulose or its derivatives,
glucose, manose, or dextrins; chelating agents such as EDTA; sugar
alcohols such as mannitol or sorbitol; counterions such as sodium;
and/or nonionic surfactants such as polysorbates, poloxamers, or
PEG. Conventional excipients include binding agents, fillers,
lubricants and wetting agents. Preservatives and other additives
may also be present such as, for example, antimicrobials, anti
oxidants, chelating agents, and inert gases and the like.
Furthermore, the pharmaceutical composition may comprise further
agents depending on the intended use of the pharmaceutical
composition. It is particularly preferred that said pharmaceutical
composition comprises further agents known in the art to affect T
cell activity, i.e. by either activating T cell activity for use in
vaccines or by neutralising T cell activity for use in T cell
neutralisation/depletion. Since the pharmaceutical preparation of
the present invention relies on the above T cell antigens, it is
preferred that the mentioned further agents are only used as a
supplement, e.g. at a reduced dose as compared to the recommended
dose when used as the only drug or in order to reduce the amount of
T cell antigen required, so as to reduce side effects conferred by
either the further agents or the T cell antigens.
[0106] The term "parenteral" as used herein refers to modes of
administration which include intravenous, intramuscular,
intra-peritoneal, intra-sternal, subcutaneous and intra-articular
injection and infusion.
[0107] Compositions comprising such carriers and/or excipients can
be formulated by well-known conventional methods. Generally, the
formulations are prepared by contacting the components of the
pharmaceutical composition uniformly and intimately with liquid
carriers/excipients or finely divided solid carriers/excipients or
both. Then, if necessary, the product is shaped into the desired
formulation.
[0108] These pharmaceutical compositions can be administered to the
subject at a suitable dose. The dosage regimen will be determined
by the attending physician and clinical factors. As is well known
in the medical arts, dosages for any one patient depends upon many
factors, including the patient's size, body surface area, age, the
particular compound to be administered, sex, time and route of
administration, general health, and other drugs being administered
concurrently. The therapeutically effective amount for a given
situation will readily be determined by routine experimentation and
is within the skills and judgment of the ordinary clinician or
physician. Generally, the regimen as a regular administration of
the pharmaceutical composition should be in the range of 1 .mu.g to
20 g units per day. However, a more preferred dosage might be in
the range of 0.01 mg to 100 mg, even more preferably 0.01 mg to 50
mg and most preferably 0.01 mg to 10 mg per day. Administration of
pharmaceutical compositions of the invention may be effected by
different ways, e.g., by intravenous, intraperitoneal,
subcutaneous, intramuscular, topical, intradermal, intranasal or
intrabronchial administration.
[0109] The components of the pharmaceutical composition to be used
for therapeutic administration must be sterile. Sterility is
readily accomplished for example by filtration through sterile
filtration membranes (e.g., 0.2 micron membranes).
[0110] The components of the pharmaceutical composition ordinarily
will be stored in unit or multi-dose containers, for example,
sealed ampoules or vials, as an aqueous solution or as a
lyophilized formulation for reconstitution. As an example of a
lyophilized formulation, 10-ml vials are filled with 5 ml of
sterile-filtered 1% (w/v) aqueous solution, and the resulting
mixture is lyophilized. The infusion solution is prepared by
reconstituting the lyophilized compound(s) using bacteriostatic
water-for-injection.
[0111] In a more preferred embodiment, the pharmaceutical
composition of the invention is for use in treating a disease
selected from the group consisting of cancer, infections and
autoimmune diseases.
[0112] "Cancer", in accordance with the present invention, refers
to a class of diseases or disorders characterized by uncontrolled
division of cells and the ability of these to spread, either by
direct growth into adjacent tissue through invasion, or by
implantation into distant sites by metastasis, where cancer cells
are transported through the bloodstream or lymphatic system.
Non-limiting examples of cancer include lymphoma, melanoma, lung
cancer, and other tumors with tumor infiltrating lymphocytes.
[0113] The term "infections", as used herein, relates to the
detrimental colonization of a host organism by a foreign species.
In an infection, the infecting organism seeks to utilize the host's
resources in order to multiply, usually at the expense of the host.
The host's response to infection is inflammation.
[0114] Bacterial infections, in accordance with the present
invention, include but are not limited to bacterial meningitis,
cholera, diphtheria, listeriosis, pertussis (whooping cough),
pneumococcal pneumonia, salmonellosis, tetanus, typhus,
tuberculosis, Streptococcus pyogenes, Staphylococcus aureus or
urinary tract infections by various microbial pathogens.
[0115] Viral infections, in accordance with the present invention,
include but are not limited to mononucleosis, human
immunodeficiency virus infection (HIV), chickenpox, common cold,
cytomegalovirus infection, dengue fever, ebola haemorrhagic fever,
hand-foot and mouth disease, hepatitis, influenza, mumps,
poliomyelitis, rabies, smallpox, viral encephalitis, viral
gastroenteritis, viral meningitis, viral pneumonia or yellow
fever.
[0116] Fungal infections, in accordance with the present invention,
include but are not limited to aspergillosis, blastomycosis,
candidiasis, coccidioidomycosis, cryptococcosis, histoplasmosis or
tinea pedis.
[0117] The term "autoimmune disease", in accordance with the
present invention, refers to diseases which arise from overactive
T-cell mediated immune responses of the body against substances and
tissues normally present in the body. Autoimmune diseases are well
known to the person skilled in the art and include, but are not
limited to rheumatoid arthritis, multiple sclerosis, inflammatory
bowel disease, diabetes mellitus type 1, psoriasis, autoimmune
uveitis, autoimmune (Hashimoto) thyroiditis and Behcet's
syndrome.
[0118] In another preferred embodiment, the composition of the
invention is a diagnostic composition.
[0119] In accordance with the present invention, the term
"diagnostic composition" relates to a composition for diagnosing
individual patients for their potential response to or curability
by the pharmaceutical compositions of the invention. The diagnostic
composition of the invention comprises the T cell antigens recited
above. The diagnostic composition may further comprise an
appropriate carrier and/or excipient, as defined above. The
diagnostic compositions may be packaged in a container or a
plurality of containers.
[0120] As discussed above, the T cell antigens may be useful in a
diagnostic composition as biomarkers to detect infections and
tumours and to improve the diagnosis of autoimmune diseases.
Furthermore, they are of prognostic value for monitoring the
progression of such diseases.
[0121] Thus, in a further preferred embodiment, the diagnostic
composition of the invention is for use in diagnosing a disease
selected from the group consisting of cancer, infections and
autoimmune diseases.
[0122] The present invention further relates to a peptide library,
wherein the peptide library comprises a plurality of vectors
comprising nucleic acid sequences encoding peptides, wherein the
peptides are potential target antigens of T cells and wherein the
nucleic acid sequences are randomised nucleic acid sequence.
[0123] The definitions as well as the preferred embodiments
provided herein above with regard to the methods of the invention
apply mutatis mutandis also to this peptide library.
[0124] The term "a plurality of vectors" relates to any number of
vectors greater than 1. Thus, a plurality of vectors may be, for
example, at least two vectors, such as five vectors, at least 10
vectors, at least 15 vectors, at least 20 vectors, at least 30
vectors, at least 40 vectors, at least 50 vectors, at 100 vectors,
at least 200 vectors, at least 300 vectors, at least 500 vectors,
at least 1.000 vectors, at least 5.000 vectors, at least 10.sup.4
vectors, at least 10.sup.5 vectors, at least 10.sup.6 vectors, at
least 10.sup.7 vectors, at least 10.sup.8 vectors, at least
10.sup.9 vectors, at least 10.sup.10 vectors or at least 10.sup.11
vectors.
[0125] The present invention also relates to a method of preparing
antigen-presenting cells, comprising transfecting or transforming
cells with a peptide library of the invention.
[0126] Again, all definitions as well as the preferred embodiments
provided herein above with regard to the methods of the invention
and the peptide library apply mutatis mutandis also to this method
of preparing an antigen-presenting cell.
[0127] Methods of transfecting or transforming cells are well known
in the art and include, without being limiting, electroporation
(using for example Multiporator (Eppendorf), Genepulser (BioRad)),
viral transfer (e.g. using adenoviral, retroviral or lentiviral
vectors), calcium-mediated transfection such as e.g. calcium
phosphate precipitation, cationic lipids, PEI (Polysciences Inc.
Warrington, Eppelheim), liposomes such as for example "Fugene"
(Roche) or "Lipofectamine" (Invitrogen) or phage vectors. Such
methods have been described in the art as well as in the examples
below.
[0128] This method of preparing antigen-presenting cells thus
provides a library of such antigen-presenting cells. Such a library
may be employed for example in the methods of the present
invention.
[0129] The present invention further relates to antigen-presenting
cells obtainable by this method of the invention.
[0130] Furthermore, the present invention also relates to a primer
or a set of primers selected from the group consisting of SEQ ID
NOs: 1 to 9 and/or SEQ ID NOs: 39 to 47 and/or SEQ ID NOs: 11 and
14.
[0131] In accordance with the present invention, a number of
primers have been identified that are suitable for the
amplification and identification of nucleic acid molecules encoding
T cell receptor .beta. chains from individual cells or cell clones.
As described above in detail, these primers have been optimised to
minimize potential interactions with each other or with primers for
the .alpha. chains during PCR reaction.
[0132] The term "set", as defined herein above, relates to a
combination of primers. The set of primers of the present invention
requires that more than one molecular species of primer is present.
Thus, at least two different primers, such as at least three, at
least four, at least five, at least six, at least seven, at least
eight, at least nine, at least ten, at least 11, at least 12, at
least 13, at least 14, at least 15, at least 16, at least 17, at
least 18, at least 19 or at least 20 different primers selected
from the primers represented by SEQ ID NOs: 1 to 9 and/or SEQ ID
NOs: 39 to 47 and/or SEQ ID NOs: 11 and 14 are comprised in the set
of primers.
[0133] The present invention further relates to a kit comprising
the peptide library of the invention and/or the primer or set of
primers of the invention and/or an/the antigen-presenting cell(s)
of the invention.
[0134] The various components of the kit may be packaged in one or
more containers such as one or more vials. The containers or vials
may, in addition to the components, comprise preservatives or
buffers for storage.
[0135] Preferably, the kit of the invention comprises the peptide
library of the invention and the primer or set of primers of the
invention.
