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,
,
*
Department of Pathology,
Committee on Immunology, and
Department of Medicine, University of Chicago, Chicago, IL 60637
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and to proliferate. These results suggest that
CTLA-4 controls the threshold of productive TCR signaling. Biochemical
analysis comparing stimulated naive and primed TCR-transgenic cells
revealed no obvious differences in expression of total CTLA-4,
tyrosine-phosphorylated CTLA-4, and associated Src homology domain
2-containing protein tyrosine phosphatase. Thus, the biochemical
mechanism explaining the differential inhibitory effect of CTLA-4 on
naive and primed CD8+ T cells remains
unclear. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CTLA-4 is an alternative counter-receptor for CD80 and CD86 that is
expressed on T cells only after activation and binds these ligands with
higher avidity than does CD28 (3, 4). CTLA-4 expression is
induced transcriptionally upon TCR/CD3 ligation, but accumulates
predominantly as an intracellular pool in activated cells. A fraction
of CTLA-4 molecules is constantly transported to the surface and is
subsequently endocytosed by a clathrin-mediated mechanism (5, 6). In contrast to CD28, ligation of CTLA-4 appears to inhibit T
cell activation events (7). Most experimental models
analyzing CTLA-4 function have used stimulation of normal lymph node T
cells with specific mAbs against CD3 and CD28, with or without
antagonism by anti-CTLA-4 mAb (8, 9). Under these
conditions, CTLA-4 ligation inhibits IL-2 production and proliferation,
prevents sustained IL-2R induction, and induces arrest in the
G1 phase of the cell cycle. We have observed that
differentiated Th1 and Th2 clones are both susceptible to inhibition by
CTLA-4 (10). Cross-linking of CTLA-4 inhibited production
of all cytokines measured by both T cell subsets, suggesting that the
biochemical mechanism by which CTLA-4 antagonizes T cell activation
involves a signaling pathway central to multiple TCR-mediated
functions. Recent observations using transient transfection systems
suggest that CTLA-4 may mediate its inhibitory activity following
tyrosine phosphorylation by Src family kinases, binding and recruitment
of the Src homology domain 2-containing protein tyrosine phosphatase
(SHP-2),3 and subsequent
dephosphorylation of the CD3 
-chain (11, 12, 13). However,
this model has not been validated in normal T cells, and it has been
challenged by evidence that a T cell line lacking detectable SHP-2 is
still inhibited by CTLA-4 ligation and by the failure to demonstrate
direct interaction between CTLA-4 and SHP-2 proteins in vitro
(14). Moreover, transfection of a mutant CTLA-4 that
cannot be phosphorylated retains the ability to inhibit T cell
activation (15). It has also been reported that
extracellular signal-related kinase 1/extracelluar signal-related
kinase 2 and c-Jun N-terminal kinase (Jnk)1/Jnk2 activation can be
inhibited by CTLA-4 signaling in the absence of detectable effects on
CD3 phosphorylation (16). Thus, the early biochemical
events that mediate CTLA-4 inhibitory function remain
controversial.
Perhaps the best evidence that CTLA-4 is a negative regulator of T cell activation has come from the generation of CTLA-4-deficient mice, which exhibit a profound lymphoproliferative syndrome resulting in death of the animals within 3–5 wk of age (17, 18). However, the activated phenotype of T cells from these mice makes it difficult to study the role of CTLA-4 in controlling the activation of naive T cells. CTLA-4-deficient mice have also been intercrossed with TCR-transgenic mice, but endogenous TCR gene rearrangements generate activated T cells of other specificities that still can mediate autoimmunity and can confound the results being sought regarding naive T cells. In addition, the lymphoproliferation observed in CTLA-4-/- mice is eliminated upon in vivo depletion of CD4+ cells (19), raising questions concerning a potential role for CTLA-4 in modulating the activity of CD8+ T cells.
We sought to examine whether both naive and primed CD8+ cells could be inhibited by CTLA-4, whether CTLA-4 ligation decreased the number of responding cells or the magnitude of response per individual cell, and to seek biochemical evidence in normal T cells supporting the model of CTLA-4 phosphorylation and SHP-2 recruitment in delivery of an inhibitory signal. To this end, CTLA-4-deficient mice were intercrossed with 2C TCR-transgenic x recombinase-activating gene 2 (RAG2)-deficient mice (generating 2C/RAG2-/-/CTLA-4+/+ and 2C/RAG2-/-/CTLA-4-/- mice). This model system enabled functional and biochemical comparisons of naive vs primed CTLA-4-/- CD8+ T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2C TCR-transgenic mice were developed as described previously (20) and obtained originally from Dr. Dennis Loh (Washington University, St. Louis, MO). These were intercrossed with RAG2-deficient mice (kindly provided by Dr. Celeste Simon while at the University of Chicago) to obtain 2C/RAG2-/- mice (21). CTLA-4-deficient mice (generously provided by Dr. Craig Thompson while at the University of Chicago) were generated as described previously (17) and also were bred to homozygosity with RAG2-deficient mice. These were then bred with 2C/RAG2-/- mice to obtain 2C/RAG2-/-/CTLA-4+/+, 2C/RAG2-/-/CTLA-4+/-, or 2C/RAG2-/-/CTLA-4-/- mice. All transgenic mice were on a C57BL/6 x 129 (H-2b) background and were bred in a specific pathogen-free barrier facility at the University of Chicago. Mice were used at 6–8 wk of age for experiments.
