U.S. patent application number 11/185273 was filed with the patent office on 2005-12-22 for t cell activation.
This patent application is currently assigned to Chiron S.p.A.. Invention is credited to Abrignani, Sergio.
Application Number | 20050281793 11/185273 |
Document ID | / |
Family ID | 10760006 |
Filed Date | 2005-12-22 |
United States Patent
Application |
20050281793 |
Kind Code |
A1 |
Abrignani, Sergio |
December 22, 2005 |
T cell activation
Abstract
A method for antigen independent activation of T cells
comprising contacting T cells with a combination of cytokines such
as two or more of interleukin-2, interleukin-6 and tumor necrosis
factor .alpha..
Inventors: |
Abrignani, Sergio;
(Vagliagli, IT) |
Correspondence
Address: |
Chiron Corporation
Intellectual Property - R440
P.O. Box 8097
Emeryville
CA
94662-8097
US
|
Assignee: |
Chiron S.p.A.
Siena
IT
1-53100
|
Family ID: |
10760006 |
Appl. No.: |
11/185273 |
Filed: |
July 19, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11185273 |
Jul 19, 2005 |
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09520248 |
Mar 7, 2000 |
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09520248 |
Mar 7, 2000 |
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08776259 |
Jan 21, 1997 |
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6074635 |
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08776259 |
Jan 21, 1997 |
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PCT/IB95/00691 |
Aug 17, 1995 |
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Current U.S.
Class: |
424/93.71 ;
424/85.1; 424/85.2 |
Current CPC
Class: |
C12N 5/0636 20130101;
A61P 31/18 20180101; A61P 31/04 20180101; A61P 37/04 20180101; A61K
38/2013 20130101; C12N 2501/25 20130101; C07K 14/52 20130101; A61P
31/00 20180101; A61K 2300/00 20130101; C12N 2501/23 20130101; A61P
31/12 20180101; A61K 2035/124 20130101; A61K 38/2013 20130101; A61P
33/00 20180101; A61P 43/00 20180101 |
Class at
Publication: |
424/093.71 ;
424/085.1; 424/085.2 |
International
Class: |
A61K 045/00; A61K
038/19; A61K 038/20 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 17, 1994 |
GB |
9416657.6 |
Claims
1. A method for activating T cells, in vivo, comprising contacting
T cells independent of antigen with a combination of interleukin-2,
interleukin-6, and tumor necrosis factor alpha, or functionally
equivalent fragments thereof.
2. The method of claim 1, wherein the T cells are naive T cells
and/or memory resting T cells.
3. The method of claim 1, wherein the T cells are naive CD45RA+
cells and/or memory resting CD45RO+ cells.
4. The method of any of the preceding claims, wherein the
activation of the T cells in vivo leads to an enhanced
immunological response.
5. A method of therapy comprising activating in a human or animal
subject T cells using the method of claim 4.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/520,248, filed Mar. 7, 2000, which is a
continuation of U.S. patent application Ser. No. 08/776,259, filed
Jan. 21, 1997, which is a 371 National Stage application of
International Patent Application No. PCT/IB95/00691, filed Aug. 17,
1995, which claims priority to Great Britain Patent Applicatio No.
GB 9416657.6, filed Aug. 17, 1994, all of which are incorporated
herein in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to an antigen independent
method for the activation of T cells. The invention also relates to
a method for increasing lymphokine production in a T cell culture
and a method for increasing the immune response at specific sites
in vivo which has therapeutic applications in the treatment of
disease.
BACKGROUND TO THE INVENTION
[0003] T cells are involved in the immune response and are
primarily involved in cellular immunity, such as guarding against
virally infected cells, fungi, parasites and foreign tissue.
[0004] Briefly, T cells are activated by binding to
antigen-displaying macrophages. However, the T cell receptor must
specifically complex with the antigen and a Major
Histocompatibility Complex (MHC) protein displayed on the surface
of the macrophage.
[0005] The binding induces the macrophage to release interleukin-1,
a polypeptide growth factor, which stimulates the bound T cell to
proliferate and differentiate. This proliferation and
differentiation is enhanced by the T cells autostimulatory
secretion interleukin-2. The T cell can differentiate into a number
of different phenotypes, such as cytotoxic T cells which are
specifically targeted to antigen displaying host cells and are
capable of lysing the cell, helper T cells which are involved in
activating cytotoxic T cells and in co-operating with B cells to
produce antibodies and memory T cells which upon re-encountering
their cognate antigen proliferate at a faster rate than non-memory
T cells.