[0136] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. In case
of conflict, the patent specification, including definitions, will
prevail.
[0137] The figures show:
[0138] FIG. 1: Schematic overview over the transfectants used.
580.alpha..sup.-.beta..sup.- T hybridoma cells were successively
transfected with the TCR .alpha.- and .beta.-chains of TCR JM22 in
plasmids pRSVneo and pRSVhygro, respectively (termed 58-JM22
cells), the human CD8.alpha.- and .beta.-chains, which were
connected by an IRES-2 sequence, in a retrovirus generated by
plasmid pLPC-CD8.alpha.IRESI.beta. (termed 58-JM22-CD8 cells), and
the packaging line GP+E and sGFP under the control of NFAT in
pcDNA6 (termed 58-JM22-CD8-sGFP cells).
[0139] As antigen-presenting cells (APC), COS-7 cells were used
that were transiently co-transfected with plasmid pRSV-A2, which
codes for HLA-A*0201 in plasmid pRSV, and pcDNArc-library, which
codes for either the control peptide flu(58-66)
(pcDNArc-flu(58-66)), or for a combinatorial random peptide library
where the HLA-A2 anchor positions 2, 6, and 9 are fixed
(pcDNArc-A2-269).
[0140] FIG. 2: Fluorescence microscopy of co-cultures of
58-JM22-CD8-sGFP cells with COS-7 cells that were transfected with
HLA-A*0201.
[0141] 58-JM22-CD8-sGFP cells show green fluorescence upon
activation. COS-7 cells were either used as untransfected cells
(COS-7empty) or were transfected with HLA-A*0201, either stably
(COS-7-A2stable) or transiently (COS-7-A2trans). The peptide
flu(58-66) was added either as chemically synthesized peptide
(flu(58-66)-peptide), or was transfected as a plasmid encoding the
peptide (pcDNArc-flu(58-66)). The synthetic peptide hCMV(184-192),
which binds to HLA-A*0201, but is not recognized by TCR JM22, and
the empty plasmid pcDNA-empty served as negative controls. In
addition, also the full length influenza matrix protein
(pcDNA-flu(1-252)) was transfected. The following transfectants are
shown: FIG. 2a: COS-7-A2stable+flu(58-66)-peptide; FIG. 2b:
COS-7empty+flu(58-66)-peptide; FIG. 2c:
COS-7-A2stable+pcDNA-flu(58-66); FIG. 2d:
COS-7empty+pcDNA-flu(58-66); FIG. 2e:
COS-7-A2trans+pcDNA-flu(58-66); FIG. 2f: COS-7-A2trans+pcDNA-empty;
FIG. 2g: COS-7-A2trans+pcDNA-flu(1-252); FIG. 2h:
COS-7-A2stable+hCMV-peptide
[0142] FIG. 3: FACS scans of 58-JM22-CD8-sGFP cells after
co-culture with COS-7 cells transfected with HLA and peptide. The
same combinations as in FIG. 2 are shown.
[0143] FIG. 4: Transfection of COS-7 cells with pcDNA-sGFP and
pRSV-HLA-A2. The transfection yields were measured by the intrinsic
sGFP fluorescence and by staining of HLA-A2 with the antibody BB7.2
(Proimmune) (black lines, black areas). All transfections were
transient. In the case of sGFP analysis, cells that were not
transfected served as negative controls. In the case of HLA-A2
analysis, the isotype control antibody BD-555743 was used.
(controls: grey lines, empty areas) FIG. 4a: single transfection
with pcDNA-sGFP; FIG. 4b: single transfection with pRSV-HLA-A2;
FIG. 4c: co-transfection with pcDNA-sGFP and pRSV-HLA-A2; sGFP
analysis; FIG. 4d: co-transfection with pcDNA-sGFP and pRSV-HLA-A2;
HLA-A2 analysis; FIG. 4e: co-transfection with pcDNA-sGFP and
pRSV-HLA-A2; analysis of double positive cells.
[0144] FIG. 5: Fluorescence microscopy of co-cultures of
58-JM22-CD8-sGFP cells with COS-7 cells that were co-transfected
with HLA-A*0201 and a library encoded by plasmid pcDNArc-A2-269. A
positive 58-JM22-CD8-sGFP cell shows green fluorescence upon
activation. The frequency of positive cells is about 3 per 10.sup.6
non-activated cells. The positive cell is indicated by an arrow.
FIG. 5a: Transmitted light. Cos-7 cells (irregular shape) and TCR
transfected T hybridoma cells (round shape) are shown; FIG. 5b:
sGFP fluorescence (472 nm-520 nm) indicates activated TCR
transfected T hybridoma cells; FIG. 5c: PE-channel (545 nm-605 nm),
no fluorescence is detected. This provides evidence that the
detected sGFP fluorescence is not due to autofluorescence.
[0145] FIG. 6: Schematic outline of the protocol that leads to the
identification of antigen-mimotopes. Initially an activated, green
fluorescent TCR-transfectant is picked together with the subjacent
antigen presenting cell that carries the activating plasmid. The
inserts of the plasmids are cloned, and transfected into bacteria.
To count the numbers of bacterial clones, a fraction of the
bacterial clones is plated onto agar plates. Then 30 or more
sub-pools of bacteria are created that contain 500 independent
bacterial clones. They are transfected into COS-7 cells and tested
again. From a positive sub-pool, further sub-pools are generated,
which contain less clones per sub-pool. Finally, single bacterial
clones are analyzed. Positive clones are sequenced and reveal the
antigenic mimotope.
[0146] FIG. 7: Amino acid sequences of the parent peptide
flu(58-66) and the mimotopes #1 to #4 that were discovered using
the combinatorial random peptide library pcDNArc-A2-269. Mimotope
#2 was found twice. In this library, the amino acids that code for
HLA-A2 anchors at positions 2, 6, and 9 were fixed (highlighted
grey). The amino acids at positions 4, 5 and 8, which are known to
contact the TCR JM22 (Stewart-Jones et al. 2003), are boxed with
dashed lines. The amino acids in the mimic peptides that were found
to deviate from the parent peptide are circled.
[0147] FIG. 8: Amplification of V.alpha.-/.beta.-chains by the
degenerative V Primers. The capacity of the V.alpha.- and
V.beta.-primer sets to amplify the full TCR V.beta.- and
V.alpha.-gene repertoire in a single reaction when combined with
each other was tested by multiplex RT-PCR with mRNA from peripheral
blood T cells. RNA of 500 cells was transcribed and amplified by
one step RT-PCR with the same protocol as for single cell PCR
involving the set of 9 V.beta. and 24 V.alpha. primers.
Subsequently, the PCR product was amplified with the individual
V.beta. or V.alpha. primers in individual PCR reactions. (FIG. 8a)
Using the same 9 VP primers (Vp1-9) and nested C.beta. primer, 0.5
ul aliquots of the multiplex PCR products were amplified by 35
cycles PCR (94.degree. C. 30'', 53.degree. C. 30'', 68.degree. C.
30'') with final extension of 15' at 68.degree. C. All Vp1-9
primers (lanes 1-9) produced PCR signals of the expected size.
(FIG. 8b) In parallel, the TCR V.alpha.-repertoire was amplified
from the multiplex-PCR product using the 24 different
V.alpha.-primers in individual reactions. V.alpha. semi-nested
amplification was performed the same way as for V.beta.
amplification. All V.alpha. primers (upper and lower lanes 1-12)
produced PCR signals of the expected size. (FIG. 8c) 2nd
semi-nested PCR was performed from the first multiplex PCR reaction
using FAM-labelled C.beta. in primer and primers for the individual
V.beta. gene families. PCR products were loaded on POP 6 gel and
analysed on a genetic sequencer. All V.beta. families show
polyclonal distributions and document that the multiplex V.beta.
primer set can amplify every functional V.beta. family.
[0148] FIG. 9: Three-step TCR VD-chain amplification from single T
cells. After the 1.sup.st pre-amplification a 2.sup.nd run-off PCR
step is performed to introduce a universal primer site. A 3.sup.rd
semi-nested PCR then amplifies the respective TCR V.beta.-gene
rearrangement using the universal primer and the C.beta.(2) nested
primer.
[0149] FIG. 10: Isolation of single T cells. A. For single cell
TCR-analysis T cells were labelled with monoclonal antibody-coated
magnetic beads, aspirated in invert microscopy and transferred into
a PCR vial for RT-PCR.
[0150] The examples illustrate the invention:
EXAMPLE 1: MATERIALS AND METHODS
Plasmids and Transfections
[0151] Table 6 lists primer sequences employed for plasmid
constructions. All constructs were confirmed by full length
sequencing of the inserts.
TABLE-US-00006 TABLE 6 Linkers and PCR primers. SEQ ID NAME
SEQUENCE NO: Plasmid recovery Outer pcDNA-for-1 5'-CAC TGC TTA CTG
GCT TAT CG 84 PCR pcDNA-rev-1 5'-ACT AGA AGG CAC AGT CGA GG 85
inner pcDNA-for-2- 5'-CACCCGA CTC ACT ATA GGG AGA CC 86 PCR
TOPO(Mimo 1) dTOPO pcDNA-2nd-for- 5'-CACCTCCGGCGCGCCACCATG 87
TOPO(Mimo 2,3) pcDNA-rev- 5'-CTG ATC AGC GGG TTT AAA CTC 88 2(Mimo
1) pcDNA-rev-3 5'-TGG TGA TGG TGA TGA TGA CC 89 (Mimo 2, 3) inner
pcDNA-2nd-for 5'-TCC GGC GCG CCA CCA TG 90 PCR RE digest
pcDNA-2nd-rev- 5'-CTA GAC TCG AGC GGC CGC 91 10 JM22
JM22-Valpha-for 5'-TGT GTC GAC ATG GTC CTG AAA TTC 92 TCC GTG
JM22-VJalpha- 5'-TTG GGA TCC CGC TCC TGC ACA GAG 93 rev GTA GTG GC
JM22-Jalpha-for 5'-GAG GGA TCC CAA GGA AAT CTC ATC 94 TTT GGA AAA
Calpha PvuII-rev 5'-AGC ACT GTT GCT CTT GAA GTC 95 JM22-Vbeta-
5'-CAT GTA CTG GTA CCG ACA GG 96 Kpn-for JM22-VJbeta- 5'-CCG AAG
TAC TGC TCG TAG GAG CTC 97 Sca-rev CTC GAA CTA CTG GCA CA GAGA TAG
AAA G CD8 hCD8a-Not-for 5'-ATA AGA ATG CGG CCG CAT GGC CTT 98 ACC
AGT GAC CGC hCD8a-EcoRI- 5'-GGA ATT CTT AGA CGT ATC ACG CCG 99 rev
AAA G hCD86-Mlu-Mut 5'-TCG ACG CGT ATG CGA CCG CGG CTG 100 TGG
hCD86-Xho-rev 5'-CCG CTC GAG TTA TTT GTA AAA TTG 101 TTT CAT GAA AC
NFAT- sGFP-Eco-for 5'-TC GAA TTC GCC ACC ATG GTG AGC 102 sGFP
sGFP-Xho-rev 4'-GA CTC GAG AGC TTA GTG ATG GTG 103 ATG HLA-
HLA-A0101-lead 5'-CTC GTC GAC ATG GCC GTC ATG GCG 104 A*0201 CCC
HLA-A0101-end 5'-TAC GGA TCC TCA CAC TTT ACA AGC 105 TGT GAG
pcDNArc pcDNArc-MCS- 5'-GAT CCG GCG CGC CCC TGC AGG GC 106 for
pcDNArc-MCS- 5'-GGCCGCCCTGCAGGGGCGCGCCG 107 rev flu(58- 66)
N27-flu-Asc-for ##STR00001## 108 ##STR00002## N27-flu-Not-43v
##STR00003## 109 ##STR00004## random peptide libraries
N27-all-lib-Asc- for ##STR00005## 110 NNK NNK NNK NNK NNK NNK NNK
NNK ##STR00006## N27-A2-269-lib-Asc-for ##STR00007## 111 ATC NNK
NNK NNK GTG NNK NNK CTA ##STR00008## N27-Not-rev ##STR00009## 112
The first column gives the purpose the primers or linkers were used
for. The second column lists the names of the primers or linkers.
In the third column the sequences are given. The regions coding for
peptide flu(58-66) or for the random peptide libraries are
highlighted in bold print. The restriction sites or
linker-overhangs are boxed. The letters N and K are defined: N = A
or T or G or C.; K = G or T. The corresponding SEQ ID Numbers are
presented in the fourth column.
Construction of Expression Plasmids pRSVhygro-JM22.alpha. and
pRSVneo-JM22.beta. and Generation of TCR-Transfected Hybridoma
Cells (58-JM22 Cells)
[0152] RNA was prepared from peripheral blood of a volunteer by the
TRIzol-RS reagent (Gibco/Invitrogen), and DNA prepared using
Superscript III reverse transcriptase (Gibco/Invitrogen) and
oligo(dT) primer. The V-regions of the .alpha.- and .beta.-chains
of TCR JM22 (Lehner et al., 1995) were amplified by PCR. The N(D)N
regions were inserted by mutagenesis. We used the plasmids pRSVneo
and pRSVhygro that contained the C-regions as cloning cassette
(Seitz 2006). Specifically, we produced two .alpha.-chain PCR
fragments: The first reached from the leader-region of V.alpha.10
to the J region (primers: JM22-Valpha-for and JM22-VJalpha-rev),
and the second reached from the J-region to the PvuII site in the
C-region (primers: JM22-Jalpha-for and Calpha-PvuII-rev). The
nucleotides coding for Gly and Ser in JM22-VJalpha-rev and
JM22-Jalpha-for were substituted by silent mutations to introduce a
BamHI site. The PCR product was digested by BamHI, ligated, and
used as template for a second PCR with JM22-Valpha-for and Calpha
PvuII-rev. The SalI and PvuII digested product was inserted into
pRSVhygro that already contained the TCR C-region as a cloning
cassette.
[0153] For cloning the JM22.beta.-chain we used a
V.beta.17-J.beta.2.7 positive clone as template which we had
previously cloned into pRSVneo. We amplified the region between the
KpnI site of V.beta.17 and the ScaI site in J.beta.2.7 with the
primers JM22-Vbeta-Kpn-for and JM22-VJbeta-Sca-rev and cloned the
PCR product into the above plasmid.
[0154] Both plasmids were transfected into the T hybridoma cell
line 58.alpha..sup.-.beta..sup.-, (Blank et al., 1993), selected by
G418 and hygromycinB (Sigma), and individual clones were isolated
as described for other TCR-chains in Seitz et al. 2006. The
resulting TCR transfected cell line is designated 58-JM22.
Construction Plasmid pLPC-hCD8.alpha.-IRES2-hCD8.beta. and
Generation of 58-JM22-CD8 Cells
[0155] The .alpha.- and .beta.-chains of human CD8 molecules were
amplified from cDNA using the primers: hCD8a-Not-for,
hCD8a-EcoRI-rev, hCD8b-MluI for, and hCD8b-Xho-rev. The
.alpha.-chain was inserted into the NotI and EcoRI sites and the
.beta.-chain into the MluI and XhoI sites of pQCXIX (Clontech). The
pIRES sequence in pQCXIX (Clontech) was replaced by pIRES2 which
was excised from pIRES2-DsRed2 (BD Biosciences). The fragment
comprising hCD8.alpha.-IRES2-hCD8.beta. was excised by NotI and
EcoRV and inserted into the NotI and the blunted ClaI sites of
pLPCX (Clontech). The resulting plasmid
pLPC-hCD8.alpha.-IRES2-hCD8.beta. was transfected into GP+E86
packaging cells (ATCC). 58-JM22 hybridoma cells, which already are
transfected with the CD3 .xi.-chain under histidinol selection
(Blank et al, 1993), were co-cultured with the
retrovirus-expressing GP+E86-CD8.alpha..beta. cells for two days.
Transformed cells were selected by 1.0 .mu.g/ml puromycin (Sigma).
CD8.alpha..beta. expression was analyzed by FACS using the
anti-CD8.alpha.- and anti-CD8.beta.-antibodies antibodies B9.11 and
2ST8-5H7 (Beckman Coulter). The resulting TCR and CD8 transfected
cell line is designated 58-JM22-CD8.
Construction of Plasmid pcDNA6-NFAT-sGFP and Generation of
58-JM22-CD8-sGFP Cells
[0156] The NFAT-response-element-hrGFP-SV40splice/pA sequence was
isolated from pNFAT-hrGFP (Stratagene). We first cut with AccI,
created blunt ends, and then digested with AatII. The fragment was
ligated into pcDNA.TM.6/V5-His C which was digested with PmeI and
AatII yielding the plasmid pcDNA-NFAT-hrGFP. sGFP(S65T) (Heim et
al., 1995) was amplified using the primers sGFP-Eco-for and
sGFP-Xho-rev and ligated into the EcoRI and XhoI sites of
pcDNA-NFAT-hrGFP which yielded plasmid pcDNA-NFAT-sGFP. 58-JM22-CD8
cells were transfected and selected by 3 .mu.g/ml blasticidin
(Invitrogen) and individual clones were picked and analyzed for GFP
expression after stimulation with the anti-CD3 antibody 145-2C11
(BD) and HLA-A2-flu(58-66). The resulting TCR, CD8, and NFAT-sGFP
transfected cell line is designated 58-JM22-CD8-sGFP.
Construction of Plasmind pRSV-A2 and pHSE3'-A2
[0157] The cDNA of the HLA-A2 heavy chain was amplified by PCR
using the primers 5'EX1-A-6 and 3'A-ex8-1 (M. P. Bettinotti et al.
(2003) J. Immunol. Meth. 279:143-148) and cDNA from patient PM16488
(Seitz 2006). After re-amplification with primers extended for SalI
and BamHI restriction sites (HLA-A0101-lead and HLA-A0101-end), the
PCR products were inserted into the SalI and BamHI sites of pRSVneo
and pHSE3' (Pircher et al., 1989). These plasmids were used to
transfect COS-7 cells as described below.
Construction of Plasmid pcDNA6-Flu(1-252)
[0158] The full length sequence of matrix protein flu(1-252) was
synthesized with the Kozak sequence CCACC directly before the start
codon, a stop signal and NheI and XbaI overhangs (GeneArt). The
fragment was inserted into the NheI and XbaI sites of
pcDNA.TM.6/V5-His A (Invitrogen).
Construction of Plasmid pcDNA6-Flu(58-66)
[0159] A nucleotide sequence coding for flu(58-66) was inserted
into the HindIII and XhoI sites of pcDNA.TM.3.1zeo(+) (Invitrogen)
by a double stranded synthetic linker that contained sticky
overhangs, the Kozak sequence CCACC directly before the start
codon, and a Stop-signal. The forward strand flu(58-66)-COS-for and
the reverse strand flu(58-66)-COS-rev were hybridized by incubation
at 95.degree. C. for 5 min, cooled slowly and ligated into the
HindIII and XhoI sites of pcDNA.TM.3.1 zeo(+). From this plasmid
the NheI-XbaI Fragment was ligated into the NheI and XbaI sites of
pcDNA.TM.6/V5-His A.
Construction of Plasmid pcDNA6rc-Spacer
[0160] The plasmid pcDNArc-spacer was designed as recipient plasmid
for complex libraries. We first introduced the restriction sites
for rare cutting enzymes AscI and NotI into the multiple cloning
site of pcDNA.TM. 6/V5-His A (Invitrogen). To this end, we digested
pcDNA.TM./V5-His A with BamHI and NotI and ligated the linker
oligonucleotides pcDNArc-MCS-for and pcDNArc-MCS-rev into the BamHI
and NotI sites of pcDNA.TM.6/V5-His A, yielding the plasmid pcDNArc
("rc" means "rare cutter").
[0161] Because the two restriction sites AscI and NotI are quite
close to each other, we then inserted an irrelevant DNA fragment to
facilitate later AscI and NotI digestions. Hence, we excised a 2514
bp fragment from plasmid pLNCX2 (Clontech) by AscI and NotI
digestion and inserted it into the AscI and NotI digested plasmid
pcDNArc. The resulting plasmid "pcDNArc-spacer" was then used as
recipient plasmid for libraries and flu(58-66).
Construction of Plasmid pcDNA6rc-Flu(58-66)
[0162] The nucleotide sequence coding for flu(58-66) was inserted
into the AscI and NotI sites of pcDNA6rc. We generated a double
stranded synthetic linker with sticky AscI and NotI overhangs, the
Kozak sequence CCACC directly before the start codon, and a
Stop-signal. The forward strand N27-flu-Asc-for and the reverse
strand N27-flu-Not-rev were hybridized by incubation at 95.degree.