Cell lines and transfectants
The DBA/2-derived mastocytoma P815 was cultured in DMEM supplemented with 10% FCS and incubated at 37°C in an 8% CO2 atmosphere. This P815 line lacks endogenous expression of B7-1 or B7-2, lacks expression of CD40, and fails to stimulate proliferation of purified naive 2C/RAG2-/- T cells in vitro. P815.B71 was generated and maintained as described (22). P1.HTR is a highly transfectable variant of P815 that grows semiadherently in tissue culture and as a solid tumor mass when implanted s.c. in vivo (23). This cell was used for immunization by in vivo tumor rejection. The C57BL/6-derived thymoma cell line EL4 also was cultured in DMEM with 10% FCS and was used as a syngeneic negative control stimulator cell.
Purification of CD8+ T cells
Splenic CD8+ cells were isolated by negative enrichment using a StemSep magnetic separation system (StemCell Technologies, Vancouver, Canada) according to the manufacturer’s protocol. The purity of the eluted fraction, determined by flow cytometry using the clonotypic Ab 1B2 or anti-CD8 mAb, ranged between 92 and 96%.
T cell thymidine incorporation assay
CD8+ T cells (3 x 104) were mixed with 3 x 104 mitomycin C-treated P815 or P815.B71 tumor cells in microtiter plates in a final volume of 200 µl. Cultures were pulsed at various times with 1 µCi of [3H]TdR/well and harvested 8 h later onto glass filters using a Packard 96-well plate harvester (Packard, Meriden, CT). Incorporated radioactivity was assessed using a Packard Top Count microplate scintillation counter (Packard).
In vivo priming of mice by tumor rejection
2C/RAG2-/-/CTLA-4+/+ or 2C/RAG2-/-/CTLA-4-/- mice were immunized s.c. in the left flank with 10 x 106 living P1.HTR tumor cells in 100 µl of sterile Dulbecco’s PBS. The animals were sacrificed 10 days after immunization, spleen cells were obtained, and CD8+ T cells were purified by negative enrichment, as described above. Following confirmation of purity by flow cytometry, cells were stimulated to assess proliferation and cytokine production.
Abs and flow cytometry
For flow cytometry, PE-coupled anti-CTLA-4 mAb and
anti-Thy1.2 mAb (BD PharMingen, San Diego, CA), FITC-coupled 1B2
(prepared in our laboratory), FITC-coupled anti-CD44 (BD
PharMingen), and biotinylated anti-CD28 and anti-CD8 mAbs (BD
PharMingen) were used. Expression of the TCR, CD44, CD28, and CD8 was
determined by normal surface staining, whereas CTLA-4 expression was
assessed after restimulation in vitro (see below) and following saponin
permeabilization, as described (6). Nonspecific surface
staining was reduced by blocking with the anti-FcR mAb 2.4G2.
Samples were analyzed on a Becton Dickinson FACScan instrument, and
data were analyzed using CellQuest software (Becton Dickinson, Mountain
View, CA). Live cells were selected for analysis using forward vs side
scatter gating.
For CFSE staining, CD8+ T cells were purified by negative enrichment as described above, and 5 x 106 cells were washed once and resuspended in Dulbecco’s PBS at a concentration of 2 x 107/ml. An equal volume of a CFSE (Molecular Probes, Eugene, OR) solution (5 µM in PBS) was added, and cells were incubated at room temperature for 8 min. The reaction was quenched by the addition of 500 µl of FCS. Cells were washed twice in complete medium, counted, and plated in 24-well plates (Costar, Cambridge, MA) at 1.8 x 105/well; each well then received 1.8 x 105 mitomycin C-treated P815.B71 cells in a total volume of 1.2 ml of complete medium. One well was harvested each day, and cells were surface-stained with PE-coupled anti-Thy1.2 mAb before analysis by flow cytometry.
Stimulation of T cells for lymphokine production and CTLA-4 expression
For lymphokine production, purified CD8+ T
cells (1.8 x 105) were stimulated with
mitomycin C-treated P815 or P815.B71 tumor cells (1.8 x
105) in a total volume of 1.2 ml. Supernatants
were collected at 24 h to determine IL-2 production and at 48
h to assess for IFN- content. To assess CTLA-4 expression, flow
cytometric analysis was performed at the indicated times on
permeabilized cells using the anti-CTLA-4 mAb 4F10.
Lymphokine assays and intracellular cytokine measurements
IFN- and IL-2 concentrations were measured by ELISA using Ab
pairs obtained from BD PharMingen. Concentrations are expressed in
U/ml, as determined using the respective recombinant cytokