U.S. patent application number 10/445391 was filed with the patent office on 2004-02-05 for direct selection of antigen-specific t cells, compositions obtained thereby and methods of use thereof.
Invention is credited to Assenmacher, Mario, Miltenyi, Stefan, Schmitz, Juergen.
Application Number | 20040023377 10/445391 |
Document ID | / |
Family ID | 22189678 |
Filed Date | 2004-02-05 |
United States Patent
Application |
20040023377 |
Kind Code |
A1 |
Assenmacher, Mario ; et
al. |
February 5, 2004 |
Direct selection of antigen-specific T cells, compositions obtained
thereby and methods of use thereof
Abstract
The invention provides a method for convenient analysis and cell
separation of antigen-specific T cells based on one or more
products secreted by these cells in response to antigen
stimulation. The T cells are provided with a capture moiety for the
product, which can then be used directly as a label in some
instances, or the bound product can be further labeled via label
moieties that bind specifically to the product and that are labeled
with traditional labeling materials such as fluorophores,
radioactive isotopes, chromophores or magnetic particles. The
labeled cells are then separated using standard cell sorting
techniques based on these labels. Such techniques include flow
cytometry, magnetic gradient separation, centrifugation, and the
like.
Inventors: |
Assenmacher, Mario;
(Bergisch Gladbach, DE) ; Miltenyi, Stefan;
(Bergisch Gladbach, DE) ; Schmitz, Juergen;
(Bergisch Gladbach, DE) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
755 PAGE MILL RD
PALO ALTO
CA
94304-1018
US
|
Family ID: |
22189678 |
Appl. No.: |
10/445391 |
Filed: |
May 23, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10445391 |
May 23, 2003 |
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09309199 |
May 10, 1999 |
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6576428 |
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60085136 |
May 11, 1998 |
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Current U.S.
Class: |
435/372 ;
435/7.21 |
Current CPC
Class: |
C12N 5/0636 20130101;
G01N 33/5005 20130101; G01N 33/505 20130101; G01N 33/56966
20130101 |
Class at
Publication: |
435/372 ;
435/7.21 |
International
Class: |
G01N 033/567; C12N
005/08 |
Claims
1. A method for obtaining a cell population enriched in
antigen-specific T cells, comprising the steps of: a) obtaining a
mixed population of cells comprising T cells; b) exposing the cell
population to at least one antigen under conditions effective to
elicit antigen-specific stimulation of at least one T cell and
allowing expression of at least one product by the stimulated T
cell, wherein the product is secreted in response to antigen
stimulation; c) modifying the surface of the cells to contain a
capture moiety specific for the product such that the capture
moiety is coupled to the cell surface; d) culturing said population
under conditions wherein said product is secreted, released and
specifically bound to the capture moiety, thereby labeling the
product-secreting cells; and e) separating the cells according to
the degree to which they are labeled with said product to obtain a
population of cells substantially enriched in antigen-specific T
cells, wherein steps (b) and (c) can be performed in any order.
2. A method according to claim 1, further comprising the step of
labeling the product prior to separation.
3. The method according to claim 2 wherein the product is labeled
with a label moiety.
4. The method according to claim 3 wherein the label moiety is an
antibody specific for the product.
5. The method according to claim 3 wherein the label moiety is
fluorochromated and the separation is conducted by cell
sorting.
6. The method according to claim 3 wherein the label moiety is
magnetizable and the separation is conducted in a magnetic field of
sufficient strength to magnetize the label moiety.
7. The method according to claim 6 wherein the label moiety
comprises colloidal magnetic particles with a typical diameter of
about 5 to 200 nm.
8. The method according to claim 1 wherein the capture moiety is an
antibody or an antigen-binding fragment thereof.
9. The method according to claim 8 wherein the antibody or antigen
binding fragment thereof is bispecific.
10. The method according to claim 1 wherein the coupling is through
a lipid anchor attached to the capture moiety optionally through a
linking moiety.
11. The method according to claim 1 wherein the coupling is through
an antibody or an antigen-binding fragment thereof attached to the
capture moiety, optionally through a linker.
12. The method according to claim 1 wherein the coupling is through
direct chemical coupling of the capture moiety to components on the
cell surface, optionally through a linker.
13. The method according to claim 9 wherein the coupling is through
specific binding of the antibody to the cell.
14. A method to label antigen-specific T cells with a product
secreted and released by the cells, wherein the product is secreted
in response to antigen stimulation, which method comprises:
exposing the cells to at least one antigen under conditions
effective to elicit antigen-specific stimulation of at least one T
cell; and modifying the surface of the cells to contain a capture
moiety specific for the product; and culturing the cells under
conditions wherein the product is secreted, released and
specifically bound to the capture moiety, thereby labeling the
product-secreting cells.
15. The method according to claim 14 wherein the product is labeled
with a label moiety.
16. The method according to claim 15 wherein the label moiety is an
antibody.
17. The method according to claim 14 wherein the capture moiety is
an antibody or an antigen-binding fragment thereof.
18. The method according to claim 17 wherein the antibody is
bispecific.
19. The method according to claim 14 wherein the coupling is
through a lipid anchor attached to the capture moiety optionally
through a linker moiety.
20. The method according to claim 14 wherein the coupling is
through an antibody or an antigen-binding fragment thereof attached
to the capture moiety optionally through a linker.
21. The method according to claim 18 wherein the coupling is
through specific binding of the antibody to the cell.
22. A composition obtained from the method according to claim
21.
23. The composition according to claim 21 wherein the capture
moiety is an antibody or an antigen-binding fragment thereof.
24. The composition according to claim 23 wherein the antibody is
bispecific.
25. The composition according to claim 22 wherein the coupling is
through a lipid anchor moiety attached to the capture moiety
optionally through a linking moiety.
27. The composition according to claim 22 wherein the coupling is
through an antibody or an antigen-binding fragment thereof attached
to the capture moiety, optionally through a linker.
28. The composition according to claim 25 wherein the coupling is
through specific binding of the antibody to the cell.
29. Cells and progeny thereof separated according to the method of
claim 1.
30. Cells separated according to the method of claim 1.
31. A method of analyzing a population of cells to identify or
enumerate antigen-specific T cells that secrete and release an
amount of product relative to other cells in the population,
wherein the product is secreted in response to antigen stimulation,
the method comprising the steps of: labeling the cells by the
method according to claim 14, labeling the cells with at least one
additional label that does not label the captured product, and
detecting the amount of product label relative to the additional
label.
32. A method of determining a distribution of secretory activity in
a cell population enriched in T cells, the method comprising the
steps of: labeling cells by the method according to claim 14, and
determining the amount of product label per cell, wherein the
product is secreted and released in response to antigen
stimulation.
33. The method according to claim 14 further comprising the steps
of: determining the amount and type of product label per cell
wherein distribution of secreted product type and secretory
activity for each secreted product type in a population of cells is
determined.
34. A method for identifying antigen-specific T cells secreting and
releasing at least one product in response to antigen stimulation,
comprising the steps of: combining a mixed population of cells
enriched for T cells with at least one first, bispecific, antibody,
each antibody, having combining sites specific for a cell surface
molecule and at least one product; exposing the cell population to
at least one antigen under conditions effective to elicit
antigen-specific stimulation of at least one T cell; incubating the
combination under conditions and for a time sufficient to allow the
cells to secrete the at least one product; adding at least one
label moiety; and detecting the at least one label moiety.
35. The method according to claim 34 further comprising the step of
separating the cells secreting the product from the mixed cell
population.
36. The method according to claim 34 wherein the cell surface
molecule is a naturally occurring cell surface protein.
37. The method according to claim 36 wherein the protein is a cell
surface marker.
38. The method according to claim 37 wherein the cell surface
molecule is selected from the group consisting of CD2, CD3, CD4,
CD5, CD8, CD11b, CD26, CD27, CD28, CD29, CD30, CD31, CD38, CD40L,
CD45RO, CD45RA, LAG3, T1/ST2, SLAM, Class I MHC molecules, Class II
MHC molecules, T cell antigen receptor, and
.beta..sub.2-microglobulin.
39. The method according to claim 34 wherein the incubation
conditions include a high viscosity or gel forming medium.
40. The method according to claim 34 wherein the label moiety is an
antibody.
41. The method according to claim 40 wherein the antibody comprises
a detectable label.
42. The method according to claim 41 wherein the label is selected
from the group consisting of fluorophores, radioactive isotopes,
chromophores and magnetic particles.
43. The method according to claim 40 wherein the label moiety is
detected by fluorescence activated cell sorting.
44. The method according to claim 43 wherein the label moiety is
detected by a third antibody.
45. The method according to claim 44 wherein the label moiety is
coupled to digoxigenin and the third antibody is specific for
digoxigenin.
46. The method according to claim 45 wherein the third antibody
comprises a detectable label.
47. The method according to claim 46 wherein the label is selected
from the group consisting of fluorophores, radioactive isotopes,
chromophores, and magnetic particles.
48. The method according to claim 47 wherein the label moiety is
detected by fluorescence activated cell sorting.
49. The method according to claim 34 wherein the label moiety
comprises a magnetizable moiety.
50. The method according to claim 49 wherein the label moiety is
detected by a third antibody coupled to a magnetizable moiety.
51. A method of treating a disease or condition related to a
population of antigen-specific T cells comprising administering to
an individual in need thereof an amount of a cell population
enriched in antigen-specific T cells effective to treat the
condition.
52. The method according to claim 51, wherein the condition is
selected from the group consisting of an autoimmune disorder, graft
rejection, and an allergic response.
53. The method according to claim 51, wherein the condition is a
result of a lack of adequate control of the condition by
antigen-specific T cells.
54. The method according to claim 53, wherein the condition is
cancer.
55. The method according to claim 53, wherein the condition is an
infection.
56. A kit for use in the detection of antigen-specific T cells that
secrete a product in response to antigen stimulation, the kit
comprising: a product capture system comprised of at least one
anchor moiety and at least one capture moiety; and at least one
label moiety.
57. The kit according to claim 56, wherein the capture moiety
comprises at least one bispecific antibody having at least one
antigen recognition site for at least one cell type and at least
one antigen recognition site specific for the product.
58. The kit according to claim 57 wherein the at least one
bispecific antibody and the at least one label moiety are in a
single vial.
59. The kit according to claim 57 wherein the at least one
bispecific antibody binds to the cell through a cell surface
molecule.
60. The kit according to claim 57 wherein the cell surface molecule
is a naturally occurring cell surface protein.
61. The kit according to claim 57 wherein the cell surface molecule
is a cell surface marker.
62. The kit according to claim 61 wherein the cell surface molecule
is selected from the group consisting of CD2, CD3, CD4, CD5, CD8,
CD11b, CD26, CD27, CD28, CD29, CD30, CD31, CD38, CD40L, CD45RO,
CD45RA, LAG3, T1/ST2, SLAM, Class I MHC molecules, Class II MHC
molecules, T cell antigen receptor, and
.beta..sub.2-microglobulin.
63. The kit according to claim 55 wherein the incubation conditions
include a high viscosity or gel forming medium.
64. The kit according to claim 63 wherein the medium is selected
from the group consisting of gelatin, agarose, alginate and
combination thereof,
65. The kit according to claim 57 wherein the label moiety is an
antibody.
66. The kit according to claim 65 wherein the antibody comprises a
detectable label.
67. The kit according to claim 66 wherein the detectable label is
selected from the group consisting of fluorophores, radioactive
isotopes, chromophores, and magnetic particles.
68. The kit according to claim 67 wherein the label moiety is
detected by fluorescence activated cell sorting.
69. The kit according to claim 65 wherein the label moiety is
detected by a third antibody.
70. The kit according to claim 69 wherein the label moiety is
coupled to digoxigenin and the third antibody is specific for
digoxigenin.
71. The kit according to claim 69 wherein the third antibody
comprises a detectable label.
72. The kit according to claim 65 further comprising a biological
modifier.
73. The kit according to claim 56 further comprising a cell-cell
cross-contamination reducing capture system.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation in part of U.S. Serial No. 60/085,136
filed May 11, 1998
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH
[0002] Not Applicable
TECHNICAL FIELD
[0003] The invention is in the field of analysis of cell
populations and cell separation and the compositions obtained
thereby. More particularly, the invention concerns analysis and
separation of antigen-specific T cells based on primary labeling of
cells with their secreted products through capture of these
products by a specific binding partner for the product anchored or
bound to the cell surface.
BACKGROUND ART
[0004] Numerous attempts have been made to analyze populations of
cells and to separate cells based on the products which they
produce. Such approaches to cell analysis and separation are
especially useful in assessing those cells which are capable of
secreting a desired product (the "product"), or which are
relatively high secretors of the product. These methods include
cloning in microtiter plates and analysis of the culture
supernatant for product, cloning in agar and analysis by methods
for identification of the product of the localized cells; the
identification methods include, for example, plaque assays and
western blotting. Most methods for analysis and selection of cells
based upon product secretion involve physically isolating the cell,
followed by incubation under conditions that allow product
secretion, and screening of the cell locations to detect the cell
or cell clones that produce the product. When cells are in
suspension, after the cells have secreted the product, the product
diffuses from the cell without leaving a marker to allow
identification of the cell from which it was secreted. Thus,
secretor cells cannot be separated from non-secretor cells with
these types of systems.
[0005] In other cases, both secretor and non-secretor cells can
associate the product with the cell membrane. An example of this
type of system are B cell derived cell lines producing monoclonal
antibodies. It has been reported that these types of cell lines
were separated by fluorescence activated cell sorting (FACS) and
other methods reliant upon the presence of antibody cell surface
markers. However, procedures that analyze and separate cells by
markers that are naturally associated with the cell surface can not
accurately identify and/or be used in the separation of secretor
cells from non-secretor cells. In addition, systems such as these
are not useful in identifying quantitative differences in secretor
cells (i.e., low level secretors from high level secretors).
[0006] A method that has been used to overcome the problems
associated with product diffusion from the cells has been to place
the cell in a medium that inhibits the rate of diffusion from the
cell. A typical method has been to immobilize the cell in a
gel-like medium (agar), and then to screen the agar plates for
product production using a system reliant upon blotting, for
example Western blots. These systems are cumbersome and expensive
if large numbers of cells are to be analyzed for properties of
secretion, non-secretion, or amount of secretion.
[0007] Kohler et al. have described a negative-selection system in
which mutants of a hybridoma line secreting IgM with
anti-trinitrophenyl (anti-TNP) specificity were enriched by
coupling the hapten (i.e., TNP) to the cell surface and incubating
the cells in the presence of complement. In this way, cells
secreting wild-type Ig were lysed, whereas cells secreting IgM with
reduced lytic activity or not binding to TNP preferentially
survived. Kohler and Schulman (1980) Eur. J. Immunol.