C. for 5 min, cooled slowly and ligated into digested
pcDNA6rc-spacer.
[0163] pcDNA6rc-flu(58-66) behaved absolutely identical in all
experiments as pcDNA6-flu(58-66).
Construction of Plasmids pcDNArc-N27-all and pcDNArc-N27-A2-269
[0164] To generate random peptide libraries, we used the following
single strand oligonucleotides: N27-all-lib-Asc-for for a
completely randomized 9 amino acid library; and
N27-A2-269-lib-Asc-for: for a 9 amino acid library with 3 fixed
anchor positions of flu(58-66): isoleucin at position 2, valine at
position 6, and leucin at position 9. All oligonucleotides
contained the restriction sites AscI and NotI, the Kozak sequence
CCACC directly before the start codon, and a stop sequence.
[0165] Double strands were generated from these plasmids by
annealing the short plasmid N27-Not-rev and a subsequent fill-in
reaction. We annealed the library coding plasmids and N27-Not-rev
at 5 .mu.M each in 10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl, pH=8.2
by incubation for 5 min at 100.degree. C. and cooling slowly within
one hour to room temperature. Then an equal volume of the above
buffer was added, containing dNTPs and Taq-polymerase (both Roche)
adjusted to final concentrations of 200 .mu.M and 5 U/100 .mu.l,
respectively, and the mixture was gradually heated for 5 min to
60.degree. C., then for 4 min to 63.degree. C., then for 4 min to
65.degree. C., and finally for 1 hour to 68.degree. C. The double
strands were then digested with AscI and NotI and inserted into the
AscI and NotI sites of pcDNArc-spacer.
Construction of Plasmid pcDNA-sGFP
[0166] To measure the efficiency of a transfection method, we
cloned sGFP without NFAT response element. We digested
pcDNA-NFAT-sGFP with EcoRI and XhoI, isolated the sGFP sequence and
ligated it into pcDNA.TM.6 N5-His A (Invitrogen).
Synthetic Peptides
[0167] The peptides representing influenza matrix protein amino
acids 58 to 66 (flu(58-66): GILGFVFTL) and human cytomegalovirus
fragment pp65 amino acids 184 to 192 (hCMV-pp65(184-192):
NLVPMVATV) were synthesized by Fmoc chemistry and purified by C8
reverse phase HPLC. Their correct sequences were verified by mass
spectrometry.
Antigen Detection Assay
Cell Transfections
[0168] Transfection and selection of 58.alpha..sup.-.beta..sup.+
with JM-22.alpha.- and .beta.-chains and with
CD8.alpha.-IRES2-.beta.were performed as described for other TCR
chains (Seitz et al., 2006). These cells were then transfected with
plasmid pcDNA-NFAT-sGFP and stable transfectants were selected by 3
.mu.g/ml Blasticidine.
[0169] Two different methods were used to transiently transfect
COS-7 cells with the plasmids containing the library:
"Fugene"-mediated transfection and electroporation. The "Fugene"
method is very effective, and delivers on average 200 individual
plasmids into a single COS-7 cell. This method is preferred for
transfecting pools of plasmids or single plasmids from individual
bacterial clones, i.e. at the late stage of the antigen search.
Electroporation, on the other hand, delivers about 3 to 5 plasmids
into a single COS-7 cell. Therefore the heterogeneity of library
plasmids is lower in a single cell when electroporation is used.
This method is therefore preferred at the early stages of antigen
search, when libraries of high complexities are used. It is of note
that each COS-7 cell amplifies the transfected plasmids to about
5.000 copies in total within 2 to 3 days. Consequently, after
electroporation, each individual plasmid-species is amplified to a
much greater extent, e.g. electroporation of e.g. 4 different
transfected plasmids yields 4 different plasmid-species with 1.250
copies each while with the Fugene method, for example 200 different
transfected plasmids yield 200 different plasmid-species with only
25 copies each.
[0170] Electroporation Protocol:
[0171] To transiently transfect COS-7 cells by electroporation,
410.sup.6 cells were washed twice in RPMI and resuspended in 0.8 ml
hypo-osmolar buffer (Eppendorf). Then 16 .mu.g of DNA (in 10 mM
Tris-HCl, pH 8.5) were added. For co-transfection plasmids pRSV-A2
and pcDNA-flu(58-66) or pcDNA-flu(1-152) or pcDNA-A2-269 or pcDNArc
were used at equal molar amounts. The electric pulse was applied at
room temperature in 0.4 cm electroporation cuevette (Eppendorf)
with 1200 V and for 40 ms using a "Multiporator" transfection
apparatus (Eppendorf). Immediately after the electric pulse the
cells were resuspended in pre-warmed complete media. In some
experiments, the cells were washed twice with 20 mM Na-phosphate
buffer, pH=7.4, 150 mM NaCl, 10 mM MgCl2. Then they were seeded on
dishes (0.5-110.sup.6 cells per 3.5 cm dish) and incubated under
normal cell culture conditions.
[0172] Fugene Protocol:
[0173] Transient transfection of COS-7 cells with FugeneHD
transfection reagent (Roche) was performed at a ratio of 2 .mu.g
DNA: 7 .mu.l Fugene. Plasmids pRSV-A2 and pcDNA-flu(58-66) or
pcDNA-A2-269 were used at equal molar amounts. To remove residual
transfection complexes, COS-7 cells were washed 4 times with PBS 24
h after transfection, incubated for one hour with 50 .mu.g/ml
herring sperm DNA to replace the plasmids in the transfection
complexes, and then and for 2 hours with 0.2 mg/ml DNase and 10 mM
MgCl2 to digest remaining DNA.
[0174] The transient transfection efficiency of COS-7 cells was
determined by FACS analysis using pcDNA6 coding for-sGFP or
pRSV-A2. Positive cells were analysed by intrinsic sGFP
fluorescence or by staining with the anti-HLA-A2 antibody BB7.2
(Proimmune). Maximum expression was observed between 48 and 72
hours post transfection.
Detection of Cells Presenting an Antigen Recognized by T Cells
[0175] To detect COS-7 cells that contained a library-plasmid
coding for a peptide that is recognized by the TCR-transfected
hybridoma cells, library-transfected COS-7 cells were plated on
tissue culture plates at a density of about 40.000 cells/cm.sup.2.
After incubation for 2 to 3 days at 37.degree. C., the almost
confluent cell layer was washed twice with RPMI medium and
overlayed with 58-JM22-CD8-sGFP T-hybridoma cells, and incubated
for additional an 12 to 18 hours. Then the plates were examined
under an inverse fluorescence microscope (AxioVert200M, Zeiss,
equipped with a CCD-Camera (CoolSNAP-HQ, Roper Scientific), a
fluorescence lamp (HXP 120, Visitron), and the objectives:
5.times., NA 0.15; .delta./0, Epiplan-NEOFLUAR; 10.times., NA 0.45
Plan Apochromat; 20.times., NA 0.4; .infin./0-1.5 Achroplan, Korr
Ph2). A Cy3-Filter (excitation/emission: 545(25)/605(70) nm, Zeiss)
was used to check for auto fluorescence and a GFP-Filter
(excitation/emmision at 472(30)/520(35) nm, Semrock, BrightLine) to
detect sGFP expression. Alternatively to manual search, an
automated scan system based on a motorized xy-stage (BioPresision2,
Visitron, Puchheim, Germany) and automated picture acquisition and
analysis (MetaMorph-Software, V7.7) was used. COS-7 cells in
contact with activated green 58-JM22-CD8-sGFP hybridoma cells were
then picked with a thin capillary (Eppendorf, customTips Type I,
inner diameter 15 .mu.m) and a micro manipulator (Mini 25, Luigs
& Neumann). Picked cells were flushed into 7 .mu.l of 25%
ammonia solution and stored for up to 4 hours on ice to preserve
plasmids and to inhibit DNases. Before the PCR-reaction, the tubes
were opened for 30 min at room temperature to facilitate
evaporation of ammonia.
Amplification of Plasmids Encoding Antigens
[0176] PCR amplification of the pcDNA inserts was performed as two
rounds of nested PCR. A first PCR reaction was performed in 50 to
100 .mu.l with 1 U Taq polymerase (Roche) (0.2 mM dNTP, 0.5 .mu.M
primer). In some experiments a single PCR using inner or outer
primers was sufficient. Subsequently, an equivalent to 0.01 .mu.l
of the first reaction product were used as template in a nested PCR
reaction with iProof polymerase (Biorad) and inner primers. The PCR
product was purified with MinElute PCR purification kit (Qiagen)
and cloned into plasmid pcDNA3.1D/V5-His-TOPO.RTM. (Invitrogen).
The ligation product was transfected into DH10B ElectroMax
(Invitrogen) bacteria by electroporation.
Additional Screening and Identification of Mimotopes
[0177] The enriched mimotopes were subjected to further rounds of
antigen screening where pools of independent bacterial clones were
used (FIG. 6). A small fraction of the transfected bacteria was
grown on agar plates to determine the number of bacteria contained.
The major fraction was grown in bulk culture. From this >30
pools of about 500 independent bacterial clones each were grown in
suspension culture, plasmids were prepared, and then transfected
into fresh COS-7 cells. These were tested again for activation of
58-JM22-CD8-sGFP cells. From positive pools again >30 subpools
with 100 bacterial clones each were created, which were tested as
above. Then the procedure was repeated with >30 sub-pools of 20
independent bacterial clones, which in addition were grown on agar
plates. From these individual bacterial colonies were picked and
tested. Plasmids that were positive in this last round of analysis
were sequenced.
Amplification of TCR .alpha.- and .beta.-Chain cDNA from the Single
Cell by One Step RT-PCR
[0178] Protocol Steps 1.1, 1.2. (RT-PCR)
[0179] mRNA of single cell was transcribed into cDNA by RT reaction
for 35 min at 50.degree. C. using a one step RT-PCR kit (QIAGEN)
and gene specific C.alpha.- and .beta.-primers (0.6 uM each)
(Cprimer=C.alpha.out Primer+C.beta.out primer, Table 2).
[0180] Protocol Step 1.3. (1.sup.st Multiplex PCR)
[0181] Pooled primers for the simultaneous amplification of
V.alpha.- and .beta.-chain (Vprimer) were added to the reaction
solution. The pool contains 24 V.alpha.-specific and 9
V.beta.-specific primers (Vp1-Vp9) (0.075 uM each) (Table 1 and 3),
which cover all functional .alpha. and .beta. TCR variable region
genes. After 15 min at 95.degree. C. for the activation of hot
start polymerase, 10 PCR cycles were performed at 94.degree. C. for
30 sec, at 60.degree. C. for 90 sec and at 68.degree. C. for 60
sec. Subsequently 30 PCR cycles were performed at 94.degree. C. for
30 sec, at 53.degree. C. for 90 sec and at 68.degree. C. for 60
sec, followed by a 15 min final extension step at 68.degree. C.
[0182] Protocol Step 2.1. (Run Off-PCR)
[0183] For the amplification of the V.beta.-chain a 1 ul aliquot of
the pre-amplification product was subjected to 1 cycle of run-off
PCR at 94.degree. C. for 5 min, 53.degree. C. for 150 sec and
68.degree. C. for 15 min using primers based on the Vp primers
supplemented with a universal primer sequence at the 5' end (Vp+
primer). 10 ul reaction solution contains 1*PCR Buffer, 0.25 U DNA
Polymerase (Roche), 0.2 mM dNTP and Primer Pool (0.1 uM each
primer).
[0184] Protocol Step 2.2. (2.sup.nd Seminested PCR)
[0185] A "semi-nested" PCR was run using a C.beta. specific nested
primer (C.beta.-in) and a universal primer (UP). PCR consists of a
2 min denaturation step at 94.degree. C., 50 cycles at 94.degree.
C. (30 sec), 58.degree. C. (60 sec), 68.degree. C. (60 sec) and a
15 min final extension step at 68.degree. C.
[0186] Protocol Step 3 (V.alpha. Amplification).
[0187] The second nested amplification of the V.alpha.-chain was
performed as described in a previous report with a little
modification (Seitz et al. (2006)). Briefly, 1 ul of the
pre-amplification probe was added to a PCR solution containing a
C.alpha.-nested primer (C.alpha.-in) and the V.alpha.-nested primer
pool (0.1 uM each primer). After denaturation (2 min at 94.degree.
C.), touch down PCR was run with each 4 cycles at 61.degree. C.,
58.degree. C., and 56.degree. C. annealing followed by 40 cycles at
53.degree. C. annealing. Annealing and extension (68.degree. C.)
times were 1 min each, and denaturation time 30 sec, followed by a
final extension at 68.degree. C. for 15 min. TCR .beta.- and
.alpha.-chain were directly sequenced using
C.beta.-/C.alpha.-nested primers and 0.5 ul of the nested PCR
product.
EXAMPLE 2: PROPERTIES OF THE READOUT SYSTEM
[0188] The T hybridoma cell line 58.alpha..sup.-.beta..sup.- was
stably transfected with the .alpha.- and .beta.-chains of the TCR
JM22, the human CD8.alpha.- and .beta.-chains, and a reporter
construct where sGFP is controlled by NFAT (termed 58-JM22-CD8-sGFP
cells) (FIG. 1). To test whether green fluorescence in these
transfectants may be detected after antigen stimulation, COS-7
cells were used as APC that were stably transfected with HLA-A*0201
in plasmid pHSE3' (pHSE3'-A2) and the synthetic peptide flu(58-66)
was added. The adherent COS-7 cells formed an almost confluent
monolayer. 58-JM22-CD8-sGFP cells were added and sGFP fluorescence
was observed after 16 hours under a fluorescence microscope (FIG.
2a) and by FACS (FIG. 3a). More than 50 percent of 58-JM22-CD8-sGFP
cells were found to be activated as evident by their bright sGFP
fluorescence. If empty COS-7 cells that have not been transfected
with HLA-A2 were used (FIG. 2b and FIG. 3b), or if flu(58-66) was
replaced by the irrelevant peptide hCMV (184-192) (FIG. 2h and FIG.
3h), not a single positive 58-JM22-CD8-sGFP cell among 250.000
cells was observed. These experiments show that 58-JM22-CD8-sGFP
cells may serve as suitable readout cells to specifically detect
HLA-peptide complexes.
[0189] To avoid the need for generating stable HLA-transfected COS
cells for each HLA-allele under question, the protocol was modified
so that all transfections into COS cells are transient. In a first
step it was tested whether flu(58-66) needs to be added as a
synthetic peptide, or whether it may be encoded in a plasmid that
is transfected into COS cells that stably express HLA-A*0201.
Hence, COS-7 cells that were stably transfected with pHSE3'-A2 were
transiently super-transfected with the plasmid pcDNA that coded for
expression of flu(58-66) (pcDNA-flu(58-66)) by electroporation
(FIG. 2c and FIG. 3c; see Tab 6 for plasmid insert sequences).
pcDNA carries the SV40 origin, which ensures intracellular
plasmid-amplification in COS-7 cells (Gluzman, 1981). The yield of
fluorescent 58-JM22-CD8-sGFP was 31 percent, which is only slightly
lower as compared to the experiment where the synthetic peptide was
added directly (FIG. 2a and FIG. 3a). Of note, this experiment
shows directly that peptides encoded by pcDNA can be expressed in
the cytosol of COS-7 cells and are efficiently transported into the
lumen of the endoplasmic reticulum where they are loaded onto
class-I MHC molecules.
[0190] Next, both pRSV-A2 and pcDNA-flu(58-66) were transiently
co-transfected at a molar ratio of 1:1 into COS cells.
58-JM22-CD8-sGFP cells were added after 56 hours and their
fluorescence was observed again 16 hours later (FIG. 2e and FIG.
3e). The yield of activated 58-JM22-CD8-sGFP was 13%, i.e., it is
slightly lower than for stable transfectants (FIGS. 2a and 2d). It
is in the same range as the transfection-rate of 14 percent in a
model system, where pRSV-A2 and GFP in pcDNA were co-transfected,
which may both be easily quantified by FACS (FIG. 4). sGFP
expression in 58-JM22-CD8-sGFP cells was observed 8 hours after
starting co-culture with library-transfected COS cells and was
stable for more than 16 hours. During that time the
58-JM22-CD8-sGFP cells maintained tight contact with their antigen
presenting COS-7 cell. In negative control experiments, where
pRSV-A2 and pcDNA without insert (pcDNA-empty) were co-transfected
(FIG. 2f and FIG. 3f) or where pcDNA-flu(58-66) was transfected
without pRSV-A2 (FIG. 2d and FIG. 3d), no activation of
58-JM22-CD8-sGFP cells was observed. Strikingly, no positive cells
were identified when pcDNA-flu(58-66) was replaced by
pcDNA-flu(1-252), i.e. by a construct that codes for the full
length influenza matrix protein sequence (FIG. 2g and FIG. 3g).
This shows that the COS-7 cells were unable to correctly process
the full-length flu(1-252) protein. It is of note that the present
method circumvents all intracellular protease cleavage requirements
for the antigens, because it takes advantage of testing with short
peptides.
EXAMPLE 3: SCREENING OF RANDOMIZED PEPTIDE LIBRARIES AND
IDENTIFICATION OF FLU(58-66) MIMOTOPES
[0191] To identify mimotopes of flu(58-66), random libraries were
generated. These libraries consisted of a series of N-nucleotides
that were flanked at the 5'-end by a Kozak sequence and a
start-codon and at the 3'-end by a stop-codon (Tab 6). The
libraries were inserted into the plasmid pcDNArc and co-transfected
with pRSV-A2 into COS-7 cells. Since antigens presented by
HLA-A*0201 were investigated, three fixed amino acids were
introduced, which provide the three HLA-A2-binding anchors in
positions 2, 6, and 9 (pcDNA-A2-269) (Rammensee et al., 1999). The
HLA-A2*0201 anchor positions isoleucine in position 2, valine in
position 6 and the main anchor leucine in position 9 of flu(58-66)
were fixed. All other positions were randomized. The library was
co-transfected together with pRSV-A2 into COS-7 cells and
activation of a small number of 58-JM22-CD8-sGFP cells was observed
(FIG. 5b). The direct contact of the fluorescent activated
58-JM22-CD8-sGFP cell with an subjacent COS-7 cell is visible (FIG.
5a). The frequency of activated 58-JM22-CD8-sGFP cells was found to
be about 3 fluorescent cells per million COS-7 cells.
[0192] To recover the pcDNA plasmid that codes for the activating
peptide mimotope, the activated 58-JM22-CD8-sGFP cells were picked
together with the subjacent COS-7 cells with a thin capillary under
a fluorescent microscope. Although COS-7 are adherent cells, they
can be recovered from the bottom of the tissue culture plate. Under
co-culture conditions of 58-JM22-CD8-sGFP cells with COS-7 cells
co-transfected with HLA-A2 and pcDNA-A2-2,6,9, both the fluorescent
58-JM22-CD8-sGFP cell and the subjacent COS cell can be picked
together. This can be observed under fluorescent or, better, under
transmitted light.
[0193] From the picked cells, the mimotope coding sequences were
amplified by PCR, cloned and co-transfected with pRSV-A2 into fresh
COS-7 cells, or transfected into COS-7 cells that previously stably
transfected with HLA-A2. Then they were tested again for
58-JM22-CD8-sGFP activation. Although in most cases it was possible
to pick single COS-7 cells, many different inserts were initially
recovered, presumably because a single COS cell typically contains
more than one library-plasmid, and because plasmids in transfection
complexes may still be present in the medium outside the cells and
are aspirated together with the cells. Independent pools of
bacterial clones were therefore analyzed until the population was
homogenous and the inserts of the plasmids could be sequenced (FIG.
6). We identified four different mimotopes represented by four
peptides (termed "mimo-1" to "mimo-4"; FIG. 7). mimo-2 was
identified twice independently. The deduced amino acid sequences
thus revealed differ from flu(58-66) in several amino acids in
position one, three, and five. Amino acids in positions one and
three are known not to interact strongly with both, HLA-A*0201 and
JM22 (Stewart-Jones et al., 2003). The amino acid replacement in
position five (mimo-2: tryptophan replaces phenylalanine) is
conservative. However, it is of interest that this amino acid
interacts with several loops in the TCR complementarity determining
regions. Using the mimotop sequences for Blast searches under
standard conditions, it was possible to unequivocally identify the
parent flu(58-66) sequence.
EXAMPLE 4: ANALYSIS .alpha..beta.-T-CELL RECEPTOR REARRANGEMENTS IN
SINGLE T CELLS
4.1 Primer Design and PCR Strategy
[0194] Molecular analysis of the paired .alpha..beta.-TCR
rearrangements of single T cells has to encompass the complete
spectrum of .about.70 TCR V.alpha.- and .about.50 TCR
V.beta.-region genes. We focussed on the development of a PCR
strategy, which can amplify all different V.beta.-gene families of
the TCR .beta.-chain repertoire together with a set of 24 TCR
V.alpha.-primers recently described for the simultaneous
amplification of the TCR V.alpha. repertoire (Seitz, Schneider et
al. 2006). The complexity of this approach results from potential
interactions between the multitude of primers, which may interfere
with specific amplification. Our efforts finally resulted in a PCR
protocol, which starts with a multiplex RT-PCR capable of
pre-amplifying all TCR V.alpha.- and V.beta.-genes in a single
reaction. Subsequently, the TCR V.alpha.- and V.beta.-PCR products
are handled separately.
4.2 Preamplification of .alpha..beta. TCR-Rearrangements
[0195] To keep the number low, TCR V.beta.-primers were designed
utilizing sequence homologies of the various V.beta.-gene families
as identified by sequence alignments. One mismatch with the primary
nucleotide sequence was allowed. Primer positions had to consider
that direct sequencing could still identify the respective TCR
V.beta.-gene. To minimize potential interactions, all primers for
the V.beta. repertoire were adjusted to each other and the 24
V.alpha.-primers by an oligoanalysis software program.
[0196] 30 different V.beta.-gene primers were tested in various
combinations together with the 24 V .alpha.-primers. This finally
led to a set of nine V.beta. primers (Vp1-9, Table 1), which
covered all functional VP genes. Except Vp1, which is located on
the leader segment, all primers are positioned on the V.beta.-gene
segment. In combination with a C.beta.(1)-primer each of them
efficiently amplified the corresponding V.beta.-gene
rearrangements, as shown by agarose gel-electrophoresis and
ethidium-bromide staining (FIG. 8a).
[0197] The capacity of the V.alpha.- and V.beta.-primer sets to
amplify the full TCR V.beta.- and V.alpha.-gene repertoire in a
single reaction when combined with each other was tested by
multiplex RT-PCR with mRNA from peripheral blood T cells. After
pre-amplification, the rearrangements of the different TCR
V.beta.-gene families were amplified from the multiplex-PCR product
in 23 different nested PCR reactions using 23 different primers
specific for the various TCR V.beta.-gene families and a
FAM-labelled C.beta.(2)-primer essentially as described. The
fragment lengths of the PCR products were analysed by spectratyping
on a genetic sequencer. Amplification of each TCR V.beta.-gene
family yielded PCR products of the expected size range with
Gaussian-like fragment-lengths distributions of the TCR
rearrangements typical of polyclonal T-cell populations (FIG.
8c).
[0198] In parallel, the TCR V.alpha.-repertoire was amplified from
the multiplex-PCR product using the 24 different V.alpha.-primers
in individual reactions. As shown by agarose-gel electrophoresis
and ethidium bromide staining each of them yielded discrete PCR
products representative of the amplified TCR
V.alpha.-rearrangements (FIG. 8b). Thus, the multiplex RT-PCR
conditions we had established for pre-amplification of the
.alpha..beta.-TCR repertoire were actually capable of amplifying
both the full TCR V.beta.- and V.alpha.-repertoire in a single
reaction.
4.3 Single Cell Analysis
[0199] The next step required amplifying the respective TCR
V.beta.-chain rearrangement from the pre-amplification product of
single T cells. Because the multitude of 23 different V.beta.-gene
subfamilies prohibited a V.beta.-specific nested PCR in one sample
we introduced a universal primer sequence at the 5' end of the TCR
.beta.-chain PCR-products. For this purpose a unique 21-nucleotide
sequence was designed lacking primer interactions or homologies
with human genes and appended to the 5' end of the nine different
Vp (Vp1-9) primers. 1 .mu.l of the pre-amplification product was
subjected to a run-off PCR using these elongated primers in a
10-.mu.l PCR volume. This was followed by a third semi-nested PCR,
which amplified the respective single cell TCR-V.beta.
rearrangement independent from the TCR V.beta.-gene family using
the universal primer together with a nested C.beta.(2)-primer. The
PCR strategy is shown in FIG. 9.
[0200] In parallel, the corresponding TCR V.alpha.-rearrangement
was amplified from the pre-amplification multiplex PCR in five
different nested PCRs using five V.alpha.-primer pools described by
Seitz et al. The amplified TCR .alpha.- and .beta.-chain
rearrangements were then characterized by direct sequencing.
[0201] To test the efficiency of this approach on a single T-cell
level, peripheral blood T cells were labelled with CD4- or
CD8-monoclonal antibody-coated magnetic beads, and single T cells
were aspirated with a 2-.mu.l pipette in invert microscopy and
transferred into the pre-amplification vial. When adjusted to the
appropriate cell density, this technique easily allows identifying
and isolating single rosette-forming T cells from cell suspensions
of various origins (FIG. 10).
[0202] A total of 96 CD4.sup.+ and 96 CD8.sup.+ peripheral blood T
cells were captured and analysed this way. In 82 CD4.sup.+ (85.4%)
and 76 (79.2%) CD8.sup.+ T cells a TCR V.beta.-rearrangement could
be amplified and characterized by direct DNA sequencing of the PCR
product, showing an efficiency of TCR .beta.-chain amplification of
greater than 80%. Among the analyzed rearrangements all functional
TCR V.beta.-gene families were represented except V.beta.16, which
is rarely rearranged in general. The prevalence of the other TCR
VP-gene families reflected the average TCR V.beta.-gene usage, with
those TCRBV gene families showing a predominance that are generally
most often observed in single cell analysis, as well. From each T
cell rendering a .beta.-chain rearrangement, a corresponding TCR
.alpha.-chain could be obtained and confirmed by direct sequencing.
Given the fact that manual single-cell aspiration likely has not be
successful in every attempt we conclude that our approach allows
the molecular characterization of the paired .alpha..beta.-TCR
rearrangements from virtually every single .alpha..beta.-T
cell.
4.4 Analysis of .alpha..beta.-TCR Rearrangements of Single T Cells
in Psoriasis
[0203] According to current concepts T-cell mediated autoimmune
diseases result from the autoantigen-specific activation and clonal
expansion of autoreactive T cells. We tested the capacity of our
experimental approach to identify such pathogenic clonal T cells
from inflammatory tissue lesions and characterize their paired
.alpha..beta.-TCR rearrangements, which encode the specificity for
the putative autoantigens.
[0204] We focussed on psoriasis vulgaris, which is a T-cell
mediated autoimmune disease of the skin. Former analyses of TCR
.beta.-chain repertoires of lesional psoriatic T-cell infiltrates
by random amplification, cloning and sequencing of TCR cDNA from
lesional biopsies had suggested dominant oligoclonal T-cell
expansions, but the precise clonality and subtype of individual T
cells has remained elusive.
[0205] A pathogenic autoimmune T-cell clone should be identifiable
ex vivo in the pathogenic T-cell infiltrate as multiple T cells
with identical .alpha..beta.-TCR rearrangements. Based on this
assumption, we established a stepwise protocol to identify lesional
psoriatic T-cell clones on a single cell level and verify their
presence within the tissue lesions.
[0206] Biopsies were taken from chronic psoriatic plaques and
divided in two pieces. One half was subjected to the preparation of
TCR .beta.-chain cDNA, while the other half was seeded into culture
to allow emigration of T cells from the inflammatory infiltrate,
which occurs within 24 to 48 hours. T cells were harvested,
labelled with CD4- or CD8-specific magnetic beads, and single
CD4.sup.+ or CD8.sup.+ T cells were isolated and subjected to TCR
.beta.-chain analysis. TCR .alpha.-chain rearrangements of single T
cells were only analysed in T-cell clones, i.e. if particular TCR
V.beta.-chain rearrangements were identified both in multiple T
cells and within the second half of the biopsy specimen.
[0207] Lesional biopsies from four patients were examined this way.
In patient #1, CD4.sup.+ T cells and in patient #2, CD8.sup.+ T
cells were analysed. Two out of 40 CD4.sup.+ (#1) or two out of 37
CD8.sup.+ T cells (#2), respectively, had identical TCR V.beta.-
and V.alpha.-rearrangements (Table 7). Using rearrangement-specific
primers together with the corresponding V.beta.-leader primers and
direct sequencing of PCR products, each of the TCR
V.beta.-rearrangements could also be amplified from the
corresponding biopsy sample, corroborating the clonal expansion of
the corresponding T cells within the tissue lesion.
TABLE-US-00007 TABLE 7 TCR .beta.-chain Identical/ CDR3 also total
Identical identified in number of .alpha..beta.-TCR TCR
V.beta.-chain rearrangements* Corresponding Pat. No. Phenotype T
cells rearrangements TCR V.beta. N(D)N J.beta. skin lesion PBL
Tonsil 1 CD4 2/40 yes 7.2 CASS PTSL TDTG 2.7 + ND NA 2 CD8 2/37 yes
12.4 CAS TPSRGIS YGYT 1.2 + ND NA 3 CD4 2/82 yes 18.1 CASS TTPGN
SGNT 1.3 + + + CD8 3/41 yes 7.2 CASSL SPVAY SNQP 1.5 + + + 2/41 yes
7.6 CASSL RPGTGGF ETQY 2.5 + + - 2/41 yes 7.7 CASSL NPS SGNT 1.3 +
- + Table 7. Repetitive TCR .beta.-chain rearrangements of single T
cells from explant culture and presence of identical TCR
rearrangements in the corresponding biopsy, blood or tonsil
specimen; *Deduced amino-acid sequence, one letter code; NA, not
available; ND, not determined; +, TCR CDR3 rearrangement
identified; -, TCR CDR3 not identified
[0208] In patient #3, CD4.sup.+ and CD8.sup.+ T cells were
analysed. This patient had been tonsillectomized due to constant
psoriasis flares in association with a recurrent streptococcal
angina, which is the main infectious psoriasis trigger. Blood
lymphocytes and fractions of the tonsils were available for
analysis, as well. While one particular .alpha..beta.-TCR
rearrangement was found in two of 82 CD4.sup.+ T cells (Table 7),
three repetitive .alpha..beta.-TCR rearrangements were identified
in two or three of 41 CD8.sup.+ T cells, respectively. All of these
TCR-rearrangements were also identified within the corresponding
second half of the biopsy. Most interestingly, the repetitive TCR
V.beta.-rearrangements of both CD4.sup.+ and CD8.sup.+ T cells
could also be amplified from the PBL and/or the tonsillar tissue of
the patient, indicating a systemic distribution of the
corresponding T-cell clones.
4.5 Differential Distribution of CD8.sup.+ T Clones in Epidermis
and Dermis
[0209] While the majority of the T-cell infiltrate is located
within the dermal compartment of psoriatic skin lesions, the
development of psoriasis is crucially dependent on the accumulation
of T cells within the epidermis, the majority of them being
CD8.sup.+. Thus, the actually pathogenic T cells may represent
CD8.sup.+ T cells, which have to enter the epidermis to promote
psoriatic skin lesions. In the next two patients we therefore
analysed the differential clonal distribution of CD8.sup.+ T-cell
clones within epidermis and dermis. For this purpose dermis and
epidermis were dissociated from each other and seeded separately
into culture.
[0210] Compartment-related analysis of single CD8.sup.+ T cells
documented that in both patients the representation of CD8.sup.+
T-cell clones was stronger in the epidermal than the dermal
compartment (Table 8). In patient #4, eight different
.alpha..beta.-TCR rearrangements were found in duplicates or
triplicate among 52 epidermal CD8.sup.+ T cells, and four
repetitive .alpha..beta.-TCR rearrangements were observed in both
epidermal and dermal T cells. In patient #5, three different
.alpha..beta.-TCR rearrangements were selectively present in three,
four or seven of 33 epidermal CD8.sup.+ T cells, and two epidermal
.alpha..beta.-TCR rearrangements were seen in dermal T cells, as
well. In the dermis, only one (patient #4) or two (patient #5)
T-cell clones with identical .alpha..beta.-TCR rearrangements were
seen. Thus, epidermal T cells showed a clonal predominance over
dermal T cells.
TABLE-US-00008 TABLE 8 Differential representation of CD8.sup.+
T-cell clones in dermis and epidermis. Frequency in Frequency TCR
V.beta.-chain epidermis in dermis rearrangements* Identical/
Identical/ Patient Clone No. V.beta. N(D)N J total total Patient #
4 1 4.1 CASSQ ENRG GYAV 2.7 2/52 1/36 .sup. 2.sup.+ 6.5 CASSY SEGED
EAFF 1.1 3/52 1/36 .sup. 3.sup.+ 9.2 CASS PRGGE NTIY 1.3 2/52 NO 4
11.2 CASS STLAGGP DTQY 2.3 2/52 NO 5 11.2 CASS LGRL QETQ 2.5 2/52
NO 6 11.3 CASS PAQ -- -- 2/52 1/36 7 18.1 CAS AGTGYF QPQH 1.5 2/52
NO 8 19.1 CAS TLRSSG NEKL 1.4 2/52 NO 9 6.1 CAS TELAGD YNEQ 2.1
1/52 1/36 10 7.9 CA SWTGELG GYTF 1.2 NO 2/36 Patient # 5 1 11.3
CASS PRTSGG YNEQ 2.1 3/33 NO 2 20.1 CSAR DQGQHR TDTQ 2.3 7/33 NO 3
20.1 CSAR GGLGLMP GELF 2.2 4/33 NO 4 4.1 CASSQ LTSESY SYNE 2.1 1/33
1/25 5 6.1 CAS GWDRGT FFGQ 1.1 1/33 1/25 1 3.1 CASSQ DLWTGGWG TDTQ
2.3 NO 2/25 2 12.3 CASSL ILGGD EQYF 2.7 NO 2/25 (4) *deduced
amino-acid sequence, one letter code; .sup.+T-cells in direct
contact with APC; NO, not observed; directly rearranged to C.beta.
without J.beta. gene.
4.6 Generation of TCR Hybridomas
[0211] The paired .sigma..beta. TCR rearrangements of the clonally
expanded CD8.sup.+ T cells likely carry the antigen-specificity of
the lesional psoriatic T-cell response. To use them for the
identification of potential psoriatic antigens, recombinant TCR
hybridomas were generated with the TCR rearrangements of several
lesional CD8.sup.+ T-cell clones. For this purpose, the .alpha. and
.beta.-chain rearrangements of three different CD8.sup.+ clones
were cloned and stably expressed as recombinant .alpha..beta.-TCR
clones in the mouse 58.sup.-/- T cell hybridoma cell line together
with the human CD8 molecule and a green fluorescent protein under
the control of NFAT. Upon activation by CD3 monoclonal antibodies
these hybridomas produced ample amounts of interleukin 2 (IL-2), as
measured by an IL-2 Elisa, and became green fluorescent in
UV-microscopy. Furthermore, they were activated by cells of the
keratinocyte cell line, HaCaT, when this cell line was transfected
with HLA-Cw6, which is the main risk allele conferring
susceptibility to psoriasis.
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Sequence CWU 1
1
112120DNAArtificial sequence/note="Description of artificial
sequence primer Vp1"variation2/replace="g"variation3/replace="t"
1tcctttgtct cctgggagca 20220DNAArtificial
sequence/note="Description of artificial sequence primer Vp2"
2cctgaagtcg cccagactcc 20322DNAArtificial
sequence/note="Description of artificial sequence primer
Vp3"variation6/replace="g"variation19/replace="t" 3gtcatccaga
acccaagaca cc 22424DNAArtificial sequence/note="Description of
artificial sequence primer
Vp4"variation3/replace="t"variation11/replace="c"variation14/repla-
ce="c" 4ggatatctgt aagagtggaa cctc 24522DNAArtificial
sequence/note="Description of artificial sequence primer
Vp5variation21/replace="c" 5atgtactggt atcgacaaga tc
22623DNAArtificial sequence/note="Description of artificial
sequence primer Vp6" 6cactgtggaa ggaacatcaa acc 23721DNAArtificial
sequence/note="Description of artificial sequence primer
Vp7"variation11/replace="g" 7tctccactct caagatccag c
21826DNAArtificial sequence/note="Description of artificial
sequence primer
Vp8"variation4/replace="g"variation10/replace="g"variation13/replace="t"
8cagaatgtaa atctcaggtg tgatcc 26923DNAArtificial
sequence/note="Description of artificial sequence primer
Vp9"variation7/replace="t"variation12/replace="g"variation14/repla-
ce="t" 9ccagacacca aaacacctgg tca 231020DNAArtificial
sequence/note="Description of artificial sequence primer C alpha
out" 10gcagacagac ttgtcactgg 201121DNAArtificial
sequence/note="Description of artificial sequence primer C beta
out" 11tggtcgggga agaagcctgt g 211220DNAArtificial
sequence/note="Description of artificial sequence primer C alpha
in" 12agtctctcag ctggtacacg 201322DNAArtificial
sequence/note="Description of artificial sequence primer UP"
13acagcacgac ttccaagact ca 221420DNAArtificial
sequence/note="Description of artificial sequence primer C beta in"
14tctgatggct caaacacagc 201519DNAArtificial
sequence/note="Description of artificial sequence primer V
alpha-1(14)-for-out"variation3/replace="g" 15agcagcctca ctggagttg
191620DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-1(235)-for-out" 16ctgaggtgca actactcatc
201719DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-2-for-out"variation3/replace="g"
17caatgttcca gagggagcc 191821DNAArtificial
sequence/note="Description of artificial sequence primer V
alpha-3,25-for-out"variation4/replace="g"variation8/replace="t"-
variation10/replace="t" 18gaaaatgcca ccatgaactg c
211919DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-4,20-for-out"variation1/replace="t"
19atgctaagac cacccagcc 192021DNAArtificial
sequence/note="Description of artificial sequence primer V
alpha-5-for-out" 20agatagaaca gaattccgag g 212121DNAArtificial
sequence/note="Description of artificial sequence primer V
alpha-6,14-for-out"variation1/replace="g"variation2/replace="t"
21actgcacata tgacaccagt g 212219DNAArtificial
sequence/note="Description of artificial sequence primer V
alpha-7-for-out" 22cacgtaccag acatctggg 192318DNAArtificial
sequence/note="Description of artificial sequence primer V
alpha-8,21-for-out"variation7/replace="t"variation16/replace="g"
23cctgagcgtc caggaagg 182421DNAArtificial
sequence/note="Description of artificial sequence primer V
alpha-9-for-out" 24gtgcaactat tcctattctg g 212519DNAArtificial
sequence/note="Description of artificial sequence primer V
alpha-10,24-for-out"variation2/replace="g"variation13/replace="t"
25actggagcag agccctcag 192619DNAArtificial
sequence/note="Description of artificial sequence primer V
alpha-11-for-out" 26tcttcagagg gagctgtgg 192719DNAArtificial
sequence/note="Description of artificial sequence primer V
alpha-12-for-out" 27ggtggagaag gaggatgtg 192819DNAArtificial
sequence/note="Description of artificial sequence primer V
alpha-13,19,26-for-out"variation1/replace="g"variation4/replace-
="g" 28caactggagc agagtcctc 192918DNAArtificial
sequence/note="Description of artificial sequence primer V
alpha-15-for-out" 29cctgagtgtc cgagaggg 183021DNAArtificial
sequence/note="Description of artificial sequence primer V
alpha-16-for-out" 30atgcacctat tcagtctctg g 213120DNAArtificial
sequence/note="Description of artificial sequence primer V
alpha-17-for-out" 31tgatagtcca gaaaggaggg 203220DNAArtificial
sequence/note="Description of artificial sequence primer V
alpha-18-for-out" 32gtcactgcat gttcaggagg 203320DNAArtificial
sequence/note="Description of artificial sequence primer V
alpha-22,31-for-out"variation5/replace="t" 33ccctaccctt ttctggtatg
203419DNAArtificial sequence/note="Description of artificial
sequence primer V
alpha-23,30-for-out"variation5/replace="g"variation8/replace="t"
34ggcaagaccc tgggaaagg 193519DNAArtificial
sequence/note="Description of artificial sequence primer V
alpha-27-for-out" 35ctgttcctga gcatgcagg 193620DNAArtificial
sequence/note="Description of artificial sequence primer V
alpha-28-for-out" 36agacaaggtg gtacaaagcc 203719DNAArtificial
sequence/note="Description of artificial sequence primer V
alpha-29-for-out" 37caaccagtgc agagtcctc 193820DNAArtificial
sequence/note="Description of artificial sequence primer V
alpha-32-for-out" 38gcatgtacaa gaaggagagg 203941DNAArtificial
sequence/note="Description of artificial sequence primer
Vp1+"variation24/replace="t" 39acagcacgac ttccaagact cacctttgtc
tcctgggagc a 414042DNAArtificial sequence/note="Description of
artificial sequence primer Vp2+" 40acagcacgac ttccaagact cacctgatgt
cgcccagact cc 424144DNAArtificial sequence/note="Description of
artificial sequence primer
Vp3+"variation28/replace="g"variation41/replace="t" 41acagcacgac
ttccaagact cagtcatcca gaacccaaga cacc 444246DNAArtificial
sequence/note="Description of artificial sequence primer
Vp4+"variation25/replace="t"variation33/replace="c"variation36/re-
place="c" 42acagcacgac ttccaagact caggatatct gtaagagtgg aacctc
464344DNAArtificial sequence/note="Description of artificial
sequence primer Vp5+"variation43/replace="t" 43acagcacgac
ttccaagact caatgtactg gtatcgacaa gacc 444445DNAArtificial
sequence/note="Description of artificial sequence primer Vp6+"
44acagcacgac ttccaagact cacactgtgg aaggaacatc aaacc
454543DNAArtificial sequence/note="Description of artificial
sequence primer Vp7+"variation33/replace="g" 45acagcacgac
ttccaagact catctccact ctcaagatcc agc 434648DNAArtificial
sequence/note="Description of artificial sequence primer
Vp8+"variation26/replace="g"variation32/replace="g"variation35/replace="t-
" 46acagcacgac ttccaagact cacagaatgt aaatctcagg tgtgatcc
484745DNAArtificial sequence/note="Description of artificial
sequence primer
Vp9+"variation29/replace="t"variation34/replace="g"variation36/re-
place="t" 47acagcacgac ttccaagact catcagacac caaaacacct ggtca
454821DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-4/1-for-in" 48acagaagaca gaaagtccag c
214920DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-4/2-for-in" 49gtccagtacc ttgatcctgc
205021DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-6-for-in" 50gcaaaatgca acagaaggtc g
215120DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-8/1-for-in" 51cagtgcctca aactacttcc
205221DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-8/2-for-in" 52gcctcagact acttcatttg g
215321DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-14-for-in" 53acagaatgca acggagaatc g
215420DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-24-for-in" 54ccttcagcaa cttaaggtgg
205519DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-28-for-in" 55tctctggttg tccacgagg
195620DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-2/1-for-in" 56tggaaggttt acagcacagc
205720DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-2/2-for-in" 57tggaaggttt acagcacagg
205821DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-5-for-in" 58cagcatactt acagtggtac c
215920DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-10-for-in" 59tcactgtgta ctgcaactcc
206020DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-12-for-in" 60tacaagcaac caccaagtgg
206120DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-22-for-in" 61aggctgatga caagggaagc
206220DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-31-for-in" 62gtggaatacc ccagcaaacc
206320DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-7-for-in" 63ctccagatga aagactctgc
206419DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-13-for-in" 64ttaagcgcca cgactgtcg
196521DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-17-for-in" 65ctgtgcttat gagaacactg c
216620DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-18-for-in" 66ccttacactg gtacagatgg
206720DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-21-for-in" 67tgctgaaggt cctacattcc
206820DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-23-for-in" 68gtggaagact taatgcctcg
206920DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-32-for-in" 69tcaccacgta ctgcaattcc
207020DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-3-for-in" 70ttcaggtaga ggccttgtcc
207120DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-11-for-in" 71agggacgata caacatgacc
207220DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-15-for-in" 72cctccaccta cttatactgg
207319DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-19-for-in" 73cctgcacatc acagcctcc
197420DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-25-for-in" 74agactgactg ctcagtttgg
207519DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-26-for-in" 75cctgcatatc acagcctcc
197619DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-29-for-in" 76actgcagttc ctccaaggc
197721DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-1/235-for-in" 77aaggcatcaa cggttttgag g
217819DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-1/14-for-in" 78ctgaggaaac cctctgtgc
197921DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-9-for-in" 79atctttccac ctgaagaaac c
218020DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-16-for-in" 80tccttccacc tgaagaaacc
208120DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-20-for-in" 81acgtggtacc aacagtttcc
208221DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-27-for-in" 82acttcagaca gactgtattg g
218320DNAArtificial sequence/note="Description of artificial
sequence primer V alpha-30-for-in" 83ctcttcaccc tgtattcagc
208420DNAArtificial sequence/note="Description of artificial
sequence primer pcDNA-for-1" 84cactgcttac tggcttatcg
208520DNAArtificial sequence/note="Description of artificial
sequence primer pcDNA-rev-1" 85actagaaggc acagtcgagg
208624DNAArtificial sequence/note="Description of artificial
sequence primer pcDNA-for-2-TOPO(Mimo 1)" 86cacccgactc actataggga
gacc 248721DNAArtificial sequence/note="Description of artificial
sequence primer pcDNA-2nd-for-TOPO(Mimo 2,3)" 87cacctccggc
gcgccaccat g 218821DNAArtificial sequence/note="Description of
artificial sequence primer pcDNA-rev-2(Mimo 1)" 88ctgatcagcg
ggtttaaact c 218920DNAArtificial sequence/note="Description of
artificial sequence primer pcDNA-rev-3(Mimo 2, 3)" 89tggtgatggt
gatgatgacc 209017DNAArtificial sequence/note="Description of
artificial sequence primer pcDNA-2nd-for" 90tccggcgcgc caccatg
179118DNAArtificial sequence/note="Description of artificial
sequence primer pcDNA-2nd-rev-10" 91ctagactcga gcggccgc
189230DNAArtificial sequence/note="Description of artificial
sequence primer JM22-Valpha-for" 92tgtgtcgaca tggtcctgaa attctccgtg
309332DNAArtificial sequence/note="Description of artificial
sequence primer JM22-VJalpha-rev" 93ttgggatccc gctcctgcac
agaggtagtg gc 329433DNAArtificial sequence/note="Description of
artificial sequence primer JM22-Jalpha-for" 94gagggatccc aaggaaatct
catctttgga aaa 339521DNAArtificial sequence/note="Description of
artificial sequence primer Calpha PvuII-rev" 95agcactgttg
ctcttgaagt c 219620DNAArtificial
sequence/note="Description of artificial sequence primer
JM22-Vbeta-Kpn-for" 96catgtactgg taccgacagg 209752DNAArtificial
sequence/note="Description of artificial sequence primer
JM22-VJbeta-Sca-rev" 97ccgaagtact gctcgtagga gctcctcgaa ctactggcac
agagatagaa ag 529836DNAArtificial sequence/note="Description of
artificial sequence primer hCD8a-Not-for" 98ataagaatgc ggccgcatgg
ccttaccagt gaccgc 369928DNAArtificial sequence/note="Description of
artificial sequence primer hCD8a-EcoRI-rev" 99ggaattctta gacgtatcac
gccgaaag 2810027DNAArtificial sequence/note="Description of
artificial sequence primer hCD8b-Mlu-Mut" 100tcgacgcgta tgcgaccgcg
gctgtgg 2710135DNAArtificial sequence/note="Description of
artificial sequence primer hCD8b-Xho-rev" 101ccgctcgagt tatttgtaaa
attgtttcat gaaac 3510223DNAArtificial sequence/note="Description of
artificial sequence primer sGFP-Eco-for" 102tcgaattcgc caccatggtg
agc 2310326DNAArtificial sequence/note="Description of artificial
sequence primer sGFP-Xho-rev" 103gactcgagag cttagtgatg gtgatg
2610427DNAArtificial sequence/note="Description of artificial
sequence primerHLA-A0101-lead" 104ctcgtcgaca tggccgtcat ggcgccc
2710530DNAArtificial sequence/note="Description of artificial
sequence primer HLA-A0101-end" 105tacggatcct cacactttac aagctgtgag
3010623DNAArtificial sequence/note="Description of artificial
sequence primer pcDNArc-MCS-for" 106gatccggcgc gcccctgcag ggc
2310723DNAArtificial sequence/note="Description of artificial
sequence primer pcDNArc-MCS-rev" 107ggccgccctg caggggcgcg ccg
2310844DNAArtificial sequence/note="Description of artificial
sequence primer N27-flu-Asc-for" 108cgcgccacca tgggcattct
tgggtttgtg ttcactctgt gagc 4410944DNAArtificial
sequence/note="Description of artificial sequence primer
N27-flu-Not-rev" 109ggccgctcac agagtgaaca caaacccaag aatgcccatg
gtgg 4411067DNAArtificial sequence/note="Description of artificial
sequence primer
N27-all-lib-Asc-for"variation22/replace="t"variation23/replace="t"variati-
on24/replace="t"variation25/replace="t"variation26/replace="t"variation27/-
replace="t"variation28/replace="t"variation29/replace="t"variation30/repla-
ce="t"variation31/replace="t"variation32/replace="t"variation33/replace="t-
"variation34/replace="t"variation35/replace="t"variation36/replace="t"vari-
ation37/replace="t"variation38/replace="t"variation39/replace="t"variation-
40/replace="t"variation41/replace="t"variation42/replace="t"variation43/re-
place="t"variation44/replace="t"variation45/replace="t"variation46/replace-
="t"variation47/replace="t"variation48/replace="t" 110cagggaaggc
gcgccaccat gaagaagaag aagaagaaga agaagaagtg agcggccgct 60aaactat
6711167DNAArtificial sequence/note="Description of artificial
sequence primer
N27-A2-269-lib-Asc-for"variation22/replace="t"variation23/replac-
e="t"variation24/replace="t"variation28/replace="t"variation29/replace="t"-
variation30/replace="t"variation31/replace="t"variation32/replace="t"varia-
tion33/replace="t"variation34/replace="t"variation35/replace="t"variation3-
6/replace="t"variation40/replace="t"variation41/replace="t"variation42/rep-
lace="t"variation43/replace="t"variation44/replace="t"variation45/replace=-
"t" 111cagggaaggc gcgccaccat gaagatcaag aagaaggtga agaagctatg
agcggccgct 60aaactat 6711218DNAArtificial
sequence/note="Description of artificial sequence primer
N27-Not-rev" 112tagtttagcg gccgctca 18
* * * * *
References