U.S. patent application number 12/735741 was filed with the patent office on 2011-05-12 for cd4+cells with cytolytic properties.
Invention is credited to Jean-Marie Saint-Remy.
Application Number | 20110111395 12/735741 |
Document ID | / |
Family ID | 40735985 |
Filed Date | 2011-05-12 |
United States Patent
Application |
20110111395 |
Kind Code |
A1 |
Saint-Remy; Jean-Marie |
May 12, 2011 |
CD4+CELLS WITH CYTOLYTIC PROPERTIES
Abstract
The present invention relates to CD4+ T cells, more specifically
cytolytic or cytotoxic CD4+ T-cells and methods of obtaining and
identifying them.
Inventors: |
Saint-Remy; Jean-Marie;
(Grez-Doiceau, BE) |
Family ID: |
40735985 |
Appl. No.: |
12/735741 |
Filed: |
February 16, 2009 |
PCT Filed: |
February 16, 2009 |
PCT NO: |
PCT/EP2009/051807 |
371 Date: |
August 13, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61035908 |
Mar 12, 2008 |
|
|
|
Current U.S.
Class: |
435/6.17 ;
435/325; 435/377; 435/7.92 |
Current CPC
Class: |
A61K 2035/124 20130101;
C12N 5/0636 20130101; C12N 2501/23 20130101; A61K 2039/57 20130101;
A61K 2039/627 20130101 |
Class at
Publication: |
435/6 ; 435/325;
435/377; 435/7.92 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12N 5/0783 20100101 C12N005/0783; G01N 33/68 20060101
G01N033/68 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 14, 2008 |
EP |
08447006.1 |
Claims
1. An isolated population of cytotoxic CD4+ T-cells characterised,
when compared to natural CD4+ regulatory T-cells, by undetectable
expression of the transcription repressor Foxp3.
2. The population of cytotoxic CD4+ T-cells according to claim 1,
further characterised, when compared to natural CD4+ regulatory
T-cells, by an increased activity of the serine-threonine kinase
AKT.
3. The population of cytotoxic CD4+ T-cells according to claim 1,
further characterised, when compared to natural CD4+ regulatory
T-cells, by undetectable production of TGF-beta and undetectable or
very low production of IL-10.
4. The population of cytotoxic CD4+ T-cells according to claim 1,
further characterised, when compared to natural CD4+ regulatory
T-cells, by production of high concentrations of IFN-gamma.
5. The population of cytotoxic CD4+ T-cells according to claim 1,
fruther characterised, when compared to natural CD4+ regulatory
T-cells, by production of high concentrations of soluble Fas ligand
(FasL).
6. An isolated population of cytotoxic CD4+ T-cells characterised,
when compared to CD4+ effector cells, by co-expression of the
transcription activators T-bet and GATA3 after antigenic
stimulation.
7. The population of cytotoxic CD4+ T-cells according to claim 6
further characterised, when compared to CD4+ effector cells, by
constitutive expression of cell surface proteins CD25 and GITR, and
of intracellular CTLA-4.
8. The population of cytotoxic CD4+ T-cells according to claim 6,
further characterised, when compared to CD4+ effector cells, by
expression of NKG2D.
9. The population of cytotoxic CD4+ T-cells according to claim 6,
or further characterised, when compared to CD4+ effector cells, by
production of high concentrations of soluble Fas ligand (FasL).
10. An isolated population of cytotoxic CD4+ T-cells characterised,
when compared to NK cells, by expression of the CD4
co-receptor.
11. The isolated population of cytotoxic CD4+ T-cells according to
claim 10 further characterised, when compared to Natural Killer
cells (NK), by undetectable expression of CD49b.
12. An isolated population of cytotoxic CD4+ T-cells characterised,
when compared to Natural Killer T cells (NKT), by absence of
expression of the invariant alpha chain of the T cell receptor.
13. The isolated population of cytotoxic CD4+ T-cells according to
claim 12 further characterised, when compared to NKT cells, by
expression of re-arranged beta chain of the T cell receptor.
14. The isolated population of cytotoxic CD4+ T-cells according to
claim 12, or further characterised, when compared to NKT cells, by
lack of CD1d restriction.
15. A method for obtaining in vivo or ex vivo the population of
cytotoxic CD4+ T-cells according to claim 1, said method comprising
the steps of: providing isolated natural naive or memory CD4+
T-cells; contacting said cells with an immunogenic peptide
comprising a T-cell epitope and, adjacent to said T-cell epitope or
separated therefrom by a linker of at most 7 amino acids, a
C-(X)2-[CST] or [CST]-(X)2-C motif; and expanding said cells in the
presence of IL-2.
16. A method of identifying a population of cytotoxic CD4+ T-cells,
said method comprising the steps of: providing isolated natural
CD4+ regulatory T-cells; providing isolated CD4+ regulatory T-cells
suspected of being cytotoxic; and determining that the T-cells
provided in (ii) display, compared to the T-cells provided in (i),
undetectable expression of the transcription repressor Foxp3
17. The method according to claim 16, said method further
comprising determining in step (iii) that the T-cells provided in
(ii) display, compared to the T-cells provided in (i), an increased
activity of the serine-threonine kinase AKT.
18. The method according to claim 16, said method further
comprising determining in step (iii) that the T-cells provided in
(ii) display, compared to the T-cells provided in (i), undetectable
production of TGF-beta and undetectable or very low production of
IL-10.
19. The method according to claim 16, said method further
comprising determining in step (iii) that the T-cells provided in
(ii) display, compared to the T-cells provided in (i), production
of high concentrations of IFN-gamma.
20. The method according to claim 16, said method further
comprising determining in step (iii) that the T-cells provided in
(ii) display, compared to the T-cells provided in (i), production
of high concentrations of soluble Fas ligand (FasL).
21. A method of identifying a population of cytotoxic CD4+ T-cells,
said method comprising the steps of: providing isolated natural
CD4+ effector T-cells; providing isolated CD4+ effector T-cells
suspected of being cytotoxic; and (iii) determining that the
T-cells provided in (ii) display, compared to the T-cells provided
in (i), co-expression of the transcription activators T-bet and
GATA3.
22. The method according to claim 21, said method further
comprising determining in step (iii) that the T-cells provided in
(ii) display, compared to the T-cells provided in (i) constitutive
expression of cell surface proteins CD25 and GITR, and of
intracellular CTLA-4.
23. The method according to claim 21, said method further
comprising determining in step (iii) that the T-cells provided in
(ii) display, compared to the T-cells provided in (i) expression of
NKG2D.
24. The method according to claim 21, said method further
comprising determining in step (iii) that the T-cells provided in
(ii) display, compared to the T-cells provided in (i) high
concentrations of soluble Fas ligand (FasL).
25. A method of identifying a population of cytotoxic CD4+ T-cells,
said method comprising the steps of: (i) providing isolated natural
killer (NK) cells; (ii) providing isolated CD4+ regulatory T-cells
suspected of being cytotoxic; and (iii) determining that the
T-cells provided in (ii) display, compared to the T-cells provided
in (i), the CD4 co-receptor.
26. The method according to claim 25, said method further
comprising determining in step (iii) that the T-cells provided in
(ii) display, compared to the T-cells provided in (i), an
undetectable expression of CD49b.
27. A method of identifying a population of cytotoxic CD4+ T-cells,
said method comprising the steps of: (i) providing isolated natural
killer T (NKT) cells; (ii) providing isolated CD4+ regulatory
T-cells suspected of being cytotoxic; and (iii) determining that
the T-cells provided in (ii) display, compared to the T-cells
provided in (i), absence of expression of the invariant alpha chain
of the T cell receptor.
28. The method according to claim 27, said method further
comprising determining in step (iii) that the T-cells provided in
(ii) display, compared to the T-cells provided in (i), expression
of re-arranged beta chain of the T cell receptor.
29. The method according to claim 27, said method further
comprising determining in step (iii) that the T-cells provided in
(ii) display, compared to the T-cells provided in (i), a lack of
CD1d restriction.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to CD4+ T cells, more
specifically cytolytic or cytotoxic CD4+ T-cells and methods of
obtaining and identifying them.
BACKGROUND OF THE INVENTION
[0002] Natural regulatory T cells (Tregs) are actively selected in
the thymus and exert potent suppressive activity in the periphery
for the induction and maintenance of tolerance. These cells are
characterised by a distinct phenotype including high expression of
cell surface proteins CD25 and GITR, high intracellular expression
of CTLA-4, but absence of IL-7R. They are anergic and
hyporesponsive in the absence of exogenous growth factors, and do
not produce IL-2. Expression of the transcription repressor Foxp3
is the hallmark of such natural Tregs. The suppressive activity of
natural Tregs was shown to be linked to a defect in phosphorylation
of AKT, a serine-threonine kinase dependent of
phosphatidylinositide-3 kinase (PI3K; Crellin et al. (2007) Blood
109: 2014-2022).
[0003] The use of such natural Tregs in controlling immune
disorders by adoptive cell transfer is severely limited by the very
low frequency of cells of defined specificity, the difficulty to
expand them in vitro and by the absence of efficient methods by
which they can be expanded in vivo. Besides, the functional
activity of natural regulatory T cells is non-specific, as they
produce suppressive cytokines such as IL-10 and TGF-beta. Hence,
there is a need for suppressor T cells with increased specificity
that are in addition more amenable to expansion.
SUMMARY OF THE INVENTION
[0004] In one aspect, the current invention encompasses isolated
populations of cytotoxic CD4+ T-cells (in either resting or
activated state) characterised, when compared to natural CD4+
regulatory T-cells, by absence of expression or undetectable
expression of the transcription repressor Foxp3. In a further
embodiment, not excluding the previous embodiment, said population
of cytotoxic CD4+ T-cells, in activated state/upon antigenic
stimulation, is, compared to natural CD4+ regulatory T-cells,
further characterised by strong phosphorylation of PI3K and of AKT.
In a further embodiment, not excluding the previous embodiments,
said population of cytotoxic CD4+ T-cells is yet further
characterised by production, upon antigenic stimulation, of high
concentrations of IFN-gamma with variable concentrations of IL-4,
IL-5, IL-10 and TNF-alpha (depending of the cytokine commitment of
the corresponding effector clone), but no or undetectable
production of IL-17 or TGF-beta, all when compared to natural CD4+
regulatory T-cells. More specifically, IL-10 concentrations of
activated CD4+ T-cells according to the invention are significantly
and drastically reduced compared to IL-10 concentrations in
activated natural CD4+ regulatory T-cells. In a further embodiment,
not excluding the previous embodiments, said population of
cytotoxic CD4+ T-cells is yet further characterised by production,
upon antigenic stimulation, of high concentrations of soluble FasL
(Fas ligand) compared to natural CD4+ regulatory T-cells.