10:467-476.
[0008] More recently, a system has been described for labeling and
separating cells based on secreted product. PCT/US93/10126. In this
system, a specific binding partner for a secreted product is
coupled to the surface of cells. The product is secreted, released,
and bound to the cell by the specific binding partner. Cells are
then separated based on the degree to which they are labeled with
the bound product.
[0009] Other systems allow the cells to secrete their products in
the context of microdroplets of agarose gel which contain reagents
that bind the secretion products, and encapsulation of the cells.
Such methods have been disclosed in publications by Nir et al.
(1990) Applied and Environ. Microbiol. 56:2870-2875; and Nir et al.
(1991) Applied and Environ. Microbiol. 56:3861-3866. These methods
are unsatisfactory for a variety of reasons. In the process of
microencapsulation, statistical trapping of numbers of cells in the
capsules occurs, resulting in either a high number of empty
capsules when encapsulation occurs at low cell concentrations, or
multiple cells per capsule when encapsulation occurs at high cell
concentrations. Secreted product is trapped in the agarose drop by
the capture antibody and detected by a second fluorochromated
antibody. This process, while allowing for the detection and
isolation of cells based on secreted product, is complicated,
requires special equipment, and is not suited to all types of
sorting methods. In order to analyze and separate single cells or
single cell clusters by this technique, large volumes must be
handled to work with relatively small numbers of cells because of
the numbers of empty capsules and because of the size of the
microcapsules (50-100 .mu.m). The large volume of droplets results
in background problems using flow cytometry analysis and
separation. In addition, the capsules do not allow separation using
magnetic beads or panning for cell separation.
[0010] Various methods have been used to couple labels to cell
surfaces where the label such as a fluorochrome is intended for
direct detection. For example, hydrophobic linkers inserted into
the cell membrane to couple fluorescent labels to cells have been
described in PCT WO 90/02334, published 8 Mar. 1990. Antibodies
directed to HLA have also been used to bind labels to cell
surfaces. Such binding results in a smaller dimension than the
encapsulated droplets described above and such cells can be
conveniently used in standard separation procedures including flow
cytometry and magnetic separations.
[0011] ELISpot assays and methods for intracellular cytokine
staining have been used for enumeration and characterization of
antigen-specific CD4.sup.+ and CD8.sup.+ T cells. Lalvani et al.
(1997) J. Exp. Med. 186:859-865; and Waldrop et al. (1997) J. Clin
Invest. 99:1739-1750. These methods can be quite useful for T-cell
epitope mapping or for monitoring immunogenicity in vaccine trials,
but they do not allow isolation of live antigen-specific T cells,
e.g., for clinical trials of specific adoptive immunotherapy of
cancer or infections. Kern et al. (1998) Nat. Med. 4:975-978; El
Ghazali et al. (1993) Curr. Opin Immunol. 23:2740-2745; and Yee et
al (1997) Curr. Opin. Immund. 9:702-708. Soluble multivalent
complexes of peptide-loaded major histocompatibility complex (MHC)
molecules have been exploited recently to detect and also isolate
antigen-specific T cells. Altman et al. (1996) Science 274:94-96;
Dunbar et al. (1998) Curr. Biol. 8:413-416; Ogg et al. (1998)
279:2103-2106; Luxembourg et al. (1998) Nat. Biotechnol.
16:281-285; Murali-Krishna et al. (1998) Immunity 8:177-187;
Gallimore et al. (1998) J. Exp. Med. 187:1383-1393; and Flynn et
al. (1998) Immunity 8:683-691. These reagents are highly specific
but the approach is limited to well defined combinations of
antigenic peptides and restricting HLA alleles.
[0012] The immune system comprises two types of antigen-specific
cells: B cells and T cells. T cells can be characterized
phenotypically by the manner in which they recognize antigen, by
their cell surface markers, and by their secreted products. Unlike
B cells, which recognize soluble antigen, T cells recognize antigen
only when the antigen is presented to them in the form of small
fragments bound to major histocompatibility complex (MHC) molecules
on the surface of another cell. Any cell expressing MHC molecules
associated with antigen fragments on its surface can be regarded as
an antigen-presenting cell (APC). In most situations, however, more
than the mere display of an MHC-bound antigen fragment on a cell
surface is required to activate a T lymphocyte. In addition to the
signal delivered via the T cell receptor (TCR) engaged by MHC
molecule plus antigen, the T cell must also receive co-stimulatory
signals from the APC. Typically APCs are dendritic cells,
macrophages or activated B lymphocytes.
[0013] T cells express distinctive membrane molecules. Included
among these are the T cell antigen receptor (TCR), which appears on
the cell surface in association with CD3; and accessory molecules
such as CD5, CD28 and CD45R. Subpopulations of T cells can be
distinguished by the presence of additional membrane molecules.
Thus, for example, T cells that express CD4 recognize antigen
associated with class II MHC molecules and generally function as
helper cells, while T cells that express CD8 recognize antigen
associated with class I MHC molecules and generally function as
cytotoxic cells. The CD4.sup.+ subpopulation of T cells can be
categorized further into at least two subsets on the basis of the
types of cytokines secreted by the cell. Thus, while both subsets
secrete IL-3 and GM-CSF, TH1 cells generally secrete IL-2,
IFN-.gamma., and TNF-.lambda., whereas TH2 cells generally secrete
IL-4, IL-5, IL-10, and IL-13.
[0014] Minor changes in the peptide bound to the MHC molecule can
not affect the affinity of the peptide-MHC molecule interaction,
yet they can generate partial signals that lead to a halfway
response characterized by proliferation and secretion of only a
fraction of the cytokines produced during a full T cell response.
Some modified peptides can even block proliferation and cytokine
secretion altogether and induce a state of T cell anergy or
unresponsiveness. There are thus three different types of peptides:
agonist (those that stimulate a full response), partial agonist
(those that stimulate a partial response) and antagonist (those
that induce unresponsiveness). When a single APC presents a mixture
of an agonist and an antagonist on its surface, the negative effect
of the latter can overcome the positive effect of the former, even
if the antagonist is present in much smaller amounts than the
agonist. Some viruses seem to use mutations in their proteins to
produce antagonist peptides capable of suppressing the activity of
the T cell clones that recognize agonist peptides derived from the
original wild-type virus.
[0015] Secretion by a T cell of a particular cytokine is generally
associated with a particular function. For example, differences in
the cytokines secreted by the TH1 and TH2 subsets of CD4.sup.+ T
cells are believed to reflect different biological functions of
these two subsets. The TH1 subset is responsible for classical
cell-mediated functions such as delayed-type hypersensitivity and
activation of cytotoxic T cells, whereas the TH2 subset functions
more effectively as a helper for B-cell activation. The TH1 subset
can be particularly suited to respond to viral infections and
intracellular pathogens because it secretes IL-2 and IFN-.gamma.,
which activate cytotoxic T cells. The TH2 subset can be more suited
to respond to extracellular bacteria and helminthic parasites and
can mediate allergic reactions, since IL-4 and IL-5 are known to
induce IgE production and eosinophil activation, respectively.
There is also considerable evidence suggesting that preferential
activation of TH1 cells plays a central role in the pathogenesis of
a number of autoimmune diseases. Secretion of IL-10 by TH2 cells is
thought to suppress, in an indirect manner, cytokine production by
TH1 cells, and, accordingly, has a general immunosuppressive
effect. A shift in the TH1/TH2 balance can result in an allergic
response, for example, or, in an increased cytotoxic T cell
response.
[0016] The changes initiated by the TCR in the first few minutes to
hours of activation lead to transition of the cell from the G0 to
G1 phase of the cell cycle. Several hours after stimulation of the
T cell begins to express IL-2 and high-affinity IL-2 receptor. IL2
gene expression is effected by a set of transcription factors that
are activated by the converging signaling pathways triggered by the
ligation of TCR, CD28 and possibly other T cell surface
molecules.
[0017] The transcription factors also induce expression of the CD25
gene, which encodes the .alpha.-subunit of the high-affinity IL-2
receptor. The interaction of IL-2 with the high-affinity receptor
initiates signaling pathways that cause the T cell to transit from
the G1 to the S phase of the cell cycle and progress to cell
division. The signaling pathways control the expression and
activity of several key proteins necessary for cell division. Some
of these are also activated directly by TCR- and CD28-dependent
signals while others are energized only by signals provided via the
IL-2 receptor.
[0018] The stimulated T cell undergoes a sequence of phenotypic
changes beginning with its progression from the resting state to
mitosis and later to differentiation into effector and memory
cells. Among the earliest (immediate) changes, observable within
15-30 minutes of stimulation, are the expression of genes encoding
transcription factors such as c-Fos, NF-AT, c-Myc and NF-.kappa.B,
protein kinases such as Jak-3 and protein phosphatases such as
Pac-1. The subsequent early changes, occurring within several hours
of stimulation, mark the beginning of the expression of genes
encoding activation antigens. These include several cytokines (IL-2
and others), IL-2 receptor subunit .alpha. (CD25), insulin
receptor, transferrin receptor and several other surface molecules
such as CD 26, CD30, CD54, CD69 and CD70.
[0019] Activation antigens reach a maximum level of expression just
before the first division, 24 hours after stimulation. During this
period the level of expression of several other molecules already
expressed on resting T cells increases. At a later point, some days
after activation commenced, late activation antigens become
expressed on the T cells. These include MHC class II molecules and
several members of the .beta.1 integrin family. Expression of late
activation antigens marks the differentiation of the activated cell
into effector or memory T cells.
[0020] T cells play important roles in autoimmunity, inflammation,
cytotoxicity, graft rejection, allergy, delayed-type
hypersensitivity, IgE-mediated hypersensitivity, and modulation of
the humoral response. Disease states can result from the activation
of self-reactive T cells, from the activation of T cells that
provoke allergic reactions, or from the activation of autoreactive
T cells following certain bacterial and parasitic infections, which
can produce antigens that mimic human protein, rendering these
protein "autoantigens." These diseases include, for example, the
autoimmune diseases, autoimmune disorders that occur as a secondary
event to infection with certain bacteria or parasites, T
cell-mediated allergies, and certain skin diseases such as
psoriasis and vasculitis. Furthermore, undesired rejection of a
foreign antigen can result in graft rejection or even infertility,
and such rejection can be due to activation of specific T
lymphocyte populations. Pathological conditions can also arise from
an inadequate T cell response to a tumor or a viral infection. In
these cases, it would be desirable to increase an antigen-specific
T cell response in order to reduce or eliminate the tumor or to
eradicate an infection.
[0021] Autoimmune diseases have a variety of causes. For instance,
autoimmune reactions can be provoked by injury or immunization with
collagen, by superantigens, by genetic factors, or errors in immune
regulation. Superantigens are polyclonal activators that can, among
other things, stimulate clones previously anergized by an encounter
with an autoantigen or clones that ignored the potential
autoantigens because of their low expression or availability.
Certain autoimmune disease are caused mainly by autoantibodies,
others are T cell-mediated. Autoreactive T cells cause tissue
damage in a number of autoimmune diseases including rheumatoid
arthritis and multiple sclerosis.
[0022] In the treatment of autoimmune disorders, nonspecific immune
suppressive agents have been used to slow the disease; these
therapies often cause a general immunosuppression by randomly
killing or inhibiting immunocompetent cells. Attempts to treat
autoimmune disorders by modulating the activity of autoreactive T
cells have included immunization with TCR peptides, treatment with
interferon-.beta. (IFN-.beta.) and T lymphocyte vaccination. Ebers
(1994) Lancet 343:275-278; Hohlfeld (1997) Brain 120:865-916; and
Hafler et al. (1992) Clin. Immunol. Immunopathol. 62:307-313.
[0023] The development of allergic sensitization, contact
sensitivity and inflammation is dependent on activation and
stimulation of T cells that exhibit proallergic functions.
Allergen-specific T cells are believed to play an important role in
the pathophysiology of atopic allergies. Elimination or suppression
of allergen-specific T cells could help ameliorate allergic
diseases caused by such T cells.
[0024] In the initial phase of an allergic reaction, antigen
(allergen) enters the body, is picked up by APCs, displayed by them
in the context of class II MHC molecules and recognized by helper T
cell precursors. These are stimulated to proliferate and
differentiate mainly into TH2 cells, which help B lymphocytes
differentiate into antibody-producing plasma cells. As in any other
antibody-mediated response, the B cells that receive specific help
from TH cells are those that recognized the allergen via their
surface receptors. Some of the cytokines produced by the TH2 cells,
especially IL-4 and IL-13, stimulate the B cells to effect an
immunoglobulin isotype switch and to produce IgE antibodies. The
antibodies bind to high-affinity Fc receptors on the surface of
mast cells in the connective tissue and mucosa, as well as to those
on the surface of basophils in the circulation and mucosa and
initiate the manifestations of allergic reaction.
[0025] Allograft rejection is caused principally by a cell-mediated
immune response to alloantigens (primarily MHC molecules) expressed
on cells of the graft. Analysis of the T lymphocyte subpopulations
involved in allograft rejection has implicated both CD4.sup.+ and
CD8.sup.+ populations. TH1 cells initiate the inflammatory reaction
of delayed-type hypersensitivity, leading to the recruitment of
monocytes and macrophages into the graft. Natural kill (NK) cells,
presumably alerted by the absence in the graft of MHC molecules
present in the recipient, can also attack the graft in the early
phases of the response. Neutrophils are mainly responsible for
clearing the wound or removing damaged cells and cellular debris in
the late phase of the allograft reaction.
[0026] Most immunosuppressive treatments developed have the
disadvantage of being non-specific; that is, they result in
generalized immunosuppression, which places the recipient at
increased risk for infection. Immunosuppressive agents employed to
prevent organ rejection include mitotic inhibitors such as
azathioprine, cyclophosphamide and methotrexate; corticosteroids;
and drugs, such as cyclosporin, FK506 and rapamycin, which inhibit
the transcription of the genes encoding IL-2 and the high-affinity
receptor for IL-2.
[0027] In the treatment of cancers, cellular immunotherapy has been
employed as an alternative, or an adjunct to, conventional
therapies such as chemotherapy and radiation therapy. For example,
cytotoxic T lymphocyte (CTL) responses can be directed against
antigens specifically or preferentially presented by tumor cells.