[0006] It will be apparent to one skilled in the art that the
activation of T cells is an important step in the immunological
response. By manipulating the activation of T cells it will be
possible to obtain useful immunological products and develop more
efficient treatment techniques.
[0007] Previously, to achieve T cell activation, a macrophage
displaying an antigen and an MHC protein was required. A number of
problems and drawbacks are associated with this, a major drawback
being that only T cells specific for the antigen are activated.
Other T cells not specific for the antigen remain unactivated.
Other problems may arise if the desired antigen is difficult to
obtain or hazardous to work with. Additionally, if an antigen is
used in cell culture to achieve activation and it is not easy to
remove, contamination problems may occur.
[0008] The same problems will occur in vivo and it is obviously
undesirable to infect an individual with an antigenic
substance.
[0009] By achieving antigen independent T cell activation it will
be possible to activate a population of T cells without the need to
isolate and display an antigen on the surface of macrophage.
[0010] It is known that interleukin-2 is potent T-lymphocyte growt
enhancer and the use of interleukin-2 as an adjuvant h been
described. In this role interleukin-2 was thought function as an
expander of the population of alrea activated T-lymphocytes.
However, it was not known that interleukin-2 (in combination with
other cytokines) coul act specifically to activate T-lymphocytes in
an antige independent manner.
SUMMARY OF THE INVENTION
[0011] According to the present invention there is provided a
method for antigen independent activation of T cells comprising
contacting T cells with a combination of cytokines.
[0012] Preferably, the T cells are contacted with at least two of
the following:
[0013] i) interleukin-2;
[0014] ii) interleukin-6; and
[0015] iii) tumour necrosis factor .alpha.
[0016] or functionally equivalent fragments thereof.
[0017] The T cells may be naive T cells and/or memory resting T
cells, most suitably naive CD45RA.sup.+ cells and/or memory resting
CD45RO.sup.+ cells.
[0018] Suitably, the concentration of interleukin-2 is from 100 to
400 U/ml, the concentration of interleukin-6 is from 400 to 600
U/ml and the concentration of tumour necrosis factor .alpha. is
from 15 to 35 ng/ml. More preferably, the concentration of
interleukin-2 is from 200 to 300 U/ml, the concentration of
interleukin-6 is about 500 U/ml and the concentration of tumour
necrosis factor .alpha. is about 25 ng/ml.
[0019] The T cells may be activated in vitro, for example, in a
method for obtaining increased lymphokine production from a T cell
culture, comprising activating the T cells according to the
invention.
[0020] The T cells wherein T cells may be activated in vivo,
leading to an enhanced immunological response which may be used in
a method of therapy comprising activating in a human or animal
subject T cells using the method according to the invention.
[0021] In this aspect of the invention, the combination of
cytokines acts as an adjuvant enhancing the T-cell response and
thereby enhancing the immune response.
[0022] T cells can be activated to produce desirable lymphokines
useful in cell-mediated immune responses, such as interleukins,
interferons and colony stimulating factors, without the problems
associated with antigen dependent activation.
[0023] Additionally, it will be possible to achieve isolated T cell
activation and effector T cell recruitment in areas of specific
immunological interest without the use of antigens. This will thus
be extremely useful for the in vivo treatment of numerous diseases
and infections such as HIV and Hepatitis.
[0024] The present invention has the advantages of activating
"by-stander" T cells, not just specifically one particular
stimulating antigen, thus a bigger immune response is produced
leading to the production of more lymphokines and subsequently
greater immunoglobulin production by B cells.
[0025] Another advantage of the present invention is the
maintenance of the peripheral pool of memory T cells as memory T
cells can be expanded (proliferated) without the need of specific
antigenic stimulation to maintain the clonal size. Also the naive T
cell repertoire can be maintained, as the present invention allows
the proliferation of naive T cells without them switching to the
memory phenotype, unlike in antigenic stimulation.
[0026] According to a further aspect of the invention there is
provided a pharmaceutical composition comprising two or more of the
following:
[0027] i) interleukin-2:
[0028] ii) interleukin-6; and
[0029] iii) tumour necrosis factor .alpha.
[0030] or functionally equivalent fragments thereof optionally in
association with one or more pharmaceutically acceptable
excipients.
[0031] The pharmaceutical composition may itself be useful for the
therapeutic activation of T-cells or may be administered with a
further therapeutic agent such as a vaccine. Administration may be
simultaneous or sequential.
[0032] According to the present invention there is provided a
method of gene therapy comprising the step of administering a
vector carrying a genes encoding two or more of
[0033] i) interleukin-2;
[0034] ii) interleukin-6; and
[0035] iii) tumour necrosis factor .alpha.
[0036] or functionally equivalent fragments thereof.
[0037] Suitable such vectors are well known in the art.
[0038] According to a further aspect of the invention, there is
provided a combined method of therapy comprising coadministration
of a vector carrying a gene encoding one or more of
[0039] i) interleukin-2;
[0040] ii) interleukin-6; and
[0041] iii) tumour necrosis factor .alpha.
[0042] or functionally equivalent fragments thereof
[0043] and one or more of
[0044] i) interleukin-2;
[0045] ii) interleukin-6; and
[0046] iii) tumour necrosis factor .alpha.
[0047] proteins or functionally equivalent fragments thereof.
[0048] Such maintenance of specific T cell types is extremely
advantageous when working with T cell cultures.
[0049] Many other uses and advantages can be seen for the present
invention and such uses and advantages would be apparent to one
skilled in the art.
BRIEF DESCRIPTION OF THE DRAWINGS
[0050] FIG. 1. Phenotypic and cell cycle analysis of purified
CD4.sup.+ resting T cells. (A) forward and side scatter profile.
(B) Cell cycle analysis. (C) FITC- or PE-conjugated control
antibodies. (D-F) Purity of CD4.sup.+ cells and expression of
activation markers. (G) Expression of CD45RA and CD45RO Ags on
sorted CD4.sup.+ cells. (H and I) CD4.sup.+ cells purified as
CD45RO.sup.+ or CD45RA.sup.+ subpopulations.
[0051] FIG. 2. Activation of resting CD4.sup.+ T cells by soluble
factors. (A and B) Expression of activation markers on resting T
cells cultured with supernatant from T cell clones cultured with
autologous macrophages prepulsed with Ag (hatched bars) or medium
(solid bars), or rIL-2 (open bars). Expression of CD69 or CD25 was
analyzed in double staining with anti-CD4. (C) [.sup.3H]Thymidine
incorporation of the same cells in A and B, cultured with medium
alone (triangles), rIL-2 (squares), or supernatant from a T cell
clone cultured with macrophages prepulsed with Ag (closed circle)
or medium (open circle). (D) [.sup.3H]Thymidine incorporation of
resting CD45RO.sup.+ (squares) or CD45RA.sup.+ (circles) T cells in
the presence of different concentration of IL-2 plus 1 .mu.g/ml LPS
(open symbols), or IL-2 with supernatant from LPS-activated
macrophages (closed symbols).
[0052] FIG. 3. Combination of IL-2, TNF-.alpha., and IL-6 activates
resting T cells. CD45RO.sup.+ (A) or CD45RA.sup.+ (B) resting T
cells were cultured for 8 d with various combinations of the
following: rIL-2, rIL-6, TNF-.alpha., and supernatant from
LPS-stimulated macrophages. Thymidine incorporation and CD69
expression were measured as described in FIG. 1. (C) Cell cycle
analysis of resting CD45RO.sup.+ (squares) or CD45RA.sup.+
(circles) T cells in the presence of IL-2 alone (open symbols) or
in combination with TNF-.alpha. and IL-6 (closed symbols).
[0053] FIG. 4. CD45RA.sup.+ T cells activated by cytokines do not
switch their phenotype to CD45RO. CD45RA.sup.+ T cells were
activated by combination of IL-2, TNF-.alpha., and IL-6, and after
23 days were double stained with anti-CD45RA-FITC and
anti-CD45RO-PE antibodies.
[0054] FIG. 5. Expression of IFNc and IL-4 mRNA by
cytokine-activated T cells. Purified CD4.sup.+ CD45RO.sup.+ resting
T cells are cultured with IL-2 alone for 60 (lane 1) and 100 h
(lane 3) or with IL-2, TNF-.alpha., and IL-6 for 60 (lane 2) and
100 h (lane 4) as described in Materials and Methods. (Lane 5)
Positive template; (lane 6) negative control.
[0055] FIG. 6. Frequency of resting T cells that grow in response
to cytokine combination. CD45RO.sup.+ resting T cells were plated
in the presence of purified autologous macrophages, anti-DR mAb
with IL-2 alone (closed circles) or in combination (open circles)
with TNF-.alpha. and IL-6. (Dotted lines) 95% confidence
limits.
DETAILED DESCRIPTION OF EMBODIMENT
[0056] Materials and Methods
[0057] Purification of Resting T Cells. After Ficoll-Hypaque
(pharmacia) separation of PBMC from buffy coats of healthy donors,
most macrophages were removed by plastic adherence. To obtain a
pure resting CD4.sup.+ T cell population, cells were incubated with
a cocktail of mAbs against HLA-DR (L-243; American Type Culture
Collection [ATCC], Rockville, Md.), CD19 (4GT), CD16 (B73.1), CD56
(MY31), CD57 (HNK-1, ATCC), CD8 (OKT8, ATCC), CD11b (OKM-1, ATCC),
CD14 (MO-P9), TCR-c/.delta. (B1, a gift of G. De Libero, ZLF Basel,
Switzerland), CD25 (2A3), CD69 (L78), and CD71 (L01.1). After
30-min incubation on ice, cells were washed twice and incubated
with magnetic beads (Dynabeads; Dynal, Oslo, Norway) conjugated
with goat anti-mouse IgG and rat anti-mouse IgM, at a 1:4
target/bead ratio. After 30-min incubation, bead-bound cells were
removed using rare earth magnet (Advanced Magnetics, Inc.,
Cambridge, Mass.). Remaining cells were further purified with four
more incubations with beads at increasing target/bead ratios (1:10
to 1:100). Final population was used as a source of resting
CD4.sup.+ T cells when >99.3% of the population was
TCR.alpha./.beta..sup.+ (WT/31) and CD4.sup.+ (Leu 3a), as
determined by immunofluorescence analyses using a FACScan.RTM. flow
cytometer (Becton Dickinson & Co., Mountain View, Calif.), and
fulfilled the following criteria; (a) small size at the FACS.RTM.
scatter; (b) absence of FACS-detectable levels of the activation
markers (CD69, CD71, MHC-DR and IL-2 receptor p55 chain (CD25); (c)
absence of cells in the S and G.sub.2/M parts of the cell cycle;
and (d) no significant incorporation of [.sup.3H]thymidine when
exposed to IL-2. In some experiments resting cells were further
negatively sorted as CD45RO.sup.- (adding the mAB UCHl-1) or
CD45RA.sup.- (adding the mAB L48). If not otherwise indicated, all
the mAbs were from Becton Dickinson & Co.
[0058] Preparation of Supernatants. T cells (5.times.10.sup.3/ml)
from a tetanus toxoid (TT)-specific clone were cultured with
autologous macrophages (2.5.times.10.sup.8/ml) that had been
prepulsed with or without TT (3 .mu.g/ml) (Biocine Sclavo, Siena,
Italy). After 16 h, supernatants were collected and filtered with
0.2-.mu.m filters. Culture medium has been previously described (3)
using 5% human serum or plasma. Effective supernatants were
prepared using medium with either 5% human serum (from Florence
blood bank) or serum-free media (HL-1: Ventrex, Portland, Oreg.).
Similar results were obtained with resting T cells derived from
PBMC of six different healthy individuals and with supernatants
from activated CD4.sup.+ T cell clones, with different specificity
(purified protein derivatives [PPD] or pertussis toxin), from four
different persons (see FIG. 2 and data not shown).
[0059] Cell Cycle Analysis. This was performed as described (4)
using propidium iodide in combination with anti-CD4 mAb (FITC
labelled) staining. Analyses were performed with the FACScan.RTM.
Lysis II software and doublet discrimination program (Becton
Dickinson & Co.).
[0060] Purification of B Cells. PBMC-derived B cells were stained
with FITC-labelled anti-CD19 mAb and purified by positive sorting
with FACStar.RTM. (Becton Dickinson & Co). Purity was >98%
as determined by staining with anti-CD20 and anti-Ig.
[0061] Helper Assay. Noncognate helper assays were performed as
previously described (5). Briefly, purified autologous PBMC-derived
B cells (2.times.10.sup.3/well) were cocultured for 12 d with
CD4.sup.+CD45RO.sup.+ resting T cells (3.times.10.sup.4/well) in
the presence of cytokine combinations as described (see FIG. 3) or
on anti-CD3-coated plates. To avoid an effect of cytokines on B
cell differentiation, plates were washed after 4-d culture and
cytokine combinations were replaced with IL-2 alone. Ig in the
supernatants was measured by ELISA (5).
[0062] Activation of Resting T cells by Supernatants. Resting T
cells were cultured in 96-well flat-bottom plates
(5.times.10.sup.4/well) with supernatant (50% vol/vol) from T cell
clones cultured with autologous macrophages prepulsed with Ag,
medium or rIL-2 (Cetus Corp., Emeryville, Calif.) at a
concentration corresponding to that found in the T cell
supernatants (i.e. 200-300 U/ml). Activation was measured at
various time points as expression of CD69 and CD25 of
[.sup.3H]thymidine incorporation. In some experiments,
[.sup.3H]thymidine incoporation of resting CD45RO.sup.+ or
CD45RA.sup.+ T cells was measured in the presence of different
concentrations of IL-2 plus either 1 .mu.g/ml LPS (Difco, Detroit,
Mich.) or supernatant (50% vol/vol) from LPS-activated macrophages.
For the preparation of activated macrophage supernatant,
5.times.10.sup.8 macrophages were simulated with 1 .mu.g/ml LPS
(for 6-8 h). [.sup.3H]Thymidine incorporation experiments were
performed as described (5). The results represent the mean of
triplicate wells and SD was always 15%.
[0063] Activation of Resting T Cells by Recombinant Cytokines.
Resting T cells (5.times.10.sup.4/well) in 96-well flat-bottom
microplates were cultured for 8 d with various combinations of the
following rIL-2 (200-300 U/ml), rIL-6 (500 U/ml; Ciba-Geigy, Basel,
Switzerland; IL-6 units were determined with the B9 assay),
TNF-.alpha. (25 ng/ml; Genzyme Corp., Cambridge, Mass.), and
supernatant (50% vol/vol) from LPS-stimulated macrophages.
Thymidine incorporation and CD69 expression were measured as
described in FIG. 2. IL-1b (up to 100 ng/ml, Biocine Sclavo Siena,
Italy) in combination with IL-2 and TNF-.alpha. did not have any
activities (data not shown). Recombinant cytokines from two
different sources have been used with similar results. The optimal
concentration of cytokines was established in preliminary
dose-response experiments.
[0064] PCR-assisted mRNA Amplification. Purified resting CD4.sup.+
CD45RO.sup.+ cells were cultured with THF-.alpha. plus IL-6 plus
IL-2, or IL-2 alone. Total RNA was isolated after 60-100 h of
culture from 5.times.10.sup.8 cells, by RNAzol* B (Biotecx
Laboratories, Houston, Tex.). cDNA was synthesized with murine
reverse transcriptase as described (5). .beta.-actin, IL-4, and
IFN-c specific primer pairs were purchased from Clontech (Palo
Alto, Calif.). PCR was performed as described (5).
[0065] Limiting Dilution Analyses. CD45RO.sup.+ resting T cells
were plated at different numbers in Terasaki plates (64 wells per
condition) in 20 .mu.l vol in the presence of purified autologous
irradiated (2,500 rad) macrophages (3.times.10.sup.3/well) m
anti-DR mAb (L243, 20 .mu.g/ml) with IL-2 alone (300 U/ml) or in
combinations with TNF-.alpha. (25 ng/ml) and IL-6 (500 U/ml). On
day 14, cultures were visually inspected for growth. Randomly
selected growing wells were positively stained with anti-CD4 and
anti-TCR-.alpha./.beta. antibodies. Frequency analyses were done by
the least squared method (6).
[0066] Results and Discussion
[0067] A critical point of this study was to use a resting
population devoid of activated T cells that would respond to IL-2
alone. We chose to work with resting CD4.sup.+ T cells because, at
variant with some CD8.sup.+ or c/.delta. T cells with resting
phenotype, they do not express IL-2 receptor p75-chain in the
absence of the p55-chain (7), which may be responsible for unwanted
proliferation responses to IL-2 (8) and for which we did not have a
good antibody to sort out. We therefore performed multistep
exhaustive purifications to obtain highly purified resting
CD4.sup.+ T cells from PBMC (FIG. 1). In preliminary experiments,
resting CD4.sup.+ T cells were cultured with supernatants from
CD4.sup.+ T cell clones that had been activated with Ag-pulsed
macrophages. A representative experiment in FIG. 2 shows that a
fraction of resting CD4.sup.+ T cells is activated by the
supernatant, but not by IL-2, to express CD69 (9) (FIG. 2A) and
IL-2 receptor p55-chain (FIG. 2B), and to incorporate
[.sup.3H]thymidine (FIG. 2C).