[0005] In a further aspect, the current invention encompasses
isolated populations of cytotoxic CD4+ T-cells characterised, when
compared to CD4+ effector T-cells, by constitutive expression (i.e.
independent of whether the cytotoxic CD4+ T-cells are at rest or
activated) of cell surface proteins CD25, GITR and intracellular
CTLA-4, but no or undetectable expression of CD28 or CD127. In the
present invention constitutive expression relates to the expression
of a protein in cytotoxic CD4+ T-cells after a period of rest (i.e.
no antigenic stimulation) of about 12 to 15 days. In a further
embodiment, not excluding the previous embodiment, said population
of cytotoxic CD4+ T-cells is, when compared to CD4+ effector
T-cells, further characterised by expression of NKG2D. In a further
embodiment, not excluding the previous embodiments, said population
of cytotoxic CD4+ T-cells is, upon antigenic stimulation, yet
further characterised by production of high concentrations of
soluble FasL when compared to CD4+ effector T-cells. In a further
embodiment, not excluding the previous embodiments, said population
of cytotoxic CD4+ T-cells is, after antigenic stimulation, yet
further characterised by combined expression of transcription
factors T-bet and GATA3 when compared to CD4+ effector T-cells. In
a further embodiment, not excluding the previous embodiments, said
population of cytotoxic CD4+ T-cells is yet further characterised
by absence of (detectable) IL-2 transcription when compared to CD4+
effector T-cells. In a further embodiment, not excluding the
previous embodiments, said population of cytotoxic CD4+ T-cells is,
when compared to CD4+ effector T-cells, yet further characterised
by the capacity to induce apoptosis of APC, after antigenic
stimulation by cognate interaction with peptide presented by MHC
class II determinants. In a further embodiment, not excluding the
previous embodiments, said population of cytotoxic CD4+ T-cells is,
in comparison with CD4+ effector T-cells, yet further characterised
by the capacity to induce apoptosis of bystander T cells.
[0006] In a further aspect, the current invention encompasses
isolated populations of cytotoxic CD4+ T-cells characterised, when
compared to NK-cells, by expression of the CD4 co-receptor. In a
further embodiment, not excluding the previous embodiments, said
population of cytotoxic CD4+ T-cells is, when compared to NK-cells,
yet further characterised by absence of CD49b (as detected by
binding of antibody DX5). These characteristics relative to
NK-cells are independent of the activation status of the cytotoxic
CD4+ T-cells and thus are detectable both in resting and activated
cells.
[0007] In yet a further embodiment of the invention are comprised
isolated populations of cytotoxic CD4+ T-cells characterised, when
compared to NKT-cells by expression of an alpha-beta T cell
receptor with invariant alpha chain and re-arranged beta chain. In
a further embodiment, not excluding the previous embodiments, said
population of cytotoxic CD4+ T-cells is, when compared to
NKT-cells, further characterised by absence of (detectable)
expression of the Valpha14 (mouse) or Valpha24 (human) TCR
expression. In a further embodiment, not excluding the previous
embodiments, said population of cytotoxic CD4+ T-cells is, in
comparison with NKT-cells, yet further characterised by lack of
CD1d restriction. These characteristics relative to NKT-cells are
independent of the activation status of the cytotoxic CD4+ T-cells
and thus are detectable both in resting and activated cells.
[0008] The invention comprises in another aspect isolated
populations of cytotoxic CD4+ T-cells displaying any possible
combination of any of the characteristics as described above and
relative to natural CD4+ regulatory T-cells, CD4+ effector T-cells,
NK-cells and/or NKT-cells or characterised by a combination of all
of these characteristics.
[0009] The invention relates in another aspect to a method for
obtaining or inducing populations of cytotoxic CD4+ T-cells as
described above according to the invention, said methods comprising
the steps of: [0010] (i) providing isolated natural naive or memory
CD4+ T-cells; [0011] (ii) contacting said cells with an immunogenic
peptide comprising a T-cell epitope and, adjacent to said T-cell
epitope or separated therefrom by a linker of at most 7 amino
acids, a C-(X)2-[CST] or [CST]-(X)2-C motif; and [0012] (iii)
expanding said cells in the presence of IL-2.
[0013] In a further aspect, the invention encompasses a method of
identifying a population of cytotoxic CD4+ T-cells, said method
comprising the steps of: [0014] (i) providing isolated natural CD4+
T-cells such as natural CD4+ regulatory T-cells, CD4+ effector
cells, NK-cells or NKT-cells; [0015] (ii) providing CD4+ T-cells
suspected of being cytotoxic; and [0016] (iii) determining that the
T-cells provided in (ii) display, compared to the T-cells provided
in (i), the respective characteristics as described above.
[0017] Thus, in one embodiment thereto, said method is identifying
cytotoxic CD4+ T-cells by determining in step (iii) the absence of
or undetectable expression of the transcription receptor Foxp3 when
compared with expression of Foxp3 in natural CD4+ regulatory
T-cells. Said method may further comprise determining in step (iii)
that the T-cells provided in (ii) display, compared to natural CD4+
regulatory T-cells provided in (i), an increased kinase activity of
the serine-threonine kinase AKT. In a further embodiment, not
excluding the previous embodiment, said method is further
comprising determining in step (iii) that the T-cells provided in
(ii) display, compared to natural CD4+ regulatory T-cells provided
in (i), undetectable production of TGF-beta and undetectable or
very low production of IL-10. In a further embodiment, not
excluding the previous embodiments, said method is further
comprising determining in step (iii) that the T-cells provided in
(ii) display, compared to natural CD4+ regulatory T-cells provided
in (i), high concentrations of IFN-gamma production. In a further
embodiment, not excluding the previous embodiments, said method is
further comprising determining in step (iii) that the T-cells
provided in (ii) display, compared to natural CD4+ regulatory
T-cells provided in (i), production of high concentrations of
soluble FasL.
[0018] In a further embodiment said method identifies populations
of cytotoxic CD4+ T-cells according to the invention by comparing
them with CD4+ effector cells. Thus, such methods may comprise
determining in step (iii) that the T-cells provided in (ii)
display, compared to CD4+ effector cells provided in (i),
constitutive expression of cell surface proteins CD25, GITR and
intracellular CTLA-4, but not of CD28 or CD127. In a further
embodiment, not excluding the previous embodiments, said method is
further comprising determining in step (iii) that the T-cells
provided in (ii) display, compared to CD4+ effector cells provided
in (i), expression of NKG2D on the cell surface. In a further
embodiment, not excluding the previous embodiments, said method is
further comprising determining in step (iii) that the T-cells
provided in (ii) display, compared to CD4+ effector cells provided
in (i), co-expression of transcription factors T-bet and GATA3. In
a further embodiment, not excluding the previous embodiments, said
method is further comprising determining in step (iii) that the
T-cells provided in (ii) display, compared to CD4+ effector cells
provided in (i), a absence of IL-2 transcription. In a further
embodiment, not excluding the previous embodiments, said method is
further comprising determining in step (iii) that the T-cells
provided in (ii) display, compared to CD4+ effector cells provided
in (i), the capacity to induce apoptosis of APC, after antigenic
stimulation by cognate interaction with peptide presented by MHC
class II determinants. In a further embodiment, not excluding the
previous embodiments, said method is further comprising determining
in step (iii) that the T-cells provided in (ii) display, compared
to CD4+ effector cells provided in (i), the capacity to induce
apoptosis of bystander T cells.
[0019] In a further embodiment said method identifies populations
of cytotoxic CD4+ T-cells according to the invention by comparing
them with NK-cells. Thus, such methods may comprise determining in
step (iii) that the T-cells provided in (ii) display, compared to
NK cells provided in (i), expression of the CD4 co-receptor. In a
further embodiment, not excluding the previous embodiments, said
method is further comprising determining in step (iii) that the
T-cells provided in (ii) display, compared to NK cells provided in
(i), the absence of expression of CD49b.
[0020] In a further embodiment of the invention are included
methods for identifying populations of cytotoxic CD4+ T-cells
according to the invention by comparing them to NKT-cells. Such
methods may comprise determining in step (iii) that the T-cells
provided in (ii) display, compared to NKT-cells provided in (i),
expression of an alpha-beta T cell receptor with rearranged beta
chain. In a further embodiment, not excluding the previous
embodiments, said method is further comprising determining in step
(iii) that the T-cells provided in (ii) display, compared to
NKT-cells provided in (i), absence of expression of the Valpha14
(mouse) or Valpha24 (human) TCR expression. In a further
embodiment, not excluding the previous embodiments, said method is
further comprising determining in step (iii) that the T-cells
provided in (ii) display, compared to the NKT-cells provided in
(i), absence of CD1d restriction.
[0021] In the above methods according to the invention it is
further possible to identify CD4+ T-cells suspected to be cytotoxic
CD4+ T-cells according to the invention as provided in step (ii) by
determining in step (iii) any possible combination of any or all of
the characteristics as described above and relative to natural CD4+
regulatory T-cells, CD4+ effector T-cells, NK-cells and/or
NKT-cells provided in step (i), said combinations also being
described above.
FIGURE LEGENDS
[0022] FIG. 1. Cytolytic CD4+ T cell clones express markers
associated with regulatory T cells. See Example 2 for detailed
explanation.
[0023] FIG. 2. Cytolytic CD4+ T cell clones co-express
transcription factors T-bet and GATA3 but not Foxp3. See Example 3
for detailed explanation.
[0024] FIG. 3. Cytolytic CD4+ T cells are distinct from NK cells.
See Example 5 for detailed explanation.
[0025] FIG. 4. Cytolytic CD4+ T cells are distinct from NKT cells.
See Example 6 for detailed explanation.
[0026] FIG. 5. Cytolytic CD4+ T cells show phosphorylation of AKT
by contrast to natural CD4+ regulatory cells. See Example 7 for
detailed explanation.
[0027] FIG. 6. Cytolytic CD4+ T cells induce apoptosis of
antigen-presenting cells after cognate peptide recognition. See
Example 8 for detailed explanation.
[0028] FIG. 7. Cytolytic CD4+ T cells induce apoptosis of bystander
T cells. See Example 9 for detailed explanation.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0029] The term "peptide" when used herein refers to a molecule
comprising an amino acid sequence of between 2 and 200 amino acids,
connected by peptide bonds, but which can in a particular
embodiment comprise non-amino acid structures (like for example a
linking organic compound). Peptides according to the invention can
contain any of the conventional 20 amino acids or modified versions
thereof, or can contain non-naturally occurring amino acids
incorporated by chemical peptide synthesis or by chemical or
enzymatic modification.
[0030] The term "epitope" when used herein refers to one or several
portions (which may define a conformational epitope) of a protein
which is/are specifically recognised and bound by an antibody or a
portion thereof (Fab', Fab2', etc.) or a receptor presented at the
cell surface of a B or T cell lymphocyte, and which is able, by
said binding, to induce an immune response.
[0031] The term "antigen" when used herein refers to a structure of
a macromolecule comprising one or more hapten(s) and/or comprising
one or more T cell epitopes. Typically, said macromolecule is a
protein or peptide (with or without polysaccharides) or made of
proteic composition and comprises one or more epitopes; said
macromolecule can herein alternatively be referred to as "antigenic
protein" or "antigenic peptide".
[0032] The term "T cell epitope" or "T-cell epitope" in the context
of the present invention refers to a dominant, sub-dominant or
minor T cell epitope, i.e., a part of an antigenic protein that is
specifically recognised and bound by a receptor at the cell surface
of a T lymphocyte. Whether an epitope is dominant, sub-dominant or
minor depends on the immune reaction elicited against the epitope.
Dominance depends on the frequency at which such epitopes are
recognised by T cells and able to activate them, among all the
possible T cell epitopes of a protein. In particular, a T cell
epitope is an epitope bound by MHC class I or MHC class II
molecules.
[0033] The term "CD4+ effector cells" refers to cells belonging to
the CD4-positive subset of T-cells whose function is to provide
help to other cells, such as, for example B-cells. These effector
cells are conventionally reported as Th cells (for T helper cells),
with different subsets such as Th0, Th1, Th2, and Th17 cells.
[0034] The term "immune disorders" or "immune diseases" refers to
diseases wherein a reaction of the immune system is responsible for
or sustains a malfunction or non-physiological situation in an
organism. Included in immune disorders include e.g. allergic
disorders, autoimmune diseases, alloimmunisation reactions,
rejection of viral vectors used in gene therapy/gene
vaccination.
[0035] In the specification the following acronyms and
abbreviations are used: [0036] AKT: group of serine/protein kinases
comprising AKT 1, AKT2 and AKT3 (also known as Protein Kinase B
(PKB)) [0037] CD1d: Thymocyte Antigen CD1D [0038] CD25: Interleukin
2 Receptor, Alpha Chain (also known as IL2RA, TCGFR, and TAC
antigen) [0039] CTLA-4: Cytotoxic T-Lymphocyte Antigen 4 (also
known as CD152) [0040] CD49b: Integrin, Alpha-2 (also known as
ITGA2, VLAA2) [0041] FasL: FAS Ligand (also known as TNFSF,
APT1LG1, CD95L, CD178) [0042] Foxp3: Forkhead Box P3 (also known as
SCURFIN or JM2) [0043] GATA3: GATA-Binding Protein 3 [0044] GITR:
Glucocorticoid-Induced Tnfr-Related Gene; (also known as AITR)
[0045] IFN: Interferon [0046] NKG2D: Killer cell lectin-like
receptor subfamily K, member 1, (also known as KLRK1, CD314) [0047]
T-bet: T-Box Expressed In T Cells; (also known as T-BOX 21, TBX21)
[0048] TGF-beta: Transforming Growth Factor Beta [0049] IL:
Interleukin [0050] NK: natural killer cells [0051] NKT: natural
killer T cells
DETAILED DESCRIPTION
[0052] The present invention is based on the characterisation of a
subset of CD4+ T cells having novel characteristics. This new
subset of CD4+ T cells shares characteristics of regulatory T
cells, of effector cells and of NK/NKT cells, but carries features
and expresses properties that clearly distinguish it from
regulatory cells, effector cells and NK/NKT cells. Such new CD4+ T
cells are called cytolytic (or cytotoxic) CD4+ T cells (cCD4+ T
cells in short). cCD4+ T cells are anergic, long-living and become
activated only when recognising a cognate peptide presented by
antigen-presenting cells (APCs). These cCD4+ T cells are thus
strictly antigen-specific. Furthermore, they can easily be expanded
(such as under ex vivo or in vitro conditions) in the presence of
IL-2. An additional advantage of the cCD4+ T cells of the invention
exists therein that they do not produce suppressive cytokines,
which limits the risk of non-specific effects. Clearly, the
advantages exhibited by the cCD4+ T cells of the invention make
them excellent candidates for treating immune disorders via
adoptive cell transfer.
[0053] The cCD4+ T cells of the invention are distinct from natural
Tregs by absence of expression or undetectable expression of the
transcription repressor Foxp3, by production of high concentrations
of IFN-gamma upon stimulation with variable concentrations of IL-4,
IL-5, and TNF-alpha (depending of the cytokine commitment of the
corresponding effector clone), but undetectable, or no IL-17 or
TGF-beta (transforming growth factor beta). In cCD4+ T-cells, IL-10
concentrations are significantly or drastically reduced or lower
compared to natural CD4+ regulatory cells. Furthermore, cCD4+
T-cells according to the invention display strong phosphorylation
of PI3K and AKT, and production of high concentrations of soluble
FasL, though all these characteristics are not necessarily present
together.
[0054] The cCD4+ T cells of the invention are distinct from CD4+
effector T cells by constitutive expression of cell surface
proteins CD25, GITR and intracellular CTLA-4, but no or
undetectable expression of CD28 or CD127, by cell expression of
NKG2D, by production of high concentrations of soluble FasL, by
co-expression of transcription factors T-bet and GATA3, by absence
of IL-2 transcription, by the capacity to induce apoptosis of
antigen-presenting cells (APCs), after antigenic stimulation by
cognate recognition of peptides presented by MHC class II
determinants and by the capacity to induce apoptosis of bystander T
cells, though all these characteristics are not necessarily present
together.
[0055] The cCD4+ T cells of the invention are distinct from NK
cells by expression of the CD4 co-receptor, by constitutive
expression of cell surface proteins CD25, GITR and intracellular
CTLA-4 and by absence of (detectable) CD49b expression. NK cells do
not express the CD4 co-receptor but do express CD49b
[0056] The cCD4+ T cells of the invention are distinct from NKT
cells by expression of an alpha-beta T cell receptor with
rearranged beta chain, by absence of Valpha14 (mouse) or Valpha24
(human) TCR expression and by lack of CD1d restriction, though all
these characteristics are not necessarily present together.
[0057] Hence, in one aspect, the current invention encompasses
isolated populations of cytotoxic CD4+ T-cells (in either resting
or activated state) characterised, when compared to natural CD4+
regulatory T-cells, by absence of expression or undetectable
expression of the transcription repressor Foxp3. In a further
embodiment, not excluding the previous embodiment, said population
of cytotoxic CD4+ T-cells, in activated state/upon antigenic
stimulation, is, compared to natural CD4+ regulatory T-cells,
further characterised by strong phosphorylation of PI3K and of AKT.
In a further embodiment, not excluding the previous embodiments,
said population of cytotoxic CD4+ T-cells is yet further
characterised by production, upon stimulation, of high
concentrations of IFN-gamma with variable concentrations of IL-4,
IL-5, IL-10 and TNF-alpha (depending of the cytokine commitment of
the corresponding effector clone), but no or undetectable
production of IL-17 or TGF-beta, all when compared to natural CD4+
regulatory T-cells. More specifically, IL-10 concentrations of
activated CD4+ T-cells according to the invention are significantly
and drastically reduced compared to IL-10 concentrations in
activated natural CD4+ regulatory T-cells. In a further embodiment,
not excluding the previous embodiments, said population of
cytotoxic CD4+ T-cells is yet further characterised by production,
upon antigenic stimulation, of high concentrations of soluble FasL
(Fas ligand) compared to natural CD4+ regulatory T-cells.
[0058] In a further aspect, the current invention encompasses
isolated populations of cytotoxic CD4+ T-cells characterised, when
compared to CD4+ effector T-cells, by constitutive expression (i.e.
independent of whether the cytotoxic CD4+ T-cells are at rest or
activated) of cell surface proteins CD25, GITR and intracellular
CTLA-4, but no or undetectable expression of CD28 or CD127. In a
further embodiment, not excluding the previous embodiment, said
population of cytotoxic CD4+ T-cells is, when compared to CD4+
effector T-cells, further characterised by constitutive expression
of NKG2D. In a further embodiment, not excluding the previous
embodiments, said population of cytotoxic CD4+ T-cells is, upon
antigenic stimulation, yet further characterised by production of
high concentrations of soluble FasL when compared to CD4+ effector
T-cells. In a further embodiment, not excluding the previous
embodiments, said population of cytotoxic CD4+ T-cells is, after
antigenic stimulation, yet further characterised by co-expression
of transcription factors T-bet and GATA3 when compared to CD4+
effector T-cells. In a further embodiment, not excluding the
previous embodiments, said population of cytotoxic CD4+ T-cells is
yet further characterised by absence of (detectable) IL-2
transcription when compared to CD4+ effector T-cells. In a further
embodiment, not excluding the previous embodiments, said population
of cytotoxic CD4+ T-cells is, when compared to CD4+ effector
T-cells, yet further characterised by the capacity to induce
apoptosis of APC, after antigenic stimulation by cognate
interaction with peptide presented by MHC class II determinants. In
a further embodiment, not excluding the previous embodiments, said
population of cytotoxic CD4+ T-cells is, in comparison with CD4+
effector T-cells, yet further characterised by the capacity to
induce apoptosis of bystander T cells.
[0059] In a further aspect, the current invention encompasses
isolated populations of cytotoxic CD4+ T-cells characterised, when
compared to NK-cells, by expression of the CD4 co-receptor. In a
further embodiment, not excluding the previous embodiments, said
population of cytotoxic CD4+ T-cells is, when compared to NK-cells,
yet further characterised by absence of CD49b expression. These
characteristics relative to NK-cells are independent of the
activation status of the cytotoxic CD4+ T-cells and thus are
detectable both in resting and activated cells.
[0060] In yet a further embodiment of the invention are comprised
isolated populations of cytotoxic CD4+ T-cells characterised, when
compared to NKT-cells by expression of an alpha-beta T cell
receptor with re-arranged beta chain. In a further embodiment, not
excluding the previous embodiments, said population of cytotoxic
CD4+ T-cells is, when compared to NKT-cells, further characterised
by absence of (detectable) expression of the Valpha14 (mouse) or
Valpha24 (human) TCR expression. In a further embodiment, not
excluding the previous embodiments, said population of cytotoxic
CD4+ T-cells is, in comparison with NKT-cells, yet further
characterised by lack of CD1d restriction. These characteristics
relative to NKT-cells are independent of the activation status of
the cytotoxic CD4+ T-cells and thus are detectable both in resting
and activated cells.
[0061] The invention comprises in another aspect isolated
populations of cytotoxic CD4+ T-cells displaying any possible
combination of any of the characteristics as described above and
relative to natural CD4+ regulatory T-cells, CD4+ effector T-cells,
NK-cells and/or NKT-cells or characterised by a combination of all
of these characteristics.
[0062] The CD4+ T cells of the invention can be elicited from both
naive CD4+ T cells as well as from memory CD4+ T cells, more
particularly by incubation with T-cell epitopes modified by
attachment of a consensus motif sequence with thioreductase
activity ([CST]XX[CST]-motif, wherein [CST] is an amino acid
selected from cysteine, serine and threonine, and X can be any
amino acid except proline). After elicitation, they can be expanded
in a suitable culture medium comprising IL-2.
[0063] The invention relates in another aspect to a method for
obtaining or inducing populations of cytotoxic CD4+ T-cells as
described above according to the invention, said methods comprising
the steps of: [0064] (i) providing isolated natural naive or memory
CD4+ T-cells; [0065] (ii) contacting said cells with an immunogenic
peptide comprising a T-cell epitope and, adjacent to said T-cell
epitope or separated therefrom by a linker of at most 7 amino
acids, a C-(X)2-[CST] or [CST]-(X)2-C motif; and [0066] (iii)
expanding said cells in the presence of IL-2.
[0067] In this method the cytotoxic CD4+ T-cells can be obtained or
induced in vivo or ex vivo. When in vivo, steps (i) and (iii) in
the above method are redundant as the contacting of the cells with
the T-cell epitope as described in (ii) is occurring by
administering the T-cell epitope to the subject in need thereof,
and the resulting cytotoxic CD4+ T-cells will expand in the
subject's body.
[0068] The invention relates in another aspect to a method for
obtaining or inducing populations of cytotoxic CD4+ T-cells as
described above according to the invention, said methods comprising
the steps of: [0069] (i) administering to a subject in need thereof
an immunogenic peptide comprising a T-cell epitope and, adjacent to
said T-cell epitope or separated therefrom by a linker of at most 7
amino acids, a C-(X)2-[CST] or [CST]-(X)2-C motif, thereby inducing
cytotoxic CD4+ T-cells; and [0070] (ii) isolating or obtaining the
cytotoxic CD4+ T-cells induced in (i).
[0071] Alternatively, the cCD4+ T cells according to the invention
may be obtained by incubation in the presence of APCs presenting
the above-mentioned immunogenic peptide after transduction or
transfection of the APCs with a genetic construct capable of
driving expression of such immunogenic peptide. Such APCs may in
fact themselves be administered to a subject in need to trigger in
vivo in said subject the induction of the beneficial subset of
cCD4+ T cells. In another alternative method, the cCD4+ T cells can
be generated in vivo, i.e. by the administration of the
above-mentioned immunogenic peptide to a subject, and collection of
the cCD4+ T cells generated in vivo. Accordingly, the present
invention further relates to the generation of the cCD4+ T cells of
the invention both in vivo and in vitro (ex vivo) using the
immunogenic peptides or APCs presenting such immunogenic
peptides.
[0072] Subjects suffering from, or having an immune disorder, or
whom are diagnosed to be predestined for developing an immune
disorder can be treated by administering (a sufficient or effective
amount of) the cCD4+ T-cells according to the invention wherein
said cCD4+ T-cells are specific to a T-cell epitope relevant to the
immune disorder to be treated or prevented. Prophylactic
administration, treatment or prevention of immune disorders would
be desirable in subjects predestined for developing an immune
disorder. Such predestination may be diagnosed e.g. by a positive
diagnosis of a genetic defect known to predestine a subject to
develop an immune disorder or known to increase the likelihood of
developing an immune disorder. Alternatively, inheritable immune
disorders which have manifested themselves in one or more of the
ancestors or within the family of a subject may increase the chance
that/may be predestining said subject to develop the immune
disorder, such subjects may therefore also be eligible for
prophylactic treatment with the cCD4+ T-cells according to the
invention.
[0073] The cCD4+ T cells obtainable by the above methods are of
particular interest for use in the manufacture of a medicament for
(prophylactically) preventing, suppressing or treating an immune
disorder in a mammal. Both the use of allogeneic and autogeneic
cCD4+ T-cells is envisaged. Any method comprising the
administration of said cCD4+ T cells to a subject in need is known
as adoptive cell therapy. As mentioned before, the cCD4+ T-cells to
be included in the medicament would need to be "educated", i.e.
would need to be specific, for a T-cell epitope of an antigen known
to be relevant in the to be treated immune disorder.
[0074] The above-mentioned immunogenic peptides in general comprise
(i) at least one T-cell epitope of an antigen of choice with a
potential to trigger an immune reaction, which is coupled to (ii)
an organic compound having a reducing activity, such as a
thioreductase sequence motif. The antigen of choice will vary along
with (and be determined by) the immune disorder to be prevented or
suppressed. The T-cell epitope and the organic compound are
optionally separated by a linker sequence. In further optional
embodiments the immunogenic peptide additionally comprises an
endosome targeting sequence (e.g. late endosomal targeting
sequence) and/or additional "flanking" sequences. The immunogenic
peptides can be schematically represented as A-L-B or B-L-A,
wherein A represents a T-cell epitope of an antigen (self or
non-self) with a potential to trigger an immune reaction, L
represents a linker and B represents an organic compound having a
reducing activity. The reducing activity of an organic compound can
be assayed for its ability to reduce a sulfhydryl group such as in
the insulin solubility assay known in the art, wherein the
solubility of insulin is altered upon reduction, or with a
fluorescence-labelled insulin. The reducing organic compound may be
coupled at the amino-terminus side of the T-cell epitope or at the
carboxy-terminus of the T-cell epitope.
[0075] Generally the organic compound with reducing activity is a
peptide sequence. Peptide fragments with reducing activity are
encountered in thioreductases which are small disulfide reducing
enzymes including glutaredoxins, nucleoredoxins, thioredoxins and
other thiol/disulfide oxydoreductases They exert reducing activity
for disulfide bonds on proteins (such as enzymes) through redox
active cysteines within conserved active domain consensus
sequences: C-X(2)-C, C-X(2)-S, C-X(2)-T, S-X(2)-C, T-X(2)-C
(Fomenko et al. (2003) Biochemistry 42, 11214-11225), in which X
stands for any amino acid. Such domains are also found in larger
proteins such as protein disulfide isomerase (PDI) and
phosphoinositide-specific phospholipase C. In particular, the
immunogenic peptides comprise as redox motif the thioreductase
sequence motif [CST]-X(2)-[CST], in a further embodiment thereto,
said [CST]-X(2)-[CST] motif is positioned N-terminally of the
T-cell epitope. More specifically, in said redox motif at least one
of the [CST] positions is occupied by a Cys; thus the motif is
either [C]-X(2)-[CST] or [CST]-X(2)-[C]. In the present application
such a tetrapeptide will be referred to as "the motif" or "redox
motif". More in particular, the immunogenic peptides can contain
the sequence motif [C]-X(2)-[CS] or [CS]-X(2)-[C]. Even more
particularly, the immunogenic peptides contain the sequence motif
C-X(2)-S, S-X(2)-C or C-X(2)-C.
[0076] The above immunogenic peptides can be made by chemical
synthesis, which allows the incorporation of non-natural amino
acids. Accordingly, in the redox motif the C representing cysteine
can be replaced by another amino acids with a thiol group such as
mercaptovaline, homocysteine or other natural or non-natural amino
acids with a thiol function. In order to have reducing activity,
the cysteines present in the motif should not occur as part of a
cystine disulfide bridge. Nevertheless, the motif may comprise
modified cysteines such as methylated cysteine, which is converted
into cysteine with free thiol groups in vivo. The amino acid X in
the [CST]-X(2)-[CST] motif of particular embodiments of the
reducing compounds of the invention can be any natural amino acid,
including S, C, or T or can be a non-natural amino acid. In
particular, X can be an amino acid with a small side chain such as
Gly, Ala, Ser or Thr. More particularly, X is not an amino acid
with a bulky side chain such as Tyr; or at least one X in the
[CST]-X(2)-[CST] motif can be His or Pro.
[0077] The motif in the above immunogenic peptides is placed either
immediately adjacent to the epitope sequence within the peptide, or
is separated from the T cell epitope by a linker. More
particularly, the linker comprises an amino acid sequence of 7
amino acids or less. Most particularly, the linker comprises 1, 2,
3, or 4 amino acids. Alternatively, a linker may comprise 6, 8 or
10 amino acids. Typical amino acids used in linkers are serine and
threonine. Example of peptides with linkers in accordance with the
present invention are CXXC-G-epitope (SEQ ID NO:17),
CXXC-GG-epitope (SEQ ID NO:18), CXXC-SSS-epitope (SEQ ID NO:19),
CXXC-SGSG-epitope (SEQ ID NO:20) and the like.
[0078] The immunogenic peptides can comprise additional short amino
acid sequences N or C-terminally of the (artificial) sequence
comprising the T cell epitope and the reducing compound (motif).
Such an amino acid sequence is generally referred to herein as a
`flanking sequence`. A flanking sequence can be positioned N-
and/or C-terminally of the redox motif and/or of the T-cell epitope
in the immunogenic peptide. When the immunogenic peptide comprises
an endosomal targeting sequence, a flanking sequence can be present
between the epitope and an endosomal targeting sequence and/or
between the reducing compound (e.g. motif) and an endosomal
targeting sequence. More particularly a flanking sequence is a
sequence of up to 10 amino acids, or of in between 1 and 7 amino
acids, such as a sequence of 2 amino acids.
[0079] In particular embodiments of the invention, the redox motif
in the immunogenic peptide is located N-terminally from the
epitope.
[0080] As detailed above, the immunogenic peptides comprise a
reducing motif as described herein linked to a T cell epitope
sequence. In particular cases, the T-cell epitopes are derived from
proteins which do not comprise within their native natural sequence
an amino acid sequence with redox properties within a sequence of
11 amino acids N- or C-terminally adjacent to the T-cell epitope of
interest.
[0081] In particular embodiments, the T-cell epitope is derived
from an allergen or an auto-antigen.
[0082] Allergens that can be used for selection of T-cell epitopes
are typically allergens such as: [0083] food allergens present for
example in peanuts, fish e.g. codfish, egg white, crustacea e.g.
shrimp, milk e.g. cow's milk, wheat, cereals, fruits of the Rosacea
family, vegetables of the Liliacea, Cruciferae, Solanaceae and
Umbelliferae families, tree nuts, sesame, peanut, soybean and other
legume family allergens, spices, melon, avocado, mango, fig,
banana; [0084] house dust mites allergens obtained from
Dermatophagoides spp or D.
[0085] pteronyssinus, D. farinae and D. microceras, Euroglyphus
maynei or Blomia sp., [0086] allergens from insects present in
cockroach or Hymenoptera, [0087] allergens from pollen, especially
pollens of tree, grass and weed, [0088] allergens from animals,
especially in cat, dog, horse and rodent, [0089] allergens from
fungi, especially from Aspergillus, Alternaria or Cladosporium, and
[0090] occupational allergens present in products such as latex,
amylase, etc.
[0091] Auto-antigens that can be used for selection of T-cell
epitopes are typically antigens such as: [0092] thyroglobulin,
thyroid peroxidise or TSH receptor (thyroid autoimmune diseases);
[0093] insulin (proinsulin), glutamic acid decarboxylase (GAD),
tyrosine phosphatise IA-2, heat-shock protein HSP65, islet-specific
glucose-6-phosphate catalytic subunit related protein (IGRP) (type
1 diabetes); [0094] 21-OH hydroxylase (adrenalitis); [0095]
17-alpha hydroxylase, histidine decarboxylase, Trp hydroxylase, Tyr
hydroxylase (polyendocrine syndromes); [0096] H+/K+ ATPase
intrinsic factor (gastritis & pernicious anemia); [0097] myelin
oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP),
proteolipid protein (PLP) (multiple sclerosis); [0098]
acetyl-choline receptor (myasthenia gravis); [0099] retinol-binding
protein (RBP) (ocular diseases); [0100] type II (rheumatoid
arthritis), type II and type IX collagen (inner ear diseases);
[0101] tissue transglutaminase (celiac disease); [0102] pANCA
histone H1 protein (inflammatory bowel diseases); [0103] heat-shock
protein HSP60 (atherosclerosis); [0104] angiotensin receptor
(arterial hypertension and pre-eclampsia) [0105] nitrated
alpha-synuclein (Parkinson disease)
[0106] Other antigens that can be used for selection of T-cell
epitopes include alloantigenic proteins derived from (present
in/shed from) allografted cells or organs, soluble alloproteins
(such as in administered in replacement therapy), viral vector
proteins as used in gene therapy/gene vaccination, antigens derived
from intracellular pathogens, and antigens derived from tumours or
tumour cells.
[0107] In a further aspect, the invention encompasses a method of
identifying a population of cytotoxic CD4+ T-cells, said method
comprising the steps of: [0108] (i) providing isolated natural CD4+
T-cells such as natural CD4+ regulatory T-cells, CD4+ effector
cells, NK-cells or NKT-cells; [0109] (ii) providing CD4+ T-cells
suspected of being cytotoxic; and [0110] (iii) determining that the
T-cells provided in (ii) display, compared to the T-cells provided
in (i), the respective characteristics as described above.
[0111] In particular, the cells to be provided in step (ii) are
obtainable by or may be induced or obtained by the above-described
method of the invention. The cells provided in (i) are of a source
such that they are not comprising cytotoxic CD4+ T-cells according
to the invention. Depending on the characteristic to be determined
in step (iii), the cells provided in steps (i) and/or (ii) may need
to be activated by a cognate T-cell epitope; said need is derivable
from the characteristics of the CD4+ T-cells of the invention as
described above. The above-mentioned method of identifying a
population of cytotoxic CD4+ T-cells of the invention can thus be
formulated in a more extensive way as follows: [0112] (i) providing
isolated natural CD4+ T-cells such as natural CD4+ regulatory
T-cells, CD4+ effector cells, NK-cells or NKT-cells and,
optionally, or when required, activating these cells; [0113] (ii)
providing CD4+ T-cells suspected of being cytotoxic, said cells
being inducible or obtainable as described above, and, optionally,
or when required, activating these cells; and [0114] (iii)
determining that the T-cells provided in (ii) display, compared to
the T-cells provided in (i), the respective characteristics as
described above.
[0115] Thus, in one embodiment thereto, said method is identifying
cytotoxic CD4+ T-cells by determining in step (iii) the absence of
or undetectable expression of the transcription receptor Foxp3 when
compared with expression of Foxp3 in natural CD4+ regulatory
T-cells. Said method may further comprise determining in step (iii)
that the T-cells provided in (ii) display, compared to natural CD4+
regulatory T-cells provided in (i), an increased kinase activity of
the serine-threonine kinase AKT. In a further embodiment, not
excluding the previous embodiment, said method is further
comprising determining in step (iii) that the T-cells provided in
(ii) display, compared to natural CD4+ regulatory T-cells provided
in (i), undetectable production of TGF-beta and undetectable or
very low production of IL-10. In a further embodiment, not
excluding the previous embodiments, said method is further
comprising determining in step (iii) that the T-cells provided in
(ii) display, compared to natural CD4+ regulatory T-cells provided
in (i), high concentrations of IFN-gamma production. In a further
embodiment, not excluding the previous embodiments, said method is
further comprising determining in step (iii) that the T-cells
provided in (ii) display, compared to natural CD4+ regulatory
T-cells provided in (i), production of high concentrations of
soluble FasL.
[0116] In a further embodiment, said method identifies populations
of cytotoxic CD4+ T-cells according to the invention by comparing
them with CD4+ effector cells. Thus, such methods may comprise
determining in step (iii) that the T-cells provided in (ii)
display, compared to CD4+ effector cells provided in (i),
constitutive expression of Cell surface proteins CD25, GITR and
intracellular CTLA-4, but not of CD28 or CD127. In a further
embodiment, not excluding the previous embodiments, said method is
further comprising determining in step (iii) that the T-cells
provided in (ii) display, compared to CD4+ effector cells provided
in (i), expression of NKG2D on the cell surface. In a further
embodiment, not excluding the previous embodiments, said method is
further comprising determining in step (iii) that the T-cells
provided in (ii) display, compared to CD4+ effector cells provided
in (i), combined expression of transcription factors T-bet and
GATA3. In a further embodiment, not excluding the previous
embodiments, said method is further comprising determining in step
(iii) that the T-cells provided in (ii) display, compared to CD4+
effector cells provided in (i), a absence of IL-2 transcription. In
a further embodiment, not excluding the previous embodiments, said
method is further comprising determining in step (iii) that the
T-cells provided in (ii) display, compared to CD4+ effector cells
provided in (i), the capacity to induce apoptosis of APC, after
antigenic stimulation by cognate interaction with peptide presented
by MHC class II determinants. In a further embodiment, not
excluding the previous embodiments, said method is further
comprising determining in step (iii) that the T-cells provided in
(ii) display, compared to CD4+ effector cells provided in (i), the
capacity to induce apoptosis of bystander T cells.
[0117] In a further embodiment said method identifies populations
of cytotoxic CD4+ T-cells according to the invention by comparing
them with NK-cells. Thus, such methods may comprise determining in
step (iii) that the T-cells provided in (ii) display, compared to
NK cells provided in (i), expression of the CD4 co-receptor. In a
further embodiment, not excluding the previous embodiments, said
method is further comprising determining in step (iii) that the
T-cells provided in (ii) display, compared to NK cells provided in
(i), the absence of expression of CD49b.
[0118] In a further embodiment of the invention are included
methods for identifying populations of cytotoxic CD4+ T-cells
according to the invention by comparing them to NKT-cells. Such
methods may comprise determining in step (iii) that the T-cells
provided in (ii) display, compared to NKT-cells provided in (i),
expression of an alpha-beta T cell receptor with rearranged beta
chain. In a further embodiment, not excluding the previous
embodiments, said method is further comprising determining in step
(iii) that the T-cells provided in (ii) display, compared to
NKT-cells provided in (i), absence of expression of the Valpha14
(mouse) or Valpha24 (human) TCR expression. In a further
embodiment, not excluding the previous embodiments, said method is
further comprising determining in step (iii) that the T-cells
provided in (ii) display, compared to the NKT-cells provided in
(i), absence of CD1d restriction.
[0119] In the above methods according to the invention it is
further possible to identify CD4+ T-cells suspected to be cytotoxic
CD4+ T-cells according to the invention as provided in step (ii) by
determining in step (iii) any possible combination of any or all of
the characteristics as described above and relative to natural CD4+
regulatory T-cells, CD4+ effector T-cells, NK-cells and/or
NKT-cells provided in step (i), said combinations also being
described above.
[0120] Accordingly, the invention provides different markers and
functional properties, which can be used alone or in combination to
identify and/or select and/or to use in the quality control of
cCD4+ T cells. In particular embodiments the methods comprise a
comparison with natural regulatory T-cells, CD4+ effector cells and
NK/NKT cells. However, it is envisaged that in particular
embodiments, determining the concentration of the markers mentioned
above as such is sufficient to identify the cells (based upon the
known expression concentrations or functional properties in natural
regulatory T-cells, CD4+ effector cells and NK/NKT cells).
Accordingly determining the increased activity or expression of a
marker can optionally also involve determining `high`
concentrations of expression of such marker.
[0121] Generally, an enhanced activity of a kinase can be caused by
an increased expression of that kinase or by
phosphorylation/dephosphorylation of the kinase itself which
increases its enzymatic activity. The activity of a kinase is
determined by measuring directly or indirectly the amount of
phosphate that is incorporated in a natural or model substrate
(e.g. synthetic peptide). The activity of a kinase often depends on
the phosphorylation of that same kinase. Accordingly, the degree of
phosphorylation of a kinase can be indicative for its activity as
is the case for AKT kinase. In the above the extent of kinase
activity of the serine-threonine kinase AKT and of PI3K can be
estimated via Western blotting using an antibody specific to the
phosphorylated AKT or PI3K, respectively. The phosphorylation can
be qualified by densitometric scanning of the Western blot. Other
quantitative methods comprise methods wherein Western blots are
quantified with chemoluminescence techniques (e.g. phosphorimaging)
Alternatively phosphorylation can be determined quantitatively by
measuring the incorporation of radioactive phosphate into a
substrate. Expression of the transcription repressor Foxp3, of
transcription activators T-bet and GATA3 and IL-2 expression can be
estimated via Northern or RNA blotting using a labelled probe
specific to the respective transcript. Expression levels can
subsequently be qualified by densitometric scanning of the Northern
blot. Expression levels of certain markers can alternatively be
determined at the mRNA level by reverse transcriptase PCR methods.
Undetectable expression as determined by RT-PCR refers to
experiments wherein no signal is detected after 35 cycles of
amplification.
[0122] Expression of surface markers can be evaluated using
specific antibodies and a fluorescence-activated cell sorter
(Facs). Facs analysis allows to determine the relative amount of
cells which express a certain marker or a combination of markers.
In this context, undetectable expression of a marker (for example
of Foxp3), relates to a population of cells wherein less than 1%,
less than 0.5% or even less than 0.1% of the cells express said
marker or combination of markers.
[0123] Facs analysis is in the present invention also used to
determine whether two or more markers are co-expressed. In this
context two proteins are considered as co-expressed when at least
70, 80, 90, 95 or 99% of the cells in a cell population stain
positive for said two or more markers in a Facs analysis.
[0124] Production of cytokines such as IL-10 and TGF-beta,
IFN-gamma, IL-4, IL-5, IL-17 and IL-13, and of soluble FasL were
determined in this invention via ELISA, but can also be determined
via an ELISPOT assay. Cytokine concentrations are quantifiable via
optical density determination in solution (ELISA) or spots
indicating the presence of cytokines can be counted manually (e.g.,
with a dissecting microscope) or using an automated reader to
capture the microwell images and to analyse spot number and size
(ELISPOT). The production of cytokines as determined by ELISA is
considered to be "undetectable" when the concentration is below 50
pg/ml, below 20 pg/ml or even below 10 pg/ml, and may depend from
the type of antibody and the supplier). The production of cytokines
as determined by ELISA is considered to be "very low" when the
concentration is between 50 to 1000 pg/ml, between 100 to 1000
pg/ml, or between 200 to 1000 pg/ml. The production of cytokines as
determined by ELISA is considered to be "high" when the
concentration is above 1000 pg/ml, 2000 pg/ml, 5000 pg/ml or even
above 7500 pg/ml.
[0125] The production of transmembrane proteins (such as FasL) as
determined by ELISA is considered to be "high" when the
concentration is above 50 pg/ml, 75 pg/ml, 100 pg/ml or even above
150 pg/ml.
[0126] Generally, concentration measurements refer to conditions
wherein the protein production of about 100,000 cells is assayed in
a volume of 200 .mu.l.
[0127] Induction of apoptosis in APCs or bystander T cells can be
measured by evaluating the binding of annexin V to
phosphatidylserine exposed as the result of apoptosis.
[0128] The increase of kinase activity of the serine-threonine
kinase AKT in the cytolytic or cytotoxic CD4+ regulatory T-cells of
the invention is about 2-fold compared to natural CD4+ regulatory
T-cells, or can be up to 3-, 4-, 5-, 5.5-, 6-, 7-, 8-, 9- or
10-fold, and can be determined by methods known in the art as
explained above.
[0129] The present invention will now be illustrated by means of
the following examples, which are provided without any limiting
intention. Furthermore, all references described herein are
explicitly included herein by reference.
EXAMPLES
Example 1
List of Peptides Used in the Examples
[0130] SEQ ID NO:1, CHGSEPCIIHRGKPF (referred to in Figures as Der
p2), corresponding to amino acid sequence 21 to 35 of allergen Der
p 2 and containing a T cell epitope and a natural thioreductase
sequence (underlined).
[0131] SEQ ID NO:2, CGPCGGYRSPFSRVVHLYRNGK (referred to in Figures
as MOG+), corresponding to amino acid sequence 40-55 derived from
the myelin oligodendrocytic glycoprotein (MOG) and modified by
addition of a thioreductase motif (underlined) separated from the
first MHC class II anchoring residue by a Gly-Gly sequence.
[0132] SEQ ID NO:3, CGPCGGYVPFHIQVP (referred to in Figures as LP
HAdV5), corresponding to amino acid sequence 555-563 from Late
Protein 2 (hexon protein family) derived from human adenovirus 5
(HAdV-5) and modified by addition of a thioreductase motif
(underlined) separated from the first MHC class II anchoring
residue by a Gly-Gly sequence.
[0133] SEQ ID NO:4, CGHCGGAAHAEINEAGR (referred to in Figures as
OVA+), corresponding to amino acid sequence 330-340 derived from
chicken ovalbumin and modified by addition of a thioreductase motif
(underlined) separated from the first MHC class II anchoring
residue by a Gly-Gly sequence.
[0134] SEQ ID NO:5, CHGCGGEPCIIHRGKPF (referred to in Figures as
Der p2+), corresponding to amino acid sequence 25 to 35 of allergen
Der p 2 and modified by addition of a thioreductase motif
(underlined) separated from the first MHC class II anchoring
residue by a Gly-Gly sequence.
[0135] SEQ ID NO:6, YRSPFSRVVHLYRNGK (referred to in Figures as
MOG-), corresponding to amino acid sequence 40-55 derived from the
myelin oligodendrocytic glycoprotein (MOG).
[0136] SEQ ID NO:7, IIARYIRLHPTHYSIRST (referred to in Figures as
fVIII-), corresponding to amino acid sequence 2144-2161 derived
from the Cl domain of human Factor VIII.
[0137] SEQ ID NO:8, CGFSSNYCQIYPPNANKIR (referred to in Figures as
Der p1 +), corresponding to amino acid sequence 114 to 128 of
allergen Der p 1 and modified by addition of a thioreductase motif
(underlined) to the amino-terminal 2 0 part of the first MHC class
II anchoring residue.
[0138] SEQ ID NO:9, NACHYMKCPLVKGQQ (referred to in Figures as Der
p2*-), corresponding to amino acid sequence 71 to 85 of allergen
Der p 2.
[0139] SEQ ID NO:10, CHGAEPCIIHRGKPF (referred to in Figures as Der
p2mut), corresponding to peptide of SEQ ID1 containing a single S
to A mutation (underlined) that abolishes the thioreductase
activity of peptide.
[0140] SEQ ID NO:11, TYLRLVKIN (referred to in Figures as gD
HSV1-), corresponding to amino acid sequence 188 to 196 derived
from glycoprotein D of human herpesvirus 1.
[0141] SEQ ID NO:12, CGHCTYLRLVKIN (referred to in Figures as gD
HSV1+), corresponding to amino acid sequence 188 to 196 derived
from glycoprotein D of human herpesvirus 1 and modified by addition
of a thioreductase motif (underlined) to the amino-terminal part of
the first MHC class II anchoring residue.
[0142] SEQ ID NO:13, SNYCQIYPPNANKIR (referred to in Figures as Der
p1-), corresponding to amino acid sequence 114 to 128 of allergen
Der p 1.
[0143] SEQ ID NO:14, ISQAVHAAHAEINEAGR (referred to in Figures as
OVA-) corresponding to amino acid sequence 324-340 derived from
chicken ovalbumin.
Example 2
Cytolytic CD4+ T cell Clones Express Markers Associated with
Regulatory T Cells
[0144] The phenotype of natural regulatory T cells is characterised
by high expression of CD25 at rest, together with high expression
of intracellular CTLA-4 and surface GITR (Glucocorticoid-Induced
TNF receptor), which distinguish regulatory T cells form effector
cells.
[0145] Peptides derived from 4 distinct antigens were used: an
allergen, an autoantigen and a virus-derived surface antigen and a
common antigen.
[0146] CD4+ T cells were obtained from the spleen of BALB/c mice
immunised with peptide p21-35 (SEQ ID NO:1, CHGSEPCIIHRGKPF),
followed by purification by magnetic beads sorting. A T cell clone
was obtained by in vitro stimulation with peptide-loaded APCs
(loaded with peptide of SEQ ID NO:1). The clonal cells were
analysed on day 15 after stimulation by fluorescence-activated cell
sorting (Facs) using a FACSCalibur.COPYRGT. flow cytometer. CD4+ T
cells were stained with an antibody recognising CD25, then
permeabilised with saponin before incubation with an antibody
specific for CTLA-4. Data show strong positivity for both CD25 and
CTLA-4 (FIG. 1A). The T cell clone was also tested for expression
of GITR and CD28, showing a strong positivity for GITR (FIG. 1D),
but absence of CD28 (FIG. 1F). A CD4+ T cell clone obtained after
mouse immunisation with peptide of SEQ ID NO:2
(CGPCGGYRSPFSRVVHLYRNGK) was tested for the expression of CD25
(FIG. 1B) and CD28 (FIG. 1G), showing strong CD25 expression but
absence of CD28. Further, CD25 expression (FIG. 10) or absence of
CD28 expression (FIG. 1H) was shown for a clone obtained after
immunisation with peptide of SEQ ID NO:3 (CGPCGGYVPFHIQVP).
Expression of surface GITR, an hallmark of the 3 clones shown above
was also observed with a CD4 T cell clone specific for peptide of
SEQ ID NO:4 (CGHCGGAAHAEINEAGR), FIG. 1 E.
Example 3
Cytolytic CD4+ T Cell Clones Co-Express Transcription Factors T-Bet
and GATA3 but Not Foxp3
[0147] T-bet is considered as a marker for Th1 cells and GATA3 as a
marker for Th2 cells. In helper cells, expression of T-bet excludes
expression of GATA3 and vice-versa.
[0148] A T cell clone was obtained as described in Example 2 with a
peptide of SEQ ID NO:1. After antigenic stimulation, cells were
fixed and permeabilised before intracellular staining with specific
antibodies to either Foxp3, T-bet or GATA3 and analysed by Facs as
described in Example 2. Cells are shown to be positive for both
T-bet and GATA3 but not for Foxp3 (FIG. 2A). Dual staining with
T-bet and GATA-3 of a T cell clone obtained by immunisation with
peptide of SEQ ID NO:4 (FIG. 2B, upper panel) of by peptide of SEQ
ID NO:2 (FIG. 2C, lower panel) is also shown.
Example 4
Cytolytic CD4+ T Cell Clones Produce Soluble FasL and IFN-Gamma
[0149] The profile of cytokines produced by effector cells
characterises the subset to which cells belong. Th1 cells produce
IL-2, IFN-gamma and TNF-alpha, Th2 cells produce IL-4, IL-5, IL-13
and IL-10, and Th17 cells produce IL-17 and IL-6.
[0150] Two distinct T cell clones were obtained from 2 mice
immunised with a peptide containing a thioreductase motif (SEQ ID
NO:5, CHGCGGEPCIIHRGKPF).
[0151] In addition, T cell lines were obtained from mice immunised
with a T cell epitope in natural sequence (SEQ ID NO:6,
YRSPFSRVVHLYRNGK) derived from the myelin oligodendrocytic
glycoprotein (MOG) and were stimulated in vitro in the presence of
the same T cell epitope modified by addition of a thioreductase
motif (underlined) separated from the first MHC class II anchoring
residue by a Gly-Gly sequence (SEQ ID NO:2,
CGPCGGYRSPFSRVVHLYRNGK).
[0152] The two CD4 T cell clones specific for SEQ ID NO:5 and a CD4
T cell line specific to SEQ ID NO:6 were stimulated with peptides
for 48 h and the supernatants were assessed for the presence of
cytokines and of FasL using
[0153] ELISAs with specific antibodies. Table 1 shows that the two
CD4 T cell clones obtained from immunisation with peptide of SEQ ID
NO:5 and the CD4 T cell clone obtained with the natural sequence
SEQ ID NO:6, when stimulated in vitro with either peptide of SEQ ID
NO:5 for the first two clones or peptide of SEQ ID NO:2 for the
third clone, significant amounts of soluble FAS-L was detected in
the supernatants. By comparison, the T cell clone obtained by
immunization with peptide of SEQ ID NO:6 was stimulated in vitro
with the same peptide, no FAS-L was detected. An additional control
is shown, made from a T cell clone stimulated by peptide of SEQ ID
NO:7 (IIARYIRLHPTHYSIRST, a T cell epitope derived from human
Factor VIII), which does not contain a thioreductase motif.
TABLE-US-00001 TABLE 1 T cell Specificity sFAS-L (pg/ml) cCD4 T
(R3TB7) to SEQ ID 115.1 NO: 5 cCD4 T (22N) to SEQ ID NO: 5 176.1
cCD4 T to SEQ ID NO: 2 50.8 CD4 T to SEQ ID NO: 6 ND CD4 T (p352a)
to SEQ ID NO: 7 ND
[0154] A consistent finding with all clones obtained by
immunisation with peptides containing a thioreductase motif, or
effector CD4 T cells stimulated in vitro with peptides containing a
thioreductase motif, was the sustained production of IFN-gamma,
while IL-2, TGF-beta and IL-17 were not detected (ND). Low
concentrations of IL-4, IL-5 and IL-10 could be detected, which
correlated with the cytokine profile of the corresponding effector
cell (Table 2).
TABLE-US-00002 TABLE 2 TGF-.beta. IL-17 IFN-.gamma. IL-5 IL-4 IL-10
IL-2 cCD4 T (R3TB7) to ND ND 4151 8 ND ND ND SEQ ID NO: 5 cCD4 T
(22N) to ND ND 9139 2 ND 102 ND SEQ ID NO: 5 cCD4 T to ND ND 133 16
ND ND ND SEQ ID NO: 2 CD4 T to ND ND 4652 2538 42 5001 7 SEQ ID NO:
6 CD4 T (p352a) to ND ND 131 725 252 3847 19 SEQ ID NO: 7 cytokine
concentrations are expressed as pg/ml
Example 5
Cytolytic CD4+ T Cells are Distinct from NK Cells
[0155] NK cells are characterised by expression of CD49b and NKG2D
but not
[0156] CD4.
[0157] T cell clones were obtained from mice immunised with
peptides of SEQ ID NO:5 or of SEQ ID NO:4. Such clones were
analysed by fluorescence-activated cell sorting for the expression
of CD49b cell marker on day 14 after in vitro restimulation.
Antibodies specific to CD49b (DX5 antibody) were used in the FACS
analysis.
[0158] The results indicated that the two clones (FIGS. 3A and FIG.
3B, respectively) were uniformly negative for CD49b, thereby
distinguishing cytolytic T cell clones from NK cells.
[0159] FIGS. 3C and 3D show the expression of NKG2D on cytolytic
CD4 cells obtained from mice immunised with peptide of SEQ ID NO:5
or peptide of SEQ ID NO:8 (CGFSSNYCQIYPPNANKIR), respectively.
[0160] By comparison, expression of NKG2D was evaluated on effector
CD4 T cell clones obtained by immunisation by peptide of SEQ ID
NO:9 (NACHYMKCPLVKGQQ) or of SEQ ID NO:7, containing no
thioreductase motif (FIGS. 3E and 3F, respectively) and effector
CD4 T cells obtained by immunisation with a full allergen, Der p2
(FIG. 3G, denoted in FIG. 3G as Der p2 FL). None of these
non-cytolytic effector CD4 T cells expressed NKG2D.
Example 6
Cytolytic CD4+ T Cells are Distinct from NKT Cells
[0161] NKT cells carry an invariant alpha chain (Valpha14-Jalpha281
in the mouse, Valpha24-JQ in man) and variable but not rearranged
beta chain at the TCR level. In addition, NKT cells produce high
concentrations of IL-4, and most NKT cells are restricted by the
CD1d molecule. CD1d restriction refers to the fact that the
recognition of an antigen loaded by CD1d+ cell (antigen presenting
cell) is mediated through the recognition of the antigen by a T
cell when such antigen is presented by the CD1d molecule. In the
present example the antigen presenting cell is replaced by a
soluble from of CD1d (BDTM DimerX; Becton Dickinson)
[0162] A T cell clone obtained as in Example 5 by stimulation with
peptide of SEQ ID NO:5 was assessed on day 14 after restimulation.
Table 2 shows that such clone (R3TB7) does not produce detectable
concentrations of IL-4, distinguishing this clone from NKT cells.
Cells were further analysed by Facs using specific antibodies to
the Vbeta8-1 TCR (FIG. 4A). In addition, the sequence of the alpha
chain of the TCR was obtained by PCR. The results indicate that the
clone expressed a rearranged Vbeta chain and a Valpha sequence
belonging to the Valpha5 subfamily (and not the sequence of the
invariant Valpha14-Jalpha281 TCR chain), thereby distinguishing the
cytolytic CD4+ T cells (R3TB7) from NKT cells (FIG. 4B). The T cell
clone was further tested for staining with peptide of SEQ ID
NO:5-loaded CD1d-Ig molecule (BDTM DimerX; Becton Dickinson) and
analysed by Facs. This experiment shows that either the antigenic
peptide is not loaded on soluble CD1d and or that the cCD4+ Tcell
is not equipped with the appropriate receptor to recognize the
peptide as associated with the CD1d molecule.
[0163] The results indicate that the cytolytic CD4+ cells were not
restricted by CD1 d molecule, further distinguishing them from CD4+
NKT cells (FIG. 4C). Data is representative of different clones
with distinct antigen specificity.
Example 7
Cytolytic CD4+ T Cells Show Phosphorylation of AKT by Contrast to
Natural CD4+ Regulatory Cells
[0164] In CD4 T lymphocytes the phosphorylation of Ser473 of
serine-threonine kinase AKT is indicative of its activity. To show
that AKT kinase activity was present and/or increased when CD4+ T
cells were incubated with peptides containing a thioreductase
motif, we made use of peptide of SEQ ID NO:5 (containing such a
motif) and peptide of SEQ ID NO:10 (CHGAEPCIIHRGKPF, containing a
single S to A mutation (underlined) that abolishes the
thioreductase activity of peptide of SEQ ID NO:1).
[0165] The cytolytic CD4+ T cell clone R3TB7 was obtained by
immunisation with peptide of SEQ ID NO:1, followed by cloning, and
amplification in the presence of dendritic cells presenting the
same peptide. The R3TB7 CD4+ T cell clone was incubated for 30
minutes with antigen-presenting cells (dendritic cells) without
peptide (FIG. 5, lane 1), with APC preloaded with redox-inactive
peptide of SEQ ID NO:10 (FIG. 5, lane 2), or with APC preloaded
with a redox active peptide of SEQ ID NO:5 (FIG. 5, lane 3). Cells
were then lysed and an aliquot was run on SDS-PAGE, the proteins
were then transferred to a PVDF membrane and probed for the
phosphorylated form of AKT (Ser473) using a specific antibody. A
control containing no T cells was also included (lane 4). The
resulting phosphorylation of the serine-threonine kinase AKT in
CD4+ T-cells incubated with redox-active peptide of SEQ ID NO:5 was
5,5-fold higher as compared to AKT in the same CD4+ T-cells
incubated without unloaded APCs (FIG. 5, lane 1, mimicking natural
CD4+ regulatory T-cells), and 2-fold higher compared to AKT in the
same CD4+ T-cells incubated with the redox-inactive peptide of SEQ
ID NO:10.
[0166] Thus, a CD4+ T cell clone obtained from animals immunised
with a thioreductase containing peptide shows strong kinase
activity of AKT when incubated in vitro with a peptide containing a
thioreductase activity (peptide of SEQ ID NO:5), yet maintains AKT
kinase activity when incubated with a peptide from which the
thioreductase activity has been removed by mutation (peptide of SEQ
ID NO:10), illustrating the stable commitment of such T cell clone
under in vitro stimulation conditions. These results as illustrated
were obtained after a single incubation of cells with peptides.
This unexpected observation contrasts with the results disclosed by
Crellin et al. (2007) (Blood 109, 2014-2022) showing a decreased
kinase activity of AKT to be associated with natural CD4+
regulatory T-cells, and emphasises the difference between the
cytolytic CD4+ T-cells with suppressive properties of the invention
and natural CD4+ regulatory T-cells.
[0167] To determine whether naive CD4+ T cells showed increased AKT
kinase activity when incubated in vitro with peptides containing a
thioreductase motif, we purified CD4 T cells from splenocytes of
naive C57BL/6 mice expressing a TCR transgene specific for peptide
of SEQ ID NO:6. Cells were stimulated once for 15 minutes with T
cell depleted splenocytes preloaded with peptide of SEQ ID NO:6
(FIG. 5, lane 5) containing no thioreductase motif, or with peptide
of SEQ ID NO:2 (FIG. 5, lane 6) containing a thioreductase motif.
Cell lysis and SDS-PAGE electrophoresis were carried out as
described above. The membrane was probed with the antibody
recognising activated AKT (Ser473).
[0168] Densitometric analysis showed that phosphorylation of AKT
was 30% higher when naive cells were stimulated with peptide of SEQ
ID NO:2 than phosphorylation obtained with peptide of SEQ ID NO:6.
Thus, a single incubation of naive CD4 T cells with a peptide
containing a thioreductase motif is sufficient to elicit
significantly higher kinase activity of AKT than observed with
cells incubated with the same peptide but with no thioreductase
motif.
Example 8
Cytolytic CD4+ T Cells Induce Apoptosis of Antigen-Presenting Cells
After Cognate Peptide Recognition
[0169] Two distinct populations of APCs (WEHI cells) were loaded
for 1 hr with either peptide of SEQ ID NO:1 or with peptide of SEQ
ID NO:9. The cells loaded with peptide of SEQ ID NO:1 were labelled
with 80 nM CFSE; those loaded with peptide of SEQ ID NO:9 were
labelled with 300 nM CFSE. The two APC populations could therewith
be distinguished from each other. CFSE is a label for cytoplasmic
proteins enabling the follow-up of cell divisions based on staining
intensity, which is reduced by 50% after every cell division, but
also the identification of a cell population within complex
mixtures of cells in culture. CFSE-labelled cells were mixed and
subsequently incubated for 18 hrs with a cytolytic CD4 T cell clone
(G121) obtained by immunisation with peptide of SEQ ID NO:1.
Peptide p71-85 (SEQ ID NO:9, Der p2*-) represents an alternative
major T-cell epitope derived from Der p 2 but does not comprise a
thioreductase-active motif. Apoptosis of CFSE-labelled APCs was
measured by the binding of annexin V. WEHI cells presenting p21-35
were fully lysed, whereas only about 40% of p71-85(SEQ ID NO:1, Der
p2)-loaded cells were affected. As a control, unloaded APCs were
used. Results are depicted in FIG. 6A.
[0170] FIG. 6B shows that splenic B cells from naive C57BU6 mice
were induced into apoptosis when cultured for 18 hrs with a
cytolytic CD4 T cell clone obtained from mice immunised with
peptide of SEQ ID NO:2, as shown by dual staining with annexin V
and 7-AAD (FIG. 6B, lower panel) but not when the antigen was
absent (FIG. 6B, upper panel).
[0171] FIG. 6C shows the killing of CFSE-stained WEHI B cells
loaded with peptide of SEQ ID NO:11 (TYLRLVKIN, a T epitope derived
from the HSV-1 virus) and co-cultured for 18 hrs with a cell line
obtained from mice immunised with peptide of SEQ ID NO:12
(CGHCTYLRLVKIN), which comprises a thioreductase motif. More than
65% of the WEHI cells were stained positive for Annexin V (FIG. 6C,
lower panel), as opposed to background staining (19%) obtained when
a control T cell line derived from mice immunised with a peptide of
SEQ ID NO:11 was used (FIG. 6C, upper panel).
Example 9
Cytolytic CD4+ T Cells Induce Apoptosis of Bystander T Cells
[0172] The mechanism of bystander T cell suppression was examined
with polyclonal CD4+CD25(-) T cells and with various CD4+ effector
T cell clones.
[0173] The capacity of cytolytic CD4+ T cells to suppress the
proliferation of CFSE labelled CD4+CD25(-) T cells activated by
incubation with an antibody to CD3 in the presence of
antigen-presenting cells was assayed. Two cytolytic CD4+ T cell
clones elicited by immunisation with peptide of SEQ ID NO:1 (G121
and R3TB7, respectively; indicated in FIG. 7A as "CD4+ (Der p2)
clone") were used. The APCs were loaded with peptide of SEQ ID NO:5
(indicated in Figure 7A as "APC (Der p 2+)"). The number of
detectable CD4+CD25(-) T cells (frames), as well as the number of
observed divisions dramatically dropped within 48 h incubation when
either one of the two cytolytic clones were added (FIG. 7A, middle
and right panels). Interestingly, only activated CD4+CD25(-) T
cells were lysed. The control experiment in which the cytolytic
CD4+ T-cells were replaced by an identical number of unlabeled
CD4+CD25(-) T cells eliminated a possible artefact related to
variable numbers of total cells in the culture medium (FIG. 7A,
left panel). P1 to P3 in the Figure depict the decrease in CFSE
labelling as a function of the number of cell divisions.
[0174] An effector CD4+ T cell clone obtained from BALB/c mice
immunised with a major epitope from the allergen Der p1
(SNYCQIYPPNANKIR, SEQ ID NO:13) was labelled with CFSE and
incubated with APC loaded with the same peptide (114-128). When an
identical number of unlabelled effector cells was added, a 40%
baseline mortality of the CFSE-labelled cells was observed. When
co-cultured with a cytolytic CD4 cell clone obtained from mice
immunised with peptide of SEQ ID NO:8, more than 73% of the
effector T cells died.
[0175] Similar results were obtained when a CD4 T cell line
obtained from mice immunised with natural epitope from ovalbumin
(ISQAVHAAHAEINEAGR, SEQ ID NO:14) was used as a bystander target
for apoptosis. The cell line was labelled with CFSE (denoted in
FIG. 7B as "labelled CD4+ (OVA-)") and cultured with peptide of SEQ
ID NO:14-loaded splenic APC (denoted in FIG. 7B as "APC (OVA-)")
followed by staining with apoptosis markers annexin V and
7-AAD.
[0176] FIG. 7B shows that 27% or 24% of the bystander CD4 cells
were alive (annexin V and 7-AAD negative) when cultured with APC
alone (left panel) or with APC and the same unlabelled CD4 T cell
line (denoted in FIG. 7B as "unlabeled CD4+ (OVA-)"; right panel of
FIG. 7B), respectively. When co-cultured with a T cell line derived
from mice immunised with the ovalbumin peptide comprising a
thioreductase motif (SEQ ID NO:4; cell line denoted in FIG. 7B as
"CD4+ (OVA+)"), less than 1% of the labelled bystander cells were
detected within the double-negative region corresponding to living
cells.
Sequence CWU 1
1
20115PRTArtificial Sequencesynthetic peptide, amino acids 21-35 of
Der p2 allergen 1Cys His Gly Ser Glu Pro Cys Ile Ile His Arg Gly
Lys Pro Phe1 5 10 15222PRTArtificial Sequencesynthetic peptide,
modified MOG T-cell epitope 2Cys Gly Pro Cys Gly Gly Tyr Arg Ser
Pro Phe Ser Arg Val Val His1 5 10 15Leu Tyr Arg Asn Gly Lys
20315PRTArtificial Sequencesynthetic peptide, modified HAdV-5
T-cell epitope 3Cys Gly Pro Cys Gly Gly Tyr Val Pro Phe His Ile Gln
Val Pro1 5 10 15417PRTArtificial Sequencesynthetic peptide,
modified chicken ovalbumin T-cell epitope 4Cys Gly His Cys Gly Gly
Ala Ala His Ala Glu Ile Asn Glu Ala Gly1 5 10 15Arg517PRTArtificial
Sequencesynthetic peptide, modified Der p2 T-cell epitope 5Cys His
Gly Cys Gly Gly Glu Pro Cys Ile Ile His Arg Gly Lys Pro1 5 10
15Phe616PRTArtificial Sequencesynthetic peptide, MOG T-cell
epitope, amino acids 40-55 of MOG 6Tyr Arg Ser Pro Phe Ser Arg Val
Val His Leu Tyr Arg Asn Gly Lys1 5 10 15718PRTArtificial
Sequencesynthetic peptide, amino acids 2144-2161 of human factor
VIII 7Ile Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile
Arg1 5 10 15Ser Thr819PRTArtificial Sequencesynthetic peptide,
modified Derp1 T-cell epitope 8Cys Gly Phe Ser Ser Asn Tyr Cys Gln
Ile Tyr Pro Pro Asn Ala Asn1 5 10 15Lys Ile Arg915PRTArtificial
Sequencesynthetic peptide, amino acids 71-85 of Der p2 allergen
9Asn Ala Cys His Tyr Met Lys Cys Pro Leu Val Lys Gly Gln Gln1 5 10
151015PRTArtificial Sequencesynthetic peptide, mutated Der p2
allergen with Ser to Ala mutation at position 4 10Cys His Gly Ala
Glu Pro Cys Ile Ile His Arg Gly Lys Pro Phe1 5 10
15119PRTArtificial Sequencesynthetic peptide, amino acids 188-196
of glycoprotein D of human herpesvirus 1 11Thr Tyr Leu Arg Leu Val
Lys Ile Asn1 51213PRTArtificial Sequencesynthetic peptide,
thioreductase motif coupled to amino acids 188-196 of glycoprotein
D of human herpesvirus 1 12Cys Gly His Cys Thr Tyr Leu Arg Leu Val
Lys Ile Asn1 5 101315PRTArtificial Sequencesynthetic peptide, amino
acids 114-128 of Der p1 allergen 13Ser Asn Tyr Cys Gln Ile Tyr Pro
Pro Asn Ala Asn Lys Ile Arg1 5 10 151417PRTArtificial
Sequencesynthetic peptide, amino acids 324-340 of chicken ovalbumin
14Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile Asn Glu Ala Gly1
5 10 15Arg1528PRTMus musculus 15Cys Ala Ala Arg Ser Ser Gly Ser Trp
Gln Leu Ile Phe Gly Ser Gly1 5 10 15Thr Gln Leu Thr Val Met Pro Asp
Ile Gln Asn Pro 20 251625PRTMus musculus 16Cys Val Val Gly Asp Arg
Gly Ser Ala Leu Gly Arg Leu His Phe Gly1 5 10 15Ala Gly Thr Gln Leu
Ile Val Ile Pro 20 25176PRTArtificial Sequencesynthetic peptide,
thioreductase motif with linker sequence 17Cys Xaa Xaa Cys Gly Xaa1
5187PRTArtificial Sequencesynthetic peptide, thioreductase motif
with linker sequence 18Cys Xaa Xaa Cys Gly Gly Xaa1
5198PRTArtificial Sequencesynthetic peptide, thioreductase motif
with linker sequence 19Cys Xaa Xaa Cys Ser Ser Ser Xaa1
5209PRTArtificial Sequencesynthetic peptide, thioreductase motif
with linker sequence 20Cys Xaa Xaa Cys Ser Gly Ser Gly Xaa1 5
* * * * *