Following activation with T cell cytokines in the presence of
appropriately presented tumor antigen, tumor infiltrating
lymphocytes (TILs) proliferate in culture and acquire potent
anti-tumor cytolytic properties. Weidmann et al. (1994) Cancer
Immunol. Immunother. 39:1-14.
[0028] The introduction into a cancer patient of in vitro activated
lymphocyte populations has yielded some success. Adoptive
immunotherapy, the infusion of immunologically active cells into a
cancer patient in order to effect tumor regression, has been an
attractive approach to cancer therapy for several decades. Two
general approaches have been pursued. In the first, donor cells are
collected that are either naturally reactive against the host's
tumor, based on differences in the expression of histocompatibility
antigens, or made to be reactive using a variety of "immunizing"
techniques. These activated donor cells are then transfused to a
tumor-bearing host. In the second general approach, lymphocytes
from a cancer patient are collected, activated ex vivo against the
tumor and then reinfused into the patient. Triozzi (1993) Stem
Cells 11:204-211; and Sussman et al. (1994) Annals Surg. Oncol.
1:296.
[0029] Current methods of cancer treatment are relatively
non-selective. Surgery removes the diseased tissue, radiotherapy
shrinks solid tumors and chemotherapy kills rapidly dividing cells.
Systemic delivery of chemotherapeutic agents, in particular,
results in numerous side effects, in some cases severe enough to
preclude the use of potentially effective drugs.
[0030] Viral diseases are also candidates for immunotherapy. Heslop
et al. (1996) Nature Med. 2:551-555. Immunological responses to
viral pathogens are sometimes ineffective in eradicating or
sufficiently depleting the virus. Furthermore, the highly mutable
nature of certain viruses, such as human immunodeficiency virus,
allows them to evade the immune system.
[0031] Clearly, there is a need to identify, analyze and enrich
populations of T cells involved in the above-mentioned pathologies.
Currently, several methods for analysis and for enrichment of
antigen-specific and/or cytokine-secreting T cells exist.
Enrichment of antigen-specific T cells can be achieved using
selective culturing techniques to obtain T cell lines and T cell
clones. These techniques generally involve culturing the T cells in
vitro over a period of several weeks and using rather cumbersome
methods to select lines or clones exhibiting the desired phenotype,
such as cytokine secretion. Other attempts to detect and enrich for
antigen-specific T cells have employed defined multimeric
MHC-antigen and MHC-peptide complexes. U.S. Pat. No. 5,635,363. For
such a technique to be successful, however, MHC-antigen complexes
of the correct MHC allotype are required, and the selection is
limited to antigen specificity, i.e., no selection for cytokine
secretion is afforded by this technique.
[0032] Intracellular cytokine staining after antigen activation,
followed by FACS analysis, is the method used to obtain information
regarding the antigen specificity and kinetics of cytokine
production. Waldrop et al. (1997) J. Clin. Invest. 99:1739-1750.
This method is useful for analysis only, since the cells are not
viable after this procedure. Similarly, cytokine ELISPOT assays are
useful for analysis only. Miyahira et al. (1995) J. Immunol. Met.
181:45-54; and Lalvani et al. (1997) J. Exp. Med. 186:859-865. In
these assays, secreted cytokines are trapped in a surrounding
matrix for analysis, but there is no mechanism for identifying and
retrieving the cell which secreted the cytokine. The gel microdrop
technology is not suited to processing large numbers of cells such
as would be necessary for treatment of the above-mentioned
indications.
[0033] It is apparent from the foregoing discussion that there is a
need for reliable techniques for analyzing and separating
populations of T cells, based on secreted product, for a number of
therapeutic and diagnostic purposes. The present invention
addresses this need by providing methods for analyzing, separating
and enriching populations of antigen-specific T cells.
DISCLOSURE OF THE INVENTION
[0034] The invention provides a method for convenient analysis and
cell separation of antigen-specific T cells based on one or more
products secreted by these cells in response to antigen
stimulation. The T cells are provided with a capture moiety
specific for the product (or, "specific binding partner"), which
can then be used directly as a label. The binding of the product to
the capture moiety results in a "captured product." Alternatively,
the cells are bound to the product via the capture moiety and can
be further labeled via label moieties that bind specifically to the
product and that are, in turn, labeled either directly or
indirectly with traditional labeling materials such as
fluorophores, radioactive isotopes, chromophores or magnetic
particles.
[0035] The labeled cells can then be separated using standard cell
sorting techniques based on these labels. Such techniques include,
but are not limited to, flow cytometry, FACS, high gradient
magnetic gradient separation, centrifugation.
[0036] Thus, in one aspect, the invention encompasses a method to
stimulate and separate antigen-specific T cells from a population
of cells according to a product secreted and released by the
antigen specific T cells in response to the stimulation. The method
comprises stimulating a mixture of cells containing T cells with
antigen, and effecting a separation of antigen-stimulated cells
according to the degree to which they are labeled with the product.
Antigen stimulation is achieved by exposing the cells to at least
one antigen under conditions effective to elicit antigen-specific
stimulation of at least one T cell. Labeling with the product is
achieved by modifying the surface of the cells to contain at least
one capture moiety, culturing the cells under conditions in which
the product is secreted, released and specifically bound
("captured" or "entrapped") to said capture moiety; and labeling
the captured product with a label moiety, where the labeled cells
are not lysed as part of the labeling procedure or as part of the
separation procedure.
[0037] Another aspect of the invention is a composition of matter
containing antigen-specific T cells capable of capturing a product
secreted and released by these cells in response to antigen
stimulation, where the surface of the cells is modified to contain
a capture moiety for the product. The captured product can be
separately labeled by a label moiety.
[0038] Still another aspect of the invention is antigen-specific T
cells and progeny thereof separated by the above-described
method.
[0039] Yet another aspect of the invention is a method to label
antigen-specific T cells with a product secreted and released by
the cells in response to antigen stimulation, by modifying the
surface of these cells to contain a specific binding partner for
the product coupled to the cell surface, and culturing the cells
under conditions wherein the product is secreted and released.
[0040] An additional aspect of the invention is a method of
analyzing a population of antigen-specific T cells to determine the
proportion of cells that secrete an amount of product relative to
other cells in the population, where the product is secreted in
response to antigen stimulation. The method comprises labeling the
cells by the above-described method, further labeling the cells
with a second label that does not label the captured product, and
detecting the amount of product label relative to the second cell
label. Such methods are useful, for example, in assessing the
immune status of an individual.
[0041] A further aspect of the invention is methods for use of T
cell populations enriched in antigen-specific T cells. The methods
comprise administering to an individual in need of treatment a
composition comprising a T cell population enriched in
antigen-specific T cells. Such methods are useful to treat a
variety of pathological conditions, including cancer, allergies,
immunodeficiencies, autoimmune disorders and viral diseases.
[0042] Yet another aspect of the invention is a kit for use in
separation of antigen-specific T cells from a mixed population
comprising effector cells. The kit can contain a physiologically
acceptable medium which can be of varying degrees of viscosity up
to a gel-like consistency, a product capture system comprising
anchor and capture moieties; a label system for detecting the
captured product; and instructions for use of the reagents, all
packaged in appropriate containers. Optionally, the kit further
comprises a magnetic labeling system and/or one or more biological
modifiers.
[0043] Still another aspect of the invention is a kit for use in
the detection/separation of antigen-specific T cells that secrete a
desired product in response to antigen stimulation, the kit
comprising a product capture system comprising anchor and capture
moieties; a label system for detecting the captured product; and
instructions for use of the reagents, all packaged in appropriate
containers. Optionally, the kit further comprises a magnetic
labeling system, and/or antigen, and/or one or more biological
modifiers.
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] FIGS. 1A-P are FACS plots showing analysis of cells
subjected to the separation protocol described in Example 1. A-H
show analysis of control cells cultured with no peptide; I-P show
analysis of peptide-stimulated cells. A, C, I, and K show scatter
properties of the starting cell population (A and I) and the
enriched cell population (C and K). B, D, J and L show profiles of
PI versus PE staining of the starting cell population (B and J) and
the enriched cell population (D and L). Plots E-H and M-P show
FITC-labeled anti-CD8 versus PE-labeled anti-IFN-.gamma. staining
of the starting cell population (E and M), the first negative
population (F and N), the second negative population (G and O) and
the enriched cell population (H and P).
[0045] FIGS. 2A-N are FACS plots showing analysis of cells
subjected to the separation protocol described in Example 2. A-G
show analysis of control cells cultured with no peptide; N-R show
analysis of peptide-stimulated cells. A-D and H-K show FITC-labeled
anti-CD8 versus PE-labeled anti-IFN-.gamma. staining of the
starting cell population (A and J), the first negative population
(B and I), the second negative population (C and J) and the
enriched cell population (D and K). F and M show staining for
V.beta.17TCR of the enriched cell population.
[0046] FIG. 3 is a series of dot plots showing
IFN-.gamma.-secretion-based enrichment and detection of live
antigen-specific CD4.sup.+ and CD8.sup.+ T cells. Dot plots show
CD8-Cy5 vs. anti IFN-.gamma.-PE (A-D) or CD4-Cy5 vs. anti
IFN-.gamma.-PE (E-L) staining of PBMC from healthy adult donors
stimulated with (A,B) or without (C,D) the HLA-A0201-restricted FLU
58-66 peptide, a purified influenza A virus preparation (with (E,F)
without (G,H)) and rTT.C (with (I,J) without (K,L)) before
(A,C,E,G,I,K) and after (B,D,F,H,J,L) magnetic enrichment of
IFN-.gamma.-secreting cells. Live lymphocytes were gated according
to light-scatter properties and propidium iodide exclusion.
[0047] FIG. 4 is a series of dot plots showing a phenotypic
analysis of enriched Flu 58-66 peptide-specific CD8.sup.+ T cells.
Enriched IFN-.gamma.-secreting CD8.sup.+ T cells from FLU 58-66
peptide-stimulated PBMC (A,B,E,F) and, for control, from
non-stimulated PBMC (C,D,G,H) were stained with anti IFN-.gamma.-PE
and counterstained with FITC-conjugated antibodies against CD27,
CD28, CD57 and the TCR V.beta.17 chain. Light-scatter properties,
propidium iodide and CD8-Cy5 staining were used for gating of live
CD8.sup.+ T cells.
[0048] FIG. 5 is a graph depicting cytolytic activity of enriched
and expanded Flu 58-66 peptide-specific T cells.
IFN-.gamma.-secreting CD8.sup.+ T cells from FLU 58-66
peptide-stimulated PBMC were expanded for 18 days in tissue culture
in the presence of IL-2 and then assayed for CTL activity assay.
The diagram shows the percentage of lysed HLA-A2.1+T2 cells pulsed
with either Flu 58-66 peptide or the negative control peptide Melan
A/MART 1 27-35.
[0049] FIG. 6 is a series of dot plots depicting the isolation and
detection of TT-specific IL-4-secreting CD4+T cells. Dot plots show
CD4-Cy5 vs. anti IL-4-PE staining of PBMC from healthy adult donors
stimulated with (A,C) or without (B,D) magnetic enrichment of
IL-4-secreting cells. Live lymphocytes were gated according to
light-scatter properties and propidium iodide exclusion.
MODES FOR CARRYING OUT THE INVENTION
[0050] The present invention provides methods for detecting,
analyzing and separating antigen-stimulated T cells on the basis of
secreted product, where the product is secreted as a result of
antigen stimulation. The methods are based on capture and
relocation to the cell surface of the secreted product.
[0051] The captured product permits the cell to be detected,
analyzed and, if desired, sorted, according to the presence,
absence or amount of the product present. The means of capture
comprises a product-specific binding partner ("capture moiety")
anchored to the cell surface by a means suitable for the cell to be
sorted.
[0052] The approach presented here combines, inter alia, the
following advantages: (a) it permits rapid isolation, enumeration,
phenotyping and expansion of live antigen-specific T lymphocytes
without the need of cyclical activation of T cells with antigen and
APCs; (b) it is generally applicable for isolation of T cells
reactive to APCs that have been pulsed with synthetic peptides,
native proteins, cell extracts, nonviable pathogens, transduced
with retroviral vectors, infected with recombinant viral vectors,
transfected with RNA or DNA, etc.; (c) it can be used for the
isolation of both CD4.sup.+ antigen-specific Th cells and CD8.sup.+
antigen-specific CTLs; and (d) it enables selective isolation of
antigen specific T cells with particular cytokinemediated effector
functions, e.g., of antigen-specific Th1-, Th2-, or Th3-like
lymphocytes.
[0053] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology
(including recombinant techniques), microbiology, cell biology,
biochemistry and immunology, which are within the skill of the art.
Such techniques are explained fully in the literature, such as,
"Molecular Cloning: A Laboratory Manual", second edition (Sambrook
et al., 1989); "Oligonucleotide Synthesis" (M. J. Gait, ed., 1984);
"Animal Cell Culture" (R. I. Freshney, ed., 1987); "Methods in
Enzymology" (Academic Press, Inc.); "Handbook of Experimental
Immunology" (D. M. Weir & C. C. Blackwell, eds.); "Gene
Transfer Vectors for Mammalian Cells" (J. M. Miller & M. P.
Calos, eds., 1987); "Current Protocols in Molecular Biology" (F. M.
Ausubel et al., eds., 1987, and periodic updates); "PCR: The
Polymerase Chain Reaction", (Mullis et al., eds., 1994); and
"Current Protocols in Immunology" (J. E. Coligan et al., eds.,
1991).
[0054] Cell sorting and cell analysis methods are known in the art
and are described in, for example, The Handbook of Experimental
Immunology, Volumes 1 to 4, (D. N. Weir, editor) and Flow Cytometry
and Cell Sorting (A. Radbruch, editor, Springer Verlag, 1992).
[0055] As used herein, a "specific binding partner" or "capture
moiety" intends a member of a pair of molecules (a "specific
binding pair") that interact by means of specific non-covalent
interactions that depend on the three-dimensional structures of the
molecules involved. A "label moiety" is a detectable, either
directly or indirectly. When the capture moiety is an antibody, it
can be referred to as the "capture antibody" or "catch antibody."
The capture moieties are those which attach both to the cell,
either directly or indirectly, and the product. The label moieties
are those which attach to the product and can be directly or
indirectly labeled.
[0056] As used herein, the term "antibody" is intended to include
polyclonal and monoclonal antibodies, chimeric antibodies, haptens
and antibody fragments, and molecules which are antibody
equivalents in that they specifically bind to an epitope on the
product antigen. The term "antibody" includes polyclonal and
monoclonal antibodies of any isotype (IgA, IgG, IgE, IgD, IgM), or
an antigen-binding portion thereof, including, but not limited to,
F(ab) and Fv fragments such as sc Fv, single chain antibodies,
chimeric antibodies, humanized antibodies, and a Fab expression
library. Antibodies can also be immobilized for instance on a
polymer or a particle.
[0057] "Bispecific antibody" and "bispecific antibodies," also
known as bifunctional antibodies, intends antibodies that recognize
two different antigens by virtue of possessing at least one first
antigen combining site specific for a first antigen or hapten, and
at least one second antigen combining site specific for a second
antigen or hapten. Such antibodies can be produced by recombinant
DNA methods or include, but are not limited to, antibodies
chemically by methods known in the art. Chemically created
bispecific antibodies that have been reduced and reformed so as to
retain their bivalent characteristics and antibodies that have been
chemically coupled so that they have at least two antigen
recognition sites for each antigen. Bispecific antibodies include
all antibodies or conjugates of antibodies, or polymeric forms of
antibodies which are capable of recognizing two different antigens.
The label moiety can be a fluorochromated antiproduct antibody,
which can include, but is not limited to, magnetic bead conjugated,
colloidal bead conjugated, FITC, Phycoerythrin, PerCP, AMCA,
fluorescent particle or liposome conjugated antibodies.
Alternatively the label moiety can be any suitable label including
but not limited to those described herein. Bispecific antibodies
include antibodies that have been reduced and reformed so as to
retain their bivalent characteristics and to antibodies that have
been chemically coupled so that they can have several antigen
recognition sites for each antigen.
[0058] As used herein the term "effector cell population" intends a
cell population which comprises at least one T cell. An effector
cell population can be obtained from a starting cell population
from which antigen-specific T cells are enriched.
[0059] The terms "cell," and "cells," and "cell population," used
interchangeably, intend one or more mammalian cells. The term
includes progeny of a cell or cell population. Those skilled in the
art will recognize that "cells" include progeny of a single cell,
and the progeny can not necessarily be completely identical (in
morphology or of total DNA complement) to the original parent cell
due to natural, accidental, or deliberate mutation and/or
change.
[0060] The terms "T lymphocyte," "T cell," "T cells," and "T cell
population," used interchangeably, intends a cell or cells which
display on their surface one or more antigens characteristic of T
cells, such as, for example, CD2 and CD3. The term includes progeny
of a T cell or T cell population. A "T lymphocyte" or "T cell" is a
cell which expresses CD3 on its cell surface and a T cell antigen
receptor (TCR) capable of recognizing antigen when displayed on the
surface of autologous cells, or any antigen-presenting matrix,
together with one or more MHC molecules or, one or more
non-classical MHC molecules. The term "T cells" as used herein
denotes any T cells known in the art, for instance, lymphocytes
that are phenotypically CD3.sup.+, i.e., express CD3 on the cell
surface, typically detected using an anti-CD3 monoclonal antibody
in combination with a suitable labeling technique. The T cells
enriched by the methods of this invention are generally CD3.sup.+.
The T cells enriched by the methods of this invention are also
generally, although not necessarily, positive for CD4, CD8, or
both.
[0061] The term "substantially enriched" as used herein, indicates
that a cell population is at least about 50-fold, more preferably
at least about 500-fold, and even more preferably at least about
5000-fold or more enriched from an original mixed cell population
comprising the desired cell population.
[0062] The term "antigen-presenting matrix," as used herein,
intends a molecule or molecules which can present antigen in such a
way that the antigen can be bound by a T cell antigen receptor on
the surface of a T cell. An antigen-presenting matrix can be on the
surface of an antigen-presenting cell (APC), on a vesicle
preparation of an APC, or can be in the form of a synthetic matrix
on a bead or a plate. The term "antigen presenting cell", as used
herein, intends any cell which presents on its surface an antigen
in association with a MHC or portion thereof, or, one or more
nonclassical MHC molecules, or a portion thereof.
[0063] The term "autogeneic," "autologous," or, "self," as used
herein, indicates the origin of a cell. Thus, a cell is autogeneic
if the cell was derived from an individual (the "donor") or a
genetically identical individual and is to be readministered to the
individual. An autogeneic cell can also be a progeny of an
autogeneic cell. The term also indicates that cells of different
cell types are derived from the same donor or genetically identical
donors. Thus, an effector cell and an antigen presenting cell are
said to be autogeneic if they were derived from the same donor or
from an individual genetically identical to the donor, or if they
are progeny of cells derived from the same donor or from an
individual genetically identical to the donor.
[0064] Similarly, the term "allogeneic," or "non-self," as used
herein, indicates the origin of a cell. Thus, a cell and progeny
thereof is allogeneic if the cell was derived from an individual
not genetically identical to the recipient to whom it is
administered; in particular, the term relates to non-identity in
expressed MHC molecules. The term also indicates that cells of
different cell types are derived from genetically non-identical
donors, or if they are progeny of cells derived from genetically
non-identical donors. For example, an APC is said to be allogeneic
to an effector cell if they are derived from genetically
non-identical donors.
[0065] A "disease or condition related to a population of
antigen-specific T cells" is one which can be related to a
population of antigen-specific T cells or lack of adequate numbers
thereof, and includes, for example, autoimmune diseases in which
antigen-specific T cells are primarily responsible for the
pathogenesis of the disease; cancers, in which cancerous cell
growth is not adequately controlled by tumor-specific cytotoxic T
cells; viral diseases, in which virus-infected cells are not lysed
by cytotoxic T cells; allergies, in which T cells specific for
allergens mediate undesired effects; immunodeficiencies, in which
inadequate numbers of T cells are present in an individual due to
either infection (such as HIV) or congenitally (such as DiGeorge
syndrome). It is also one in which antigen-specific T cells
modulate or regulate the activity of another cell or cell
population which is primarily responsible for a disease state; it
is also one in which the- presence of a population of
antigen-specific T cells is not the primary cause of the disease,
but which plays a key role in the pathogenesis of the disease; it
is also one in which a population of antigen-specific T cells
mediates an undesired rejection of a foreign antigen.
[0066] An "individual" is a vertebrate, preferably a mammal, more
preferably a human. Mammals include, but are not limited to,
humans, farm animals, sport animals, and pets.
[0067] An "effective amount" is an amount sufficient to effect
beneficial or desired clinical results. An effective amount can be
administered in one or more administrations. For purposes of this
invention, an effective amount of antigen-specific T cells is an
amount that is sufficient to diagnose, palliate, ameliorate,
stabilize, reverse, slow or delay the progression of the disease
state.
[0068] As used herein, "treatment" is an approach for obtaining
beneficial or desired clinical results. For purposes of this
invention, beneficial or desired clinical results include, but are
not limited to, alleviation of symptoms, diminishment of extent of
disease, stabilized (i.e., not worsening) state of disease,
preventing spread (i.e., metastasis) of disease, delay or slowing
of disease progression, amelioration or palliation of the disease
state, and remission (whether partial or total), whether detectable
or undetectable. "Treatment" can also mean prolonging survival as
compared to expected survival if not receiving treatment.
[0069] "Palliating" a disease means that the extent and/or
undesirable clinical manifestations of a disease state are lessened
and/or time course of the progression is slowed or lengthened, as
compared to not administering enriched T cell populations of the
present invention.
[0070] The present invention provides methods for obtaining a cell
population enriched in antigen-specific T cells which secrete a
product, where the product is secreted as a result of antigen
stimulation. The methods generally involve obtaining a mixed
population of cells comprising T cells; exposing the cell
population to at least one antigen under conditions effective to
elicit antigen-specific stimulation of at least one T cell;
modifying the surface of said mixed population to contain attached
thereto a specific binding partner for the product; allowing
expression of at least one product by the stimulated T cells,
wherein the product is secreted in response to the stimulation;
allowing binding of the product to a capture moiety coupled to the
surface of the cell to form a cell bound capture moiety-product
complex, thereby labeling the cells; and separating the stimulated
T cells according to the degree to which they are labeled with said
product.
[0071] Of course, modification of the cell surface with a specific
binding partner can be carried out before, during, or after antigen
stimulation.
[0072] Antigen Presenting Matrices and Effector Cell
Populations
[0073] The present invention provides methods for obtaining a cell
population enriched in antigen-specific T cells which secrete a
product in response to antigen stimulation. The methods comprise
obtaining a mixed population of cells (i.e., an "effector cell
population"), and exposing the cell population to at least one
antigen. The mixed cell population can be obtained by any method
known in the art and is preferably enriched for T cells. Exposure
to antigen can be achieved using antigen-presenting matrices, which
can be on the surface of antigen-presenting cells (APC's).
Antigen-presenting matrices and effector cells can be obtained from
a variety of sources. The mixed population of cells can be
stimulated by antigen in vitro or in vivo, or modified in any of a
variety of ways, for example, chemically or genetically
modified.
[0074] Antigen Presenting Matrices
[0075] The T cell populations which are subjected to the methods of
the present invention are exposed to at least one antigen under
conditions effective to elicit antigen-specific stimulation. A T
cell which is stimulated by the at least one antigen is said to be
antigen specific, i.e., it displays on its cell surface an antigen
receptor which specifically recognizes and binds to an antigen in
association with a molecule capable of presenting antigen, such as
a classical or non-classical MHC molecule or a portion thereof, on
an antigen-presenting matrix, for example, a synthetic
antigen-presenting matrix or one that is present on the surface of
an APC.
[0076] The antigen-presenting molecule can be an MHC molecule,
which can be class I or class II or, a non-classical MHC molecule
such as CD1; an MHC epitope; a fusion protein comprising an MHC
epitope; or a synthetic MHC epitope. The nature of the
antigen-presenting molecule is not critical, so long as it is
capable of presenting antigen to an effector cell. Methods of
preparing MHC epitopes are known in the art.
[0077] Antigen-presenting matrices include those on the surface of
an APC as well as synthetic antigen-presenting matrices. APCs
suitable for use in the present invention are capable of presenting
exogenous peptide or protein or endogenous antigen to T cells in
association with an antigen-presenting molecule, such as an MHC
molecule. APCs include, but are not limited to, macrophages,
dendritic cells, CD40-activated B cells, antigen-specific B cells,
tumor cells, virus-infected cells and genetically modified
cells.
[0078] APCs can be obtained from a variety of sources, including
but not limited to, peripheral blood mononuclear cells (PBMC),
whole blood or fractions thereof containing mixed populations,
spleen cells, bone marrow cells, tumor infiltrating lymphocytes,
cells obtained by leukapheresis, lymph nodes, e.g., lymph nodes
draining from a tumor. Suitable donors include an immunized donor,
a non-immunized (naive) donor, treated or untreated donors. A
"treated" donor is one that has been exposed to one or more
biological modifiers. An "untreated" donor has not been exposed to
one or more biological modifiers. APC's can also be treated in
vitro with one or more biological modifiers.
[0079] The APCs are generally alive but can also be irradiated,
mitomycin C treated, attenuated, or chemically fixed. Further, the
APCs need not be whole cells. Instead, vesicle preparations of APCs
can be used.
[0080] APCs can be genetically modified, i.e., transfected with a
recombinant polynucleotide construct such that they express a
polypeptide or an RNA molecule which they would not normally
express or would normally express at lower levels. Examples of
polynucleotides include, but are not limited to, those which encode
an MHC molecule; a co-stimulatory molecule such as B7; or an
antigen. For example, expression of a polynucleotide encoding an
MHC molecule under transcriptional control of a strong promoter
such as the CMV promoter, can result in high level expression of
the MHC molecule on the cell surface, thus increasing the density
of antigen presentation. Alternatively, an APC can be transfected
with a polynucleotide construct comprising a polynucleotide
encoding an antigen under transcriptional control of a strong
promoter such as the CMV promoter such that the antigen is
expressed on the cell surface together with an MHC molecule.
[0081] The nucleotide sequence encoding a polypeptide is operably
linked to control sequences for transcription and translation. A
control sequence is "operably linked" to a coding sequence if the
control sequence regulates transcription or translation. Any method
in the art can be used for the transformation, or insertion, of an
exogenous polynucleotide into an APC, for example, lipofection,
transduction, infection or electroporation, using either purified
DNA, viral vectors, or DNA or RNA viruses. The exogenous
polynucleotide can be maintained as a non-integrated vector, for
example, a plasmid, or, can be integrated into the host cell
genome.
[0082] Cells which do not normally function in vivo in mammals as
APCs can be modified to function as APCs. A wide variety of cells
can function as APCs when appropriately modified. Examples of such
cells are insect cells, for example Drosophila or Spodoptera;
foster cells, such as the human cell line T2, which bears a
mutation in its antigen presenting pathway that restricts the
association of endogenous peptides with cell surface MHC class I
molecules. Zweerink et al. (1993) J. Immunol. 150:1763-1771. For
example, expression vectors which direct the synthesis of one or
more antigen-presenting polypeptides, such as MHC molecules, and,
optionally, accessory molecules such as B7, can be introduced into
these cells to effect the expression on the surface of these cells
antigen presentation molecules and, optionally, accessory molecules
or functional portions thereof. Alternatively, antigen-presenting
polypeptides and accessory molecules which can insert themselves
into the cell membrane can be used. For example,
glycosyl-phosphotidylinositol (GPI)-modified polypeptides can
insert themselves into the membranes of cells. Medof et al. J. Exp.
Med. 160:1558-1578; and Huang et al. Immunity 1:607-613. Accessory
molecules include, but are not limited to, co-stimulatory
antibodies such as antibodies specific for CD28, CD80, or CD86;
costimulatory molecules, including, but not limited to, B7.1 and
B7.2; adhesion molecules such as ICAM-1 and LFA-3; and survival
molecules such as Fas ligand and CD70. See, for example, PCT
Publication No. WO 97/46256.
[0083] Alternatively, a synthetic antigen-presenting matrix can be
used to present antigen to effector cells. A synthetic matrix can
include an antigen presenting molecule, preferably an MHC Class I
or MHC Class II molecule, immobilized on a solid support, for
example, beads or plates. Accessory molecules can be present, which
can be co-immobilized or soluble, the molecules including, but not
limited to, co-stimulatory antibodies such as antibodies specific
for CD28, CD80, or CD86; costimulatory molecules, including, but
not limited to, B7.1 and B7.2; adhesion molecules such as ICAM-1
and LFA-3; and survival molecules such as Fas ligand and CD70.
Portions of accessory molecules can also be used, as long as their
function is maintained. Solid supports include metals or plastics,
porous materials, microbeads, microtiter plates, red blood cells,
and liposomes. See, for example, PCT Publication No. WO 97/46256;
and WO 97/35035.
[0084] Methods for determining whether an antigen-presenting
matrix, whether it is on a cell surface or on a synthetic support,
is capable of presenting antigen to an effector cell, are known in
the art and include, for example, .sup.3H-thymidine uptake by
effector cells, cytokine production by effector cells, and
cytolytic .sup.51Cr-release assays.
[0085] Effector Cell Populations
[0086] Antigen-specific T cells can be isolated from an effector
cell population, i.e., a population of hematopoietic cells,
preferably enriched for T cells. The effector cell population is a
starting population from which antigen-specific T cells are
isolated.
[0087] An effector cell population suitable for use in the present
invention can be autogeneic or allogeneic, preferably autogeneic.
When effector cells are allogeneic, preferably the cells are
depleted of alloreactive cells before use. This can be accomplished
by any known means, including, for example, mixing the allogeneic
effector cells and a recipient cell population and incubating them
for a suitable time, then depleting CD69.sup.+ cells, or
inactivating alloreactive cells, or inducing anergy in the
alloreactive cell population.
[0088] The effector cell population can comprise unseparated cells,
i.e., a mixed population, for example, a PBMC population, whole
blood, and the like. The effector cell population can be
manipulated by positive selection based on expression of cell
surface markers, negative selection based on expression of cell
surface markers, stimulation with one or more antigens in vitro or
in vivo, treatment with one or more biological modifiers in vitro
or in vivo, subtractive stimulation with one or more antigens or
biological modifiers, or a combination of any or all of these.
[0089] Effector cells can be obtained from a variety of sources,
including but not limited to, PBMC, whole blood or fractions
thereof containing mixed populations, spleen cells, bone marrow
cells, tumor infiltrating lymphocytes, cells obtained by
leukapheresis, biopsy tissue, lymph nodes, e.g., lymph nodes
draining from a tumor. Suitable donors include an immunized donor,
a non-immunized (naive) donor, treated or untreated donors. A
"treated" donor is one that has been exposed to one or more
biological modifiers. An "untreated" donor has not been exposed to
one or more biological modifiers.
[0090] Methods of extracting and culturing effector cells are well
known. For example, effector cells can be obtained by
leukapheresis, mechanical apheresis using a continuous flow cell
separator. For example, lymphocytes and monocytes can be isolated
from the buffy coat by any known method, including, but not limited
to, separation over Ficoll-Hypaque.TM. gradient, separation over a
Percoll gradient, or elutriation. The concentration of
Ficoll-Hypaque.TM. can be adjusted to obtain the desired
population, for example, a population enriched in T cells. Other
methods based on cell-specific affinity columns are known and can
be used. These include, for example, fluorescence-activated cell
sorting (FACS), cell adhesion, magnetic bead separation, and the
like. Affinity-based methods can utilize antibodies, or portions
thereof, which are specific for cell-surface markers and which are
available from a variety of commercial sources, including, the
American Type Culture Collection (Rockville, Md.). Affinity-based
methods can alternatively utilize ligands or ligand analogs, of
cell surface receptors.
[0091] The effector cell population can be subjected to one or more
separation protocols based on the expression of cell surface
markers. For example, the cells can be subjected to positive
selection on the basis of expression of one or more cell surface
polypeptides, including, but not limited to, "cluster of
differentiation" cell surface markers such as CD2, CD3, CD4, CD8,
TCR, CD45, CD45RO, CD45RA, CD11b, CD26, CD27, CD28, CD29, CD30,
CD31, CD40L; other markers associated with lymphocyte activation,
such as the lymphocyte activation gene 3 product (LAG3), signaling
lymphocyte activation molecule (SLAM), T1/ST2; chemokine receptors
such as CCR3, CCR4, CXCR3, CCR5, homing receptors such as CD62L,
CD44, CLA, CD146, .alpha.4.beta.7, .alpha.E.beta.7; activation
markers such as CD25, CD69 and OX40; and lipoglycans presented by
CD1. The effector cell population can be subjected to negative
selection for depletion of non-T cells and/or particular T cell
subsets. Negative selection can be performed on the basis of cell
surface expression of a variety of molecules, including, but not
limited to, B cell markers such as CD19, and CD20; monocyte marker
CD14; the NK cell marker CD56.
[0092] The effector cell population can be manipulated by exposure,
in vivo or in vitro, to one or more antigens. Antigens include, but
are not limited to, peptides; proteins; glycoproteins; lipids;
glycolipids; cells; cell extracts; tissue extracts; whole
microorganisms such as protozoans, bacteria, and viruses. Antigens
can be unmodified, i.e., used in their native state. Alternatively,
an antigen can be modified by any known means, including, but not
limited to, heating, for example to denature a protein or to
inactivate a pathogen; chemical modification to denature a protein,
or to cross-link two antigen molecules; glycosylation; chemical
modification with moieties including, but not limited to
polyethylene glycol; and enzymatic digestion. If more than one
antigen is used, the exposure can be simultaneous or
sequential.
[0093] The effector cells can be cultured in the presence of at
least one antigen associated with a condition to be treated. The
antigen can be a single antigen with multiple antigenic
determinants or can be a mixture of antigens. The antigen can be an
autoantigen or a foreign antigen, depending on the condition to be
treated. Autoantigens include antigens associated with autoimmune
diseases and those associated with cancer cells. The antigen can be
a protein, cells, a tissue or a target organ. If the antigen is an
autoantigen, the autoantigen can be part of an organ, for example
the brain or the thyroid gland and need not be purified therefrom.
Purified autoantigens or mixtures of purified autoantigens can also
be used.
[0094] Co-culturing of peripheral blood leukocytes (PBL) or tumor
infiltrating lymphocytes (TIL) with autologous tumor cells is
generally accompanied by cytokine stimulation. Sporn et al.(1993)
Cancer Immunol. Immunother. 37:175-180; and Peyret et al. (1991)
Chirurgie 117:700-709.
[0095] An effector cell population can be manipulated by exposure,
in vivo or in vitro, to one or more biological modifiers. Suitable
biological modifiers include, but are not limited to, cytokines
such as IL-2, IL-4, IL-10, TNF-.alpha., IL-12, IFN-.gamma.;
non-specific modifiers such as phytohemagglutinin (PHA), phorbol
esters such as phorbol myristate acetate (PMA), concanavalin-A, and
ionomycin; antibodies specific for cell surface markers, such as
anti-CD2, anti-CD3, anti-IL-2 receptor, anti-CD28; chemokines,
including, for example, lymphotactin. The biological modifiers can
be native factors obtained from natural sources, factors produced
by recombinant DNA technology, chemically synthesized polypeptides
or other molecules, or any derivative thereof having the functional
activity of the native factor. If more than one biological modifier
is used, the exposure can be simultaneous or sequential.
[0096] The present invention provides compositions comprising T
cells enriched in antigen-specific cells, enriched according to the
methods of the invention. By "enriched" is meant that a cell
population is at least about 50-fold, more preferably at least
about 500-fold, and even more preferably at least about 5000-fold
or more enriched from an original mixed cell population comprising
the desired cell population. The proportion of the enriched cell
population which comprises the desired antigen-specific cells can
vary substantially, from less than 10% up to 100% antigen-specific
cells. The percentage which are antigen-specific can be readily
determined, for example, by a .sup.3H-thymidine uptake assay in
which the T cell population is challenged by an antigen-presenting
matrix presenting the desired antigen(s).
[0097] Cell Labeling
[0098] The methods herein are based on labeling the cells with a
product secreted by the cells, where the product is secreted in
response to antigen stimulation. To achieve labeling, the cell
surface of a cell population is modified such that a moiety that
binds specifically to a product, the "specific binding partner" is
attached to the cell surface either directly or through an
anchoring means (an "anchor moiety"), optionally through a linker
to form a capture moiety. The cell population can contain numerous
types of cells and generally made up of a mixed population.
Preferably the cell population is hematopoietic, more preferably
the cell population is effector cells, most preferably, the cell
population is T cells or a subset thereof. Subsets can be isolated
by virtue of cell surface markers, for instance, CD45 for
lymphocytes, CD8 for cytotoxic cells, etc.
[0099] Products secreted in response to antigen stimulation are
known in the art and include, but are not limited to, cytokines,
such as IL-2, IL-4, IL-10, TNF-.alpha., TGF-.beta. and
IFN-.gamma..
[0100] Specific binding partners include any moiety for which there
is a relatively high affinity and specificity between product and
binding partner, and in which the dissociation of the
product:partner complex is relatively slow so that the
product:partner complex is detected during the cell separation
technique. Specific binding partners include, but are not limited
to, substrates or substrate analogs to which a product will bind,
peptides, polysaccharides, steroids, biotin, digitoxin, digitonin
and derivatives thereof. In a preferred embodiment the specific
binding partner is an antibody or antigen-binding fragment or
derivative thereof. The term "antigen-binding fragment" includes
any peptide that binds specifically to the product. Typically,
these fragments include such immunoglobulin fragments as Fab,
F(ab').sub.2, Fab', scFv (both monomer and polymeric forms) and
isolated H and L chains. An antigen-binding fragment retains the
specificity of the intact immunoglobulin, although avidity and/or
affinity can be altered.
[0101] In the practice of the invention the capture moiety can be
attached to a cell membrane (or cell wall) by a variety of methods.
Suitable methods include, but are not limited to, direct chemical
coupling to amino groups of the protein components, coupling to
thiols (formed after reduction of disulfide bridges) of the protein
components, indirect coupling through antibodies (including pairs
of antibodies) or lectins, anchoring in the lipid bilayer by means
of a hydrophobic anchor, and binding to the negatively charged cell
surface by polycations.
[0102] In other embodiments, the capture moiety is introduced using
two or more steps, e.g., by labeling the cells with at least one
anchor moiety which allows the coupling of the capture moiety to
the anchor moiety either directly, for instance by a biotin/avidin
complex or indirectly, through a suitable linking moiety or
moieties.
[0103] Suitable anchor moieties include lipophilic molecules such
as fatty acids. Alternatively, antibodies or other specific binding
agents to cell surface markers such as the MHC antigens or
glycoproteins, can also be used.
[0104] The "capture moiety" can be coupled to the anchor moiety
through a linking agent, and can also include a linker which
multiplies the number of capture moieties available and thus the
potential for capture of product, such as branched polymers,
including, for example, modified dextran molecules, polyethylene
glycol, polypropylene glycol, polyvinyl alcohol, and
polyvinylpyrrolidone.
[0105] Methods for direct chemical coupling of antibodies to the
cell surface are known in the art, and include, for example,
coupling using glutaraldehyde or maleimide activated antibodies.
Methods for chemical coupling using multiple step procedures
include, but are not limited to, biotinylation, coupling of
trinitrophenol (TNP) or digoxigenin using for example succinimide
esters of these compounds. Biotinylation can be accomplished by,
for example, the use of D-biotinyl-N-hydroxysuccinimide.
Succinimide groups react effectively with amino groups at pH values
above 7, and preferentially between about pH 8.0 and about pH 8.5.
Biotinylation can be accomplished by, for example, treating the
cells with dithiothreitol followed by the addition of biotin
maleimide.
[0106] Coupling to the cells can also be accomplished using
antibodies against cell surface antigens ("markers"). Antibodies
directed to surface antigens generally require in the range of 0.1
to 1 .mu.g of antibody per 10.sup.7 cells. However, this
requirement will vary widely in response to the affinity of the
antibody to the product and will need to be determined empirically.
Such a determination is well within the skill of one in the art.
Thus, the appropriate amount of antibody must be determined
empirically and is within the skill of one in the art. This allows
coupling to specific cells on cell type specific marker expression.
For instance, classes of cells such as T cells or subsets thereof
can be specifically labeled. As a capture moiety, a bispecific
antibody can be used which has an antigen recognition site for the
cell or an anchor moiety placed thereon, and the product.
[0107] A capture moiety, particularly capture antibodies should be
selected based on the amount of secreted product. For example, for
cells which secrete only a few molecules, a high affinity antibody
will catch most of the secreted molecules. Alternatively, in the
case where the cell secretes many molecules during the incubation
time, a lower affinity antibody can be preferred to prevent too
early saturation of the catching matrix. Determination of suitable
affinities for the level of proteins secreted are determined
empirically and are within the skill of one in the art.
[0108] Cells carrying large amounts of N-acetylneuraminic acid on
their surface as a constituent of their lipopolysaccharides bear a
negative charge at physiological pH values. Coupling of capture
moieties can be via charge interactions. For example, moieties
bearing polycations bind to negatively charged cells. Polycations
are known in the art and include, for example, polylysine and
chitosan. Chitosan is a polymer consisting of D-glucosamine groups
linked together by .alpha.-(1-4) glucoside bonds.
[0109] Another method of coupling binding partners (which can
comprise one or more capture moieties) to the cells is via coupling
to the cell surface polysaccharides. Substances which bind to
polysaccharides are known in the art, and include, for example,
lectins, including concanavalin A, solanum tuberosum, aleuria
aurantia, datura stramonium, galanthus nivalis, helix pomatia, lens
culinaris and other known lectins supplied by, a number of
companies, including for example, Sigma Chemical Company and
Aldrich Chemical Company.
[0110] In some embodiments of the invention, the product binding
partner is coupled to the cell by hydrophobic anchoring to the cell
membrane. Suitable hydrophobic groups that will interact with the
lipid bilayer of the membrane are known in the art, and include,
but are not limited to, fatty acids and non-ionic detergents
(including, e.g., Tween-80). A drawback to attachment of the
capture moiety to the cell via the insertion of a hydrophobic
anchor is that the rate of integration of the hydrophobic moiety
into the cell is low. Thus, high concentrations of the moiety with
the hydrophobic anchor often are required. This latter situation is
often uneconomical when the capture moiety is a relatively limited
or expensive substance, for example, an antibody.
[0111] The low yield of hydrophobic molecules that embed themselves
in the membrane is relevant only when these molecules are available
in relatively limited quantities. This problem can be overcome by
using a bridging system that includes an anchoring partner and a
partner that contains the capture moiety, wherein one of the
partners is of higher availability, and wherein the two parts of
the bridging system have a high degree of specificity and affinity
for each other. For example, in one embodiment avidin or
streptavidin is attached to the cell surface via a hydrophobic
anchor, while the partner with the product capture moiety are
biotinylated anti-product antibodies. In another embodiment, the
cell surface is labeled with digoxigenin followed by conjugates of
anti-digoxigenin antibody fragments and anti-product antibodies.
This approach can be used with other pairs of molecules able to
form a link, including, for example, hapten with antihapten
antibodies, NTA with polyhistidine residues, or lectins with
polysaccharides. A preferred embodiment is one which allows
"amplification" of the system by increasing the number of capture
moieties per anchor moiety.
[0112] In one illustrative embodiment, a branched dextran is bound
to palmitic acid, thus providing a multiplicity of available
binding sites. The dextran is in turn coupled to biotin and treated
with avidin-conjugated antibody specific for the product.
[0113] It is of course contemplated within the embodiments of the
invention that bridging systems can be used between the anchor
moiety and the capture moiety when the anchor moiety is coupled in
any fashion to the cell surface. Thus, an avidin (or streptavidin)
biotin linker moiety can link an antibody anchor moiety with a
capture moiety. Bispecific antibody systems can also act as linker
moieties.
[0114] In order to analyze and, if desired, to select cells that
have the capability of secreting the product, cells modified as
above to contain the capture moiety are incubated under conditions
that allow the production and secretion of the product in a
sufficient amount to allow binding to and detection of the cells
that contain the captured product. These conditions are known to
those of skill in the art and include, inter alia, appropriate
temperature, pH, and concentrations of salts, growth factors and
substrates in the incubation medium, as well as the appropriate
concentrations of gas in the gaseous phase. When it is desirable to
distinguish between high and low producer cells, the time of
incubation is such that product secretion by the cells is still in
a linear phase. The appropriate conditions can be determined
empirically and such a determination is within the skill of one in
the art.
[0115] Additionally, cell secretion can be modified, that is,
upregulated, induced, or reduced using a biological modifier. The
biological modifiers can be added at any time but are preferably
added to the incubation medium. Alternatively, the cells can be
pretreated with these agents or cells prior to the incubation step.
Suitable biological modifiers include, but are not limited to,
molecules and other cells. Suitable molecules include, but are not
limited to, drugs, cytokines, small molecules, hormones,
combinations of interleukins, lectins and other stimulating agents,
e.g., PMA, LPS, bispecific antibodies and other agents that modify
cellular functions or protein expression.
[0116] Suitable cells include, but are not limited to, direct cell
to cell interactions such as between a tumor and T cell and
indirect cell to cell interactions such as those induced by the
proximity of other cells which secrete a biological modifier.
Suitable cells include, but are not limited to, blood cells,
peripheral bone marrow cells and various cell lines.
[0117] The incubation conditions are also such that product is
essentially not captured or is captured to a much lesser extent by
another cell, so as to distinguish non-producing cells from product
producing cells, or high producers from low producers. Generally
the incubation time is between five minutes and ten hours, and is
more usually between one and five hours. The incubation medium can
optionally include a substance that slows diffusion of the product
from the producer cell. Substances which inhibit product diffusion
in liquid media and that are non-toxic to cells are known in the
art and include a variety of substances that partially or
completely gel, including, for example, alginate, low melting
agarose and gelatin. By varying the viscosity or permeability of
the medium, the local capture by a producing cell of differently
sized products can be modulated. The molecular weight size
exclusion of the medium can be adjusted to optimize the reaction.
The optimal composition of the medium can be empirically determined
and is influenced by the cell concentration, the level of secretion
and molecular weight of the product and the affinity of the capture
moieties for the product. Such determinations are within the skill
of one in the art.
[0118] Preferably, the gels are solubilized after the incubation to
allow the isolation of the cells or groups of cells from the media
by cell sorting techniques. Thus, for example, the gels can be
linked by disulfide bonds that can be dissociated by sulfhydryl
reducing agents such as .beta.-mercaptoethanol or dithiothreitol,
or the gels can contain ion cross-linkings, including for example,
calcium ions, that are solubilized by the addition of a chelating
agent such as EDTA.
[0119] At the end of the secretion phase the cells are usually
chilled to prevent further secretion, and the gel matrix (if any)
is solubilized. This order can, of course, be reversed. As capping
can take place after the capture moiety is added due to cross
linking, an incubation step to decrease capping can be added at
this point. The cells can be incubated for instance in cytochalasin
A or B or any other suitable substance that prevents capping. The
cells containing the trapped product are then labeled with a label
moiety. Labeling can be accomplished by any method known to those
of skill in the art. For example, anti-product antibodies can be
used to directly or indirectly label the cells containing the
product. The labels used are those which are suitable for use in
systems in which cells are to be analyzed or sorted based upon the
attachment of the label moiety to the product.
[0120] In other embodiments, capture moieties that do not contain
captured product can be detected. This allows, for example, the
isolation of cells that secrete high amounts by employing a
negative separation method, i.e., detection of cells not highly
saturated with product. The cells can be labeled with other
labeling substances recognizing, e.g., cell surface markers, cell
type, cellular parameters such as DNA content, cell status, or
number of capture moieties.
[0121] The enumeration of actual capture moieties can be important
to compensate for varying amounts of these molecules due to, for
example, different conjugation potentials of the cells. It can be
especially important for the isolation of rare cells to exclude
cells with decreased or increased capability for binding the
product capture system, including the anchor and capture moieties.
Alternatively, the reactions can proceed simultaneously in a
"one-step reaction."
[0122] Cell Analysis and Cell Sorting
[0123] Analysis of the cell population and cell sorting based upon
the presence of the label can be accomplished by a number of
techniques known in the art. Cells can be analyzed or sorted by,
for example, flow cytometry or FACS. These techniques allow the
analysis and sorting according to one or more parameters of the
cells. Usually one or multiple secretion parameters can be analyzed
simultaneously in combination with other measurable parameters of
the cell, including, but not limited to, cell type, cell surface
markers, DNA content, etc. The data can be analyzed and cells
sorted using any formula or combination of the measured parameters.
Cell sorting and cell analysis methods are known in the art and are
described in, for example, The Handbook of Experimental Immunology,
Volumes 1 to 4, (D. N. Weir, editor); Flow Cytometry Cell Sorting
(A. Radbruch, editor, Springer Verlag, 1992); and Cell Separation
Methods and Applications (D. Recktenwald and A. Radbruch, eds.,
1997) Marcel Dekker, Inc. N.Y. Cells can also be analyzed using
microscopy techniques including, for example, laser scanning
microscopy, fluorescence microscopy; techniques such as these can
also be used in combination with image analysis systems. Other
methods for cell sorting include, for example, panning and
separation using affinity techniques, including those techniques
using solid supports such as plates, beads and columns.
[0124] Some methods for cell sorting utilize magnetic separations,
and some of these methods utilize magnetic beads. Different
magnetic beads are available from a number of sources, including
for example, Dynal (Norway), Advanced Magnetics (Cambridge, Mass.,
U.S.A.), Immuncon (Philadelphia, U.S.A.), Immunotec (Marseilles,
France), and Miltenyi Biotec GmbH (Germany).
[0125] Preferred magnetic labeling methods include colloidal
superparamagnetic particles in a size range of 5 to 200 nm,
preferably in a size of 10 to 100 nm. These magnetic particles
allow a quantitative magnetic labeling of cells, thus the amount of
coupled magnetic label is proportional to the amount of bound
product, and the magnetic separation methods are sensitive to
different amounts of product secretion. Colloidal particles with
various specificities are known in the art, and are available, for
example, through Miltenyi Biotec GmbH. The use of immunospecific
fluorescent or magnetic liposomes can also be used for quantitative
labeling of captured product. In these cases, the liposomes contain
magnetic material and/or fluorescent dyes conjugated with antibody
on their surfaces, and magnetic separation is used to allow optimal
separation between nonproducing, low producing, and high producing
cells.
[0126] The magnetic separation can be accomplished with high
efficiency by combining a second force to the attractive magnetic
force, causing a separation based upon the different strengths of
the two opposed forces. Typical opposed forces are, for example,
forces induced by magnetic fluids mixed in the separation medium in
the magnetic separation chamber, gravity, and viscous forces
induced by flow speed of medium relative to the cell. Any magnetic
separation method, preferably magnetic separation methods allowing
quantitative separation will be used. It is also contemplated that
different separation methods can be combined, for example, magnetic
cell sorting can be combined with FACS, to increase the separation
quality or to allow sorting by multiple parameters.
[0127] Preferred techniques include high gradient magnetic
separation (HGMS), a procedure for selectively retaining magnetic
materials in a chamber or column disposed in a magnetic field. In
one application of this technique the product is labeled by
attaching it to a magnetic particle. The attachment is generally
through association of the product with a label moiety which is
conjugated to a coating on the magnetic particle which provides a
functional group for the conjugation. The captured product thus
coupled to a magnetic "label", is suspended in a fluid which is
then applied to the chamber. In the presence of a magnetic gradient
supplied across the chamber, the magnetically labeled target cell
is retained in the chamber; if the chamber contains a matrix, it
becomes associated with the matrix. Cells which do not have or have
only a low amount of magnetic labels pass through the chamber.
[0128] The retained cells can then be eluted by changing the
strength of, or by eliminating, the magnetic field or by
introducing a magnetic fluid. The selectivity for a captured
product is supplied by the label moiety conjugated either directly
or indirectly to the magnetic particle or by using a primary
antibody and a magnetic particle recognizing the primary antibody.
The chamber across which the magnetic field is applied is often
provided with a matrix of a material of suitable magnetic
susceptibility to induce a high magnetic field gradient locally in
the camber in volumes close to the surface of the matrix. This
permits the retention of fairly weakly magnetized particles.
Publications describing a variety of HGMS systems are known in the
art, and include, for example, U.S. Pat. No. 4,452,773, U.S. Pat.
No. 4,230,685, PCT application WO85/04330, U.S. Pat. No. 4,770,183,
and PCT/EP89/01602; systems are also described in U.S. Pat. Nos.
5,411,863; 5,543,289; 5,385,707; and 5,693,539, which are commonly
owned and hereby incorporated herein by reference.
[0129] In addition, in other embodiments the processes include
labeling the cells that contain the product captured by the capture
moiety, if any. Other embodiments can also include analyzing the
cell population to detect labeled cells, if any, and if desired,
sorting the labeled cells, if any.
[0130] Diagnostic Methods for Detecting Antigen-Specific T
Cells
[0131] The present invention further provides diagnostic methods
for detecting antigen-specific T cells. These include methods for
analyzing a population of cells enriched for T cells to identify or
enumerate antigen-specific T cells, as well as methods of
determining a distribution of antigen-specific T cells that secrete
a product in response to antigen stimulation.
[0132] Methods for analyzing a population of cells enriched in T
cells to identify or enumerate antigen-specific T cells that
secrete and release an amount of product relative to other cells in
the population, wherein the product is secreted and released in
response to antigen stimulation, comprise the steps of labeling the
cells by the methods of the present invention; labeling the cells
with at least one additional label that does not label the captured
product; and detecting the amount of product label relative to the
additional label. Such methods are useful, for example, in
determining the proportion of a cell population that is specific
for a given antigen. The method can be used to provide information
regarding the immune status of an individual, including assessing
an immune response to allergens, a tumor or virus, or evaluating
the proportion of cells in an individual that are self reactive so
as to detect or monitor autoimmune diseases.
[0133] Method of Treatment Using Enriched Antigen-Specific T
Cells
[0134] The present invention provides methods of treatment of a
disease or condition related to a population of antigen-specific T
cells, using the enriched T cells of the invention.
[0135] Treatment methods include those in which an antigen-specific
T cell population is identified, enriched, and introduced into an
individual; those in which a population of antigen-specific T cells
is identified, enriched and expanded in vitro before introduction
into an individual; those in which a population of antigen-specific
T cells is identified and eliminated from a population of cells to
be introduced into an individual; ex vivo genetic modification
prior to administration; and selection of antigen-specific T cells
selected according to cytokine expression. Examples of
antigen-specific T cells selected according to cytokine expression
include, but are not limited to, IFN-.gamma. or TNF-.alpha.
secreting CD8.sup.+ T cells (cytotoxic) for treatment of cancer,
viral (e.g. CMV, EBV) and bacterial (e.g. listeria, mycobacteria)
infections; IFN-.gamma. secreting CD4.sup.+ T cells for the same
indications and also for suppression and/or counter-regulation of
allergy or vaccination against allergy, suppression of
TH2-associated autoimmune diseases or vaccination against these
autoimmune diseases; IL-10 or TGF-beta secreting CD4.sup.+ T cells,
for suppression TH1, but also TH2-associated autoimmune diseases or
vaccination against these autoimmune diseases (tolerance
induction); IL-4 secreting CD4.sup.+ T cells for suppression of
TH1-associated autoimmune diseases or vaccination against these
autoimmune diseases; and IL-4 or IL-5 secreting CD4.sup.+ T cells
for treatment of helminth infections.
[0136] T cell populations enriched according to the methods of the
present invention can be used to treat a variety of disorders.
Included among these are cancer. T cells specific for a tumor
antigen can be obtained using the methods of the present invention.
Tumor cells can be obtained from an individual, and these can be
co-cultured in vitro with T cells obtained from the same
individual. After co-culturing the cells for a suitable time,
tumor-specific T cells can be enriched according the methods of the
present invention. This enriched population can then be
re-introduced into the patient. Methods for anti-tumor
immunotherapy using autologous T cells are known in the art. See,
for example, WO 97/05239.
[0137] Alternatively, cells used in anti-tumor immunotherapy
treatments can be allogeneic. Various modes of treatment of cancer
with allogeneic T cells have been described in the art and can be
used in the methods of the present invention. See, for example, PCT
Publication No. WO 96/37208. Optionally, allogeneic T cells can be
activated prior to introduction into an individual. Activation can
be effected through contact with a biological modifier, an antibody
directed to a cell surface marker, or a ligand or analog thereof
for a cell surface receptor.
[0138] Another use of enriched T cell populations of the present
invention is in immunomodulation, for example, in the treatment of
autoimmune disorders, inflammatory disorders, allergies and
hypersensitivities such as delayed-type hypersensitivity and
contact hypersensitivity. T cells which are capable of destroying
or suppressing the activity of autoreactive cells can be enriched
in vitro, optionally expanded in vitro, then re-introduced into a
patient. In the treatment of allergic responses, the ratio of TH1
to TH2 cells can be altered, or, cells reactive toward
allergen-specific cells can be enriched and introduced into an
individual.
[0139] Inducing T cell anergy can also be used to treat, ameliorate
or prevent allograft rejection thus improving the results of organ
transplantation and increasing the range of histotypes to which a
patient can be made histocompatible.
[0140] Compositions comprising enriched T cell populations can
further be used as vaccines, to prevent or substantially reduce the
probability of the occurrence of a disease state such as a viral
infection, autoimmune disorder, allergic response, cancer, or other
disorder, or will reduce the severity or duration of the disease if
subsequently infected or afflicted with the disease.