[0068] Since the activating supernatant is produced by the
coculture of two cell types, we sought to determine the relative
contribution of soluble factors produced by T cells and APCs. For
this experiment, resting CD4.sup.+ T cells were further purified as
CD45RO.sup.+ (memory) and CD45RA.sup.+ (naive) subpopulation (10),
since they may have different activation requirements as already
reported for TCR-mediated activation (11, 12). FIG. 2D shows that
supernatant from LPS-activated macrophages alone, as IL-2 alone,
did not have any activity, whereas macrophage supernatant in
combination with IL-2 induced thymidine incorporation in both
CD45RA.sup.+ and CD45RO.sup.+ resting T cells. These results
demonstrate that IL-2 and soluble factor(s) produced by APcs are
required for the activation of resting T cells.
[0069] To identify the APC-derived factor(s), we tested the effect
of recombinant cytokines known to be produced by macrophages and to
have costimulatory activity on T cells, i.e., IL-1.beta., IL-6 and
TNF-.alpha. (13-15). In the absence of IL-2 all the possible
combinations of these cytokines did not show any activity over a
wide range of concentrations (data not shown). FIG. 3A shows that
TNF-.alpha. in combination with IL-2 induced resting CD45RO.sup.+ T
cells to express CD69 and to incorporate thymidine, whereas IL-6 in
combination with IL-2 was much less effective. Remarkably,
TNF-.alpha. and IL-6, in combination with IL-2, had a synergistic
effect leading to a stronger activation. A similar effect of IL-2,
IL-6, and TNF.alpha. was also observed on CD45RA.sup.+ resting T
cells (FIG. 38), although, in this case, ail three cytokines were
required to induce activation. Furthermore, the cell cycle analyses
in FIG. 3C show that at day 7 of culture 8% of both CD45RO.sup.+
and CD45RA.sup.+ T cells are in the S or G.sub.2/M phases of the
cell cycle. Activation of cytokines, measured as expression of
activation markers, thymidine incorporation, or entry into cell
cycle, was never inhibited by mAbs specific for DR, CD4, or CD3
(data not shown), thus confirming that TCR signalling is not
involved in this type of activation.
[0070] It is interesting to note that we have observed that
CD45RA.sup.+ T cells activated by cytokines do not switch their
phenotype to CD45RO, as was reported to occur within a few days
after TCR engagement (16). CD45RA.sup.+ T cells activated by
combination of IL-2, TNF-.alpha., and IL-6 were double stained with
anti-CD45RA and anti-CD45RO antibodies at 3-d intervals up to day
23 of culture. We never found single positive CD45RO.sup.+ cells at
any time point, and only found a few percent of double positive
CD45RA.sup.+high/CD45RO.sup.+dull. Indeed, FIG. 4 shows that naive
T cells even 23 d after cytokine activation, when most cells are
blastic and express CD69 (data not shown), are mainly CD45RA.sup.+.
The same cells activated with anti-CD3 switched in few days to the
CD45RO.sup.+ CD45RA.sup.- phenotype (data not shown).
[0071] We next asked whether resting T lymphocytes can be activated
by cytokines to display effector function. We performed
PCR-assisted mRNA amplification for lymphokines. FIG. 5 shows that
both IFN-c and IL-4 mRNA are expressed by CD45RO.sup.+ T cells
cultured with IL-2, TNF-.alpha., and IL-6, but not with IL-2 alone.
Moreover, CD45RO.sup.+ T cells activated by cytokine combination
are as effective as anti-CD3-stimulated T cells in helping B cells
to produce Ig (Table 1).