[0141] The compositions of cells can be administered by any known
route, including, but not limited to, intravenously, parenterally,
or locally. In the treatment methods of the present invention,
enriched T cells are administered to an individual. The total
number of cells, the number of doses, and the number of cells per
dose will depend upon the condition being treated. Generally, about
10.sup.6 to 10.sup.11 cells are administered in a volume ranging
from about 5 ml to 1 liter. The cells can be administered in a
single dose or in several doses over selected time intervals. Of
the cells being administered, preferably at least about 10%, more
preferably at least about 20%, more preferably at least about 50%,
are antigen-specific T cells which secrete a product.
[0142] Kits
[0143] It is contemplated that the reagents used in the detection
of secretor cells of desired products can be packaged in the form
of kits for convenience. The kits would contain, for example,
optionally one or more materials for use in preparing gelatinous
cell culture medium, the medium to be used for cell incubation for
the production of the desired secreted product; a product capture
system comprised of anchor and capture moieties; a label moiety;
and instructions for use of the reagents. All the reagents would be
packaged in appropriate containers.
[0144] The kit can also be formulated to include the following. In
this case all the reagents are preferably placed in a single vial
to which the cells are added. At least one antibody which is
bispecific for a particular cell surface structure or anchor moiety
and the product. At least one label moiety and, optionally,
biological modifiers.
[0145] Optionally, the kit can include physiologically acceptable
buffer. Such buffers are known in the art and include, but are not
limited to, PBS with and without BSA, isotonic saline, cell culture
media and any special medium required by the particular cell type.
Buffers can be used that reduce cross-labeling and increase the
local product concentration around the cells. Buffers can include
agents for increasing viscosity or decreasing permeability.
Suitable agents are described herein. The viscosity of the medium
can be reduced before analysis by any method known in the art
including, but not limited to, dissolution in a physiologically
acceptable buffer, dissolving heat, EDTA, and enzymes. In the
absence of added medium, cells already suspended in a medium can be
directly added to the vial. Suitable cell suspensions include but
are not limited to cell lines and biological samples. Biological
samples include, but are not limited to, blood, urine and
plasma.
[0146] Additional structures can be added for catching unbound
product to reduce cell cross-contamination thereby reducing the
diffusion of products away from the producing cells. These include,
but are not limited to, anti-product antibody immobilized to gel
elements, beads, magnetic beads, and polymers.
[0147] Biological modifiers can also be added to the buffer or
medium to induce specific secretion.
[0148] Additional label moieties such as antibodies (magnetically
or fluorescently labeled) can also be present, including, but not
limited to anti-cell surface marker antibodies to identify cell
types, propidium iodide to label dead cells, and magnetic beads to
label certain cell types.
[0149] In this embodiment, all materials can be placed in a single
container such as a vial and the cell sample added. The contents
are incubated to allow secretion of a product and subsequent
capture of the product and binding of the label moiety to the
product. The cells which have secreted and bound product can then
be separated and/or analyzed based on the presence, absence or
amount of the captured product. Separation can be done by any of
the methods known in the art, including, but not limited to, simple
dilution, erythrocyte lysis, centrifugation-washing step, magnetic
separation, FACS and Ficoll separation. The analysis of the cells
can be performed by a variety of methods, including, but not
limited to, FACS, image analysis, cytological labeling, and
immunoassay.
[0150] The following examples are provided solely for the purposes
of illustration and not to limit the scope of the invention. In
light of the present disclosure, numerous embodiments within the
scope of the claims will be apparent to those of ordinary skill in
the art.
EXAMPLE 1
[0151] Peripheral blood mononuclear cells (PBMC) were cultured in
complete RPMI 1640 (Gibco BRL, Grand Island, N.Y.) containing 100
U/ml penicillin, 0.1 mg/ml streptomycin, 0.3 mg/ml glutamine, 10 mM
2-mercaptoethanol and 10% human serum type AB (Sigma, St. Louis,
Mo.) at a cell concentration of 2.times.10.sup.6 cells/ml. Peptide
M1 58-66 from Influenza virus matrix protein (GILGFVFTL; Neosystem,
Strasbourg, France) was added to a final concentration of 1 .mu.M.
Control cells were cultured without peptide.
[0152] Cells were incubated at 37.degree. C. in an atmosphere
containing 7.5% CO.sub.2. After 5 hours and 30 minutes, cells were
harvested by centrifugation. Cells were incubated at a cell
concentration of 5.times.10.sup.7 cells/ml in complete RPMI 1640
with anti human interferon gamma (IFN-.gamma.) monoclonal antibody
(mAb) 4SB3 conjugated to anti-human CD45 mAb 5B1 (30 .mu.g/ml) at
8.degree. C. for 7 min. The cells were then diluted to
2.times.10.sup.6 cells/ml with complete RPMI 1640 containing 10%
FCS and incubated for 45 minutes at 37.degree. C. Then cells were
pelleted and incubated with phycoerythrin (PE)-conjugated anti
human interferon gamma (IFN-.gamma.) mAb NIB42 (4 .mu.g/ml) and
FITC-labeled anti-CD8 mAb in PBS/BSA/EDTA solution 0.05% BSA and 2
mM EDTA, for 10 minutes at 4.degree. C. Cells were then washed in
PBS/BSA/EDTA and labeled with mouse anti-PE mAb 80-5 conjugated to
MicroBeads (Miltenyi Biotec) in PBS/BSA/EDTA for 15 minutes at
8.degree. C. Cells were washed and resuspended in 500 .mu.l
PBS/BSA/EDTA.
[0153] IFN-.gamma.-secreting cells were enriched with the magnetic
cell separation system MACS. Magnetically labeled cell suspension
was pipetted onto a MiniMACS separation column in a MiniMACS
separation unit, the cell suspension was allowed to pass through
and the column was washed with 3.times.500 .mu.l buffer. The
effluent was collected as negative fraction (N1). The column was
removed from the separator, and placed on a suitable tube. 1 ml
buffer was pipetted on top of column and magnetically labeled cells
were flushed out using a plunger and applied to a second round of
MiniMACS separation.
[0154] The original cells (i.e., before MACS separation), negative
cell fractions (of first as well as second MACS separation,
designated N1 and N2, respectively) and positive cell fraction (P2)
of second MACS separation were analyzed by flow cytometry. FACScan
and CELLQuest research software (Becton Dickinson, Mountain View,
Calif.) were used for flow cytometric analysis. Dead cells and cell
debris were excluded according to scatter properties and staining
with propidium iodide (PI; 0.3 .mu.g/ml).
[0155] The results are shown in FIGS. 1A-P. While dot plots A-H
show analysis of control cells cultured without peptide, plots I-P
show analysis of peptide stimulated cells. Dot plots show-the
scatter properties of the starting cell population (A and I) and
the enriched cell populations (C and K); and PI versus PE
fluorescence of the starting cell population (B and J) and enriched
cell population (D and L).
[0156] Dot plots E-H and M-P show anti-CD8-FITC versus
anti-IFN-.gamma.-PE staining of gated cells in original (E and M),
first negative (F and N), second negative (G and O) and in the
final positive cell fraction (H and P).
[0157] The control cell population, CD8.sup.+ IFN-.gamma..sup.+
cells were enriched up to 11% among live cells (FIG. 1H), in the
peptide stimulated cell population, CD8.sup.+ IFN-.gamma..sup.+
cells were enriched up to 40% (FIG. 1P). From a starting population
of 3.5.times.10.sup.7 control cells, about 600 CD8.sup.+
IFN-.gamma..sup.+ cells were isolated, compared to 4100
CD8+IFN-.gamma..sup.+ cells isolated from a starting population of
3.5.times.10.sup.7 peptide-stimulated cells.
[0158] CD8-cells brightly stained with PE-labeled anti-IFN-.gamma.
were CD19.sup.+ B cells, most likely B cells specific for a sorting
reagent, probably PE. These cells were enriched to the same extent
from control cells compared to peptide stimulated cells.
[0159] Also the CD8.sup.- cells dimly stained with PE-labeled
anti-IFN-.gamma. (like the CD8.sup.+ IFN-.gamma..sup.+ cells) were
enriched to the same extent from control cells compared to peptide
stimulated cells. Such cells partially stain for CD4 and CD56, and
therefore are most likely T helper cells or NK cells secreting
IFN-.gamma..
[0160] Thus there is a basal level of IFN-.gamma. secretion by
(CD4+) T helper cells, (CD8.sup.+) cytotoxic T cells and
(CD56.sup.+) NK cells without intentional antigen-specific
stimulation in vitro, which reflects most likely the IFN-.gamma.
secretion induced already in vivo in ongoing immune responses at
the time of blood sampling.
[0161] However, IFN-.gamma..sup.+-secreting CD8.sup.+ cells induced
by stimulation with the HLA class I-restricted influenza peptide M1
58-66 were significantly enriched above this background level;
therefore, most of the CD8+IFN-.gamma..sup.+ cells enriched from
peptide stimulated cells are peptide-specific T cells. Specificity
of enriched cells was further confirmed by staining for the
presence of V.beta.17 TCR, which is a conserved T cell receptor
(TCR) segment in M1 58-66 specific cytotoxic T cells. Lehner et al.
(1995) J. Exp. Med. 181:79-91; and Lalvani et al. (1997) J. Exp.
Med. 186:859-865. Among IFN-.gamma..sup.+ cells isolated from
peptide stimulated cells, but not among IFN-.gamma..sup.+ cells
isolated from control cells, most express V.beta.17.sup.+ TCRs.
EXAMPLE 2
[0162] Peripheral blood mononuclear cells (PBMC) were cultured in
complete RPMI 1640 (Gibco BRL, Grand Island, N.Y.) containing 100
U/ml penicillin, 0.1 mg/ml streptomycin, 0.3 mg/ml glutamine, 10 mM
2-ME and 10% human serum type AB (Sigma, St. Louis, Mo.) at
2.times.10.sup.6 cells/ml. Peptide M1 58-66 from Influenza virus
matrix protein (GILGFVFTL; Neosystem, Strasbourg, France) was added
to a final concentration of 1 .mu.M. Control cells were cultured
without peptide.
[0163] After 5 hours and 30 minutes cells were harvested by
centrifugation. Cells were incubated at 5.times.10.sup.7 cells/ml
in complete RPMI 1640 with anti-human IFN-.gamma. mAb 4SB3
conjugated to anti-human CD45 mAb 5B1 (30 .mu.g/ml) at 8.degree. C.
for 7 minutes. The cells were then diluted to 2.times.10.sup.6
cells/ml with complete RPMI 1640 containing 10% FCS and incubated
for 45 minutes at 37.degree. C. Then cells were spun down and
incubated with phycoerythrin (PE)-conjugated anti-human-IFN-.gamma.
mAb NIB42 (4 .mu.g/ml) and FITC-labeled anti-CD8 in PBS/BSA/EDTA,
for 10 minutes at 4.degree. C. Cells were then washed in
PBS/BSA/EDTA and labeled with mouse anti-PE mAb 80-5 conjugated
MicroBeads (Miltenyi Biotec) in PBS/BSA/EDTA for 15 minutes at
8.degree. C. Cells were washed and resuspended in 500 .mu.l
PBS/BSA/EDTA.
[0164] IFN-.gamma.-secreting cells were enriched with the magnetic
cell separation system MACS. Magnetically labeled cell suspension
was pipetted on top of a MiniMACS separation column in a MiniMACS
separation unit, cell suspension was allowed to pass through and
column was washed with 3.times.500 .mu.l buffer. Effluent was
collected as negative fraction. The column was removed from
separator, and placed on a suitable tube. 1 ml buffer was pipetted
on top of column and magnetically labeled cells were flushed out
using a plunger and applied to a second round of MiniMACS
separation.
[0165] Original cells (i.e., before MACS separation), negative cell
fractions (of first as well as second MACS separation) and positive
cell fraction of second MACS separation were analyzed by flow
cytometry. FACScan and CELLQuest research software (Becton
Dickinson, Mountain View, Calif.) were used for flow cytometric
analysis. Dead cells and cell debris were excluded according to
scatter properties and staining with propidium iodide (PI; 0.3
.mu.g/ml) as shown in Example 1. The results are shown in FIG.
2.
[0166] While dot plots 2A-G show analysis of control cells cultured
without peptide, plots 2J-R show analysis of peptide stimulated
cells.
[0167] Dot plots 2A-D and 2J-M show FITC-labeled anti-CD8 versus
PE-labeled anti-IFN-.gamma. staining of gated cells in original (A,
J), first negative (B, K), second negative (C, L) and in the final
positive cell fraction (D, M).
[0168] While in the control cells CD8.sup.+ IFN-.gamma.+ cells were
enriched up to 8.2% among live cells (2D), out of peptide
stimulated cells CD8.sup.+ IFN-.gamma..sup.+ cells were enriched up
to 41.6% (2M). Out of 6.1.times.10.sup.7 control cells, about 1360
CD8.sup.+ IFN-.gamma..sup.+ cells were isolated compared to 11700
CD8.sup.+ IFN-.gamma..sup.+ cells out of 6.9.times.10.sup.7 peptide
stimulated cells.
[0169] IFN-.gamma..sup.+ secreting CD8.sup.+ cells induced by
stimulation with the HLA class I-restricted influenza peptide M1
58-66 were significantly enriched above background level, i.e.,
most of the CD8.sup.+ IFN-.gamma..sup.+ cells enriched from peptide
stimulated cells must be peptide-specific T cells. Specificity of
enriched cells was further confirmed by staining against V.beta.17
TCR, which is a conserved T cell receptor (TCR) segment in M1 58-66
specific cytotoxic T cells (Lehner 1995; Lalvani 1997). Only among
IFN-.gamma..sup.+ cells isolated from peptide stimulated cells, but
not among IFN-.gamma..sup.+ cells isolated from control cells, most
express V.beta.17.sup.+ TCRs (2F versus 2O).
[0170] The following examples show that appropriate
antigen-specific stimulation, CD4.sup.+ and CD8.sup.+ lymphocytes
rapidly express cytokines. The technique is demonstrated here for
HLA-A0201-restricted influenza matrix protein (FLU) peptide 58-66
-specific CD8.sup.+ cytotoxic T lymphocytes (CTLs), influenza A
virus- and recombinant tetanus toxin C (rTT.C)-fragment-specific T
helper type 1 (Th1) cells, and tetanus toxoid (TT) specific T
helper type 2 (Th2) cells.
EXAMPLE 3
Materials and Methods for Examples 4-8
[0171] Cells and Ex Vivo Stimulation
[0172] Buffy coats were obtained from the Institute for
Transfusions medicine, Hospital Merheim, Cologne, Germany and, if
necessary, selected on the basis of HLA-type. PBMC were prepared by
standard Ficoll-Pacque (Pharmacia, Uppsala, Sweden) density
gradient centrifugation, washed twice in PBS and resuspended at a
cell concentration of 2.times.10.sup.6 cells per ml in cell culture
medium consisting of RPMI 1640 (Life Technologies, Paisley, UK)
supplemented with 10% (wt/vol) human AB-serum (Boehringer
Ingelheim, Ingelheim, Germany), 1 mM L-alanyl-glutamine (Life
Technologies), 100 U/ml penicillin/streptomycin (Life
Technologies), 0.05 mM 2-mercaptoethanol (Life Technologies) and 1
mM sodium-pyruvate (Life Technologies). 12.5 ml of the cell
suspension were place in 100.times.20 mm tissue culture dishes
(Sarstedt, Newton, Mass.) and FLU 58-66 peptide (Neosystems,
Strasbourg, France) was added to a final concentration of 1 .mu.M,
purified influenza A virus preparation (Biodesign, Kennebunk, Me.)
was added to a final concentration of .mu.g/ml, rTT.C (Boehringer
Mannheim, Mannheim, Germany) was added to a final concentration of
7 .mu.g/ml and purified TT (Statens Serum Institut, Copenhagen,
Denmark) was added to a final concentration of 1 .mu.g/ml. Cells
were incubated at 37.degree. C. in a humidified 7.5% CO.sub.2
atmosphere for 5-10 h.
[0173] Capturing of Secreted Cytokines by Cellular Affinity
Matrices
[0174] Ab-Ab conjugates directed against CD45 and either IL-4 or
IFN-.gamma.were produced by standard protein coupling techniques.
Aslam et al. (1998) Bioconjugation, Macmillan Reference Ltd.,
London. After the ex vivo stimulation, cells were harvested using a
disposable cell scraper (Costar, Cambridge, Mass.) and labeled for
7 min at a cell concentration of 10.sup.8 cells per ml in ice-cold
medium with 50 .mu.g per ml of the Ab-Ab conjugates. Then, cells
were diluted with medium to a final cell concentration of
2.times.10.sup.6 cells per ml and allowed to secrete for 45 min at
37.degree. C. in a humidified 7.5% CO.sub.2 atmosphere.
[0175] Magnetic Enrichment and Detection of Cytokine Secreting
Cells
[0176] After the cytokine capturing period, cells were harvested
again, resuspended at a cell concentration of 10.sup.8 cells per ml
in phosphate-buffered saline containing 0.5% (w/v) bovine serum
albumin and 5 mM EDTA (buffer) and stained for 10 min at +4.degree.
C. with 5 .mu.g/ml anti IFN-.gamma.-PE or anti IL-4-PE,
respectively. Cells were washed with buffer (300.times.g, 10 min),
resuspended in 400 .mu.l buffer and magnetically labeled for 15 min
at +4.degree. C. with 100 .mu.l anti PE Ab-microbeads (Miltenyi
Biotec, Bergisch, Gladbach, Germany). After washing, the cells were
applied onto a MS+column and placed in a MiniMACS magnet (Miltenyi
Biotech). The column was rinsed with buffer and the retained cells
were eluted from the column after removing it from the magnetic
field to achieve a higher enrichment rate, the eluted cells from
the first column were applied to another MS+column and the magnetic
separation was repeated. Cell samples were analyzed on a
FACScalibur flow cytometer (Becton Dickinson, San Jose, Calif.)
using the CellQuest software package.
[0177] Magnetic Enrichment and Detection of Cytokine Secreting
Cells
[0178] For detection, enumeration and phenotyping of
cytokine-secreting cells the following reagents were used: anti
IFN-.gamma.-CD45 (anti IFN-.gamma., clone 4SB3; CD45, clone 5B1, W.
Knapp, Vienna, Austria), anti IFN-.gamma.-PE (clone 45-15), anti
IL-4-CD45 (anti IL-4, clone 1 A6-10; CD45, clone 5B1, W. Knapp
Vienna, Austria), anti IL-4-PE (clone 7A3-3), CD8-Cy5 (clone
BM135/80, Behring Diagnostics, Marburg, Germany), CD4-Cy5 (clone
M-T321, Behring), CD4-FITC (clone SK3, Becton Dickinson), CD27-FITC
(clone M-T271, Pharmingen, San Diego, Calif.), CD28-FITC (clone
CD28.2, Pharmingen) CD57-FITC (clone HNK-1, Becton Dickinson), anti
V.beta.17.FITC (clone E17.5F3.15.13, Coulter-Immunotech, Marseille,
France). Meager et al. (1984) Interferon Res. 4:619-625; Alkan et
al. (1994) J. Immunoassay 15:217-225; and Bird et al. (1991)
Cytokine 3:562-567.
[0179] Cytolytic Activity Assay
[0180] The cytotoxic activity of enriched cytokine-secreting cells
was analyzed using a flow cytometry-based assay which has been
described previously. Mattis et al. (1997) J. Immunol. Met.
204:135-142. Briefly, 1.times.10.sup.6 HLA-A2.1.sup.+ T2 cells were
labeled with 4 .mu.g per ml of the green fluorescent dye DiO
(Molecular Probes, Eugene, Oreg.) in phosphate-buffered saline
containing 5 mM EDTA and 3% fetal calf serum for 45 min at
37.degree. C. Cells were washed three times with buffer,
resuspended in cell culture medium and loaded with 1 .mu.M Flu
58-66 peptide or Melan A/MART 1 27-35 peptide (Bachem, Heidelberg,
Germany) overnight at 37.degree. C. in a humidified 7.5% CO.sub.2
atmosphere. Enriched cytokine-secreting cells were expanded for 18
d in tissue culture in the presence of recombinant human IL-2
(Peprotech, London, U.K.). Expanded cytokine-secreting cells and
peptide-loaded DiO-labeled HLA-A2.1.sup.+ T2 cells were
co-cultivated for 16 h at a ratio of 1:1 at 37.degree. C. in a
humidified 7.5% CO.sub.2 atmosphere. After the culture period,
cells were harvested and analyzed by flow cytometry. In order to
permit discrimination between live and dead DiO-labeled T2 cells,
samples were counterstained with the red fluorescent exclusion dye
propidium iodide.
EXAMPLE 4
[0181] The capability to secrete effector cytokines like
IFN-.gamma. following short-term antigenic restimulation with
synthetic peptide- or native antigen-pulsed APCs is a typical
feature of memory/effector CD4.sup.+ (Th1-type) and CD8.sup.+ T
cells. Salmon et al. (1989) J. Immunol. 143:907-912; and Hamaan et
al. (1997) 186:1407-1418. To isolate low-frequency memory/effector
antigen-specific CD4.sup.+ and CD8.sup.+ T cells directly from
peripheral blood based on antigen-induced secretion of IFN-.gamma.
and cellular affinity matrix technology, peripheral blood
mononuclear cells (PBMC) from HLA-matched adult healthy blood
donors were stimulated for 5-6 h with: (a) the HLA-A0201-restricted
FLU peptide 58-66, (b) a purified influenza A virus preparation and
(c) rTT.C. After the stimulation period, an affinity matrix for
IFN-.gamma. was created on the cell surface using antibody (Ab)-Ab
conjugates directed against CD45 and IFN-.gamma., and the cells
were allowed to secrete IFN-.gamma. in culture for 45 min. Then,
IFN-.gamma., relocated to the affinity matrix of the secreting
cells, was stained with a phycoerythrin (PE)-conjugated
IFN-.gamma.-specific Ab, and PE-labeled cells were enriched by MACS
using anti PE Ab microbeads. See, also, Brosterhus et al., 10th
Int. Congress in Immunology, New Delhi, India, 1-6 Nov. 1998, pp.
1469-1473.
[0182] Compared with the non-stimulated control samples, a
significantly higher proportion of IFN-.gamma.-secreting CD8.sup.+
cells were detectable after enrichment in the FLU 58-66
peptide-stimulated sample (FIG. 3A: 38.3% vs. 13.7%), and
significantly higher proportions of IFN-.gamma.-secreting CD4.sup.+
cells were detectable after enrichment in the samples stimulated
with the influenza A virus preparation (FIG. 3B: 35.5% vs. 1.1%)
and rTT.C (FIG. 3C: 6.1% vs. 0.3%), respectively. When looking at
the absolute numbers of enriched IFN-.gamma.-secreting T cells and
their frequencies among total PBMC, differences between the
stimulated and non-stimulated samples are even more remarkable: (a)
12,500 IFN-.gamma.-secreting CD8.sup.+ T cells were isolated from
5.3.times.10.sup.7 FLU 58-66 peptide-stimulated PBMC (frequency 1
in 4,200) and 1370 IFN-.gamma.-secreting CD8.sup.+ T cells were
isolated from 5.1.times.10.sup.7 non-stimulated PBMC (frequency: 1
in 37,000); (b) 351 IFN-.gamma.-secreting CD4.sup.+ T cells were
isolated from 5.times.10.sup.6 influenza A virus-stimulated PBMC
(frequency 1 in 14,000) and 4 IFN-.gamma.-secreting CD4.sup.+ T
cells were isolated from 5.0.times.10.sup.6 non-stimulated PBMC
(frequency 1 in 1,250,000); and (c) 132 IFN-.gamma.-secreting
CD4.sup.+ T cells were isolated from 1.8.times.10.sup.7
rTT.C-stimulated PBMC (frequency: 1 in 136,000) and 7
IFN-.gamma.-secreting CD4.sup.+ T cells were isolated from
1.9.times.10.sup.7 non-stimulated PBMC (frequency: .about.1 in
2,710,000). Considering these experimental results, it is evident
that IFN-.gamma.-secreting T cells present at frequencies of below
10.sup.-6 can be detected with our technique.
EXAMPLE 5
[0183] Both memory-and effector-type CD8.sup.+ T cells are capable
of secreting IFN-.gamma.. Hamann et al. (1997). To determine the
phenotype of FLU 58-66 peptide-specific CD8.sup.+ T cells, enriched
IFN-.gamma.-secreting CD8.sup.+ T cells from the FLU 58-66
peptide-stimulated sample and the control sample were analyzed by
three-color immunofluorescence for the expression of a panel of
leukocyte surface markers that allow to distinguish between memory
and effector-type CD8.sup.+ T cells. Hamann et al. As shown in FIG.
2, most FLU 58-66 peptide-specific CD8.sup.+ T cells were (1997)
CD27.sup.+, CD28.sup.+ and CD57.sup.-, consistent with a memory
phenotype, whereas most of the IFN-.gamma.-secreting CD8.sup.+ T
which became isolated independent of the FLU 58-66 peptide were
CD27.sup.-, CD28.sup.-, CD57.sup.+, consistent with an effector
phenotype. The latter could have been induced in vivo to secrete
IFN-.gamma. and thus might reflect ongoing immune responses.
[0184] More than 54.8% of the IFN-.gamma.-secreting CD8.sup.+ T
cells from the FLU 58-66 peptide-stimulated sample expressed the
V.beta.17 TCR chain, compared with less than 2.2% of the
IFN-.gamma.-secreting CD8.sup.+ T cells from the control sample
(FIG. 4). This confirms previous reports showing a bias of
HLA-A0201-restricted FLU peptide 58-66-specific CD8.sup.+ T cells
towards the use of V.beta.17 TCR chain, first in cloned CTLs and
later, using fluorescent tetramers of FLU 58-66 peptide-loaded
HLA-A2.1 molecules, also in PBMC. Lehner et al. (1995) J. Exp. Med.
181:79-91; and Dunbar et al. (1998).
EXAMPLE 6
[0185] To further confirm the specificity of the enriched
IFN-.gamma.-secreting CD8.sup.+ T cells from the FLU 58-66
peptide-stimulated PBMC, and to study their cytolytic activity, the
cells were expanded for 18 d in tissue culture in the presence of
IL-2, and then assayed for CTL activity at an effector: target
ratio of 1:1. As shown in FIG. 5, significant killing was observed
when target cells were loaded with FLU 58-66 peptide, but not when
target cells were loaded with a control peptide (Melan A/MART 1
27-35).
EXAMPLE 7
[0186] PBMC from 49 HLA-A2+ individuals were cultured with or
without the FLU 58-66 peptide and subjected to the enrichment
procedure for IFN-.gamma.-secreting cells as described in Example
3. In 45 cases, on average about 80-fold more IFN-.gamma.-secreting
CD8.sup.+ T cells were isolated from the FLU 58-66
peptide-stimulated sample as compared to the control sample. Only
in three cases, no significant difference was detected between both
samples. The median frequency of FLU 58-66 peptide-specific
CD8.sup.+ T cells among PBMC, as determined by subtracting the
frequencies of the control samples from the frequencies of the FLU
58-66 peptide-stimulated samples, was 1 in 30,000 (range between 1
in 600,000 and 1 in 1000). These results are completely consistent
with previous reports in which the frequencies of FLU 58-66
peptide-specific CD8.sup.+ T cells were determined using
enzyme-linked immunospot (ELISPOT) assays for single cell
IFN-.gamma. release or tetramers of FLU 58-56 peptide-loaded
HLA-A2.1 molecules. Lalvani et al. (1997; and Dunbar et al.
(1998).
EXAMPLE 8
[0187] To demonstrate that our approach isolates live
antigen-specific Th2-type CD4.sup.+ T cells, PBMC were stimulated
with purified TT and IL-4-secreting CD4.sup.+ T Cells were isolated
using an Ab-Ab conjugate directed against CD45 and IL-4. After 10 h
of TT stimulation, 150 IL-4-secreting CD4.sup.+ T cells could be
isolated from 2.2.times.10.sup.7 PBMC with a purity of 6,89% (FIG.
6). This corresponds to a frequency of TT-specific Th2 cells among
total CD4.sup.+ T cells of 1 in 94,000. The frequency of
IL-4-secreting CD4.sup.+ T Cells in the control culture without TT
was about 10 times lower.
[0188] All references cited herein, both supra and infra are hereby
incorporated herein. Although the foregoing invention has been
described in some detail by way of illustration and example for
purposes of clarity and understanding, it will be apparent to those
skilled in the art that certain changes and modifications can be
practiced. Therefore, the description and examples should not be
construed as limiting the scope of the invention, which is
delineated by the appended claims.
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