1TABLE 1 Resting CD45RO* T Cells Activated by Cytokines Can Provide
Help to B Cells IgG B cells cocultured with: IgM ng/ml IgA IL-2
plus TNF-.alpha. plus IL-6 <15 <5 <10 T cells plus medium
<15 <5 <10 T cells plus IL-2 <15 <5 <10 T cells
plus IL-2 plus TNF-.alpha. 32 23 <10 T cells plus IL-2 plus IL-6
<15 31 28 T cells plus IL-2 plus TNF-.alpha. 75 274 308 plus
IL-6 T cells plus anti-CD3 mAb 235 219 413 plus IL-2
[0072] To exclude the possibility that T cell help to B cells could
be due to activation of autoreactive cells, at the end of the
helper assay, the B cells were removed by sorting, and the
CD4.sup.+ T cells were tested in proliferation against autologous
purified B cells or macrophages. We never found any autoreactive
proliferation (data not shown).
[0073] Neither cytokines nor anti-CD3 induced CD45RA.sup.+ T cells
to produce IFN-c (<1 IU/ml) and to help B cells (data not
shown). Thus, we conclude that, similar to TCR-mediated activation
(17), cytokines recruit CD45RA.sup.+ T cells to proliferate but not
to help Ig production, whereas they activate resting CD45RO.sup.+ T
cells to proliferate and display effector functions.
[0074] To evaluate the frequency of resting T cells with memory
phenotype that could be stimulated by cytokines to grow, we
performed limiting dilution experiments. CD45RO.sup.+ CD4.sup.+
resting T cells were cultured with IL-2 alone or in combination
with TNF-.alpha. and IL-6, in the presence of autologous irradiated
macrophages and anti-DR antibodies to prevent autoreactive
responses. FIG. 6 shows that 1 of 33 resting CD45RO.sup.+ CD4.sup.+
T cells grew to a visible clone in response to IL-2, TNF-.alpha..
and IL-6. At present we do not know why only 3% of cells grew in
response to cytokines. The cells that proliferated could have been
a subset of resting T cells or could have been at a different stage
of maturation/activation. It is possible that many cells
(.apprxeq.20%) respond to cytokines and express activation markers.
Some of these cells will display effector functions and only a
minority (3%) will be able to grow in vitro to a clone of visible
size.
[0075] TNF-.alpha. and IL-6 both have been shown to upregulate
IL-2R expression on T cells (15, 18). This could be a possible
mechanism for the activation of resting T cells by this cytokine
combination. However, resting T cells cultured for 1-3 d with
TNF-.alpha. and IL-6, and washed and cultured for 4-5 d more with
IL-2, did not show FACS.RTM.-detectable levels of IL-2R (p55) (data
not shown), whereas IL-2R was expressed on .apprxeq.20% of the same
cells cultured with TNF-.alpha., IL-6, and IL-2 from the beginning
of the culture. This experiment, however, does not rule out the
possibility that low levels of IL-2R below the FACS.RTM.
sensitivity, are expressed and functionally relevant. Indeed, it
has been reported that Il-2 is required for induction of IL-2R by
TNF-.alpha. or IL-6 (19). Furthermore, IL-2 augments not only
expression of its own receptor (20) but also upregulates
THF-.alpha. receptor (21). Elucidation of the mechanism of
activation of resting T cells by cytokines will require additional
biochemical and molecular analyses.
[0076] This novel Ag-independent pathway of T cell activation may
play two important roles in vivo, by recruiting effector T cells at
the site of immune response and by maintaining the peripheral pool
of memory T cells. A scenario could be depicted where resting T
cells at sites of Ag-specific response are activated by cytokines
produced by specific T cells and macrophages to proliferate and to
secrete other lymphokines that can further amplify the response.
Indeed, the frequency of resting CD45RO.sup.+ T cells that respond
to cytokine combination is definitely higher than the usual
frequency of T cells primed by any known Ags.
[0077] It has been postulated that memory can be carried by
long-lived clones consisting of short-lived cells that require
repeated, intermittent stimulation by persisting Ag, by recurrent
infection, or by cross-reacting environmental Ags (22-24). In the
light of our results, it is tempting to speculate that memory T
cells may not require antigenic stimuli to maintain their clonal
size, since resting T cells with memory phenotype (CD45RO.sup.+)
can be expanded by cytokines secreted during responses to unrelated
antigens. On the other hand, cytokines can induce proliferation of
naive cells without switch to memory phenotype and may therefore
help to maintain the naive (CD45RA.sup.+) T cell repertoire.
[0078] It will be understood that the invention is described above
by way of example only and modifications within the scope and
spirit of the invention may be made.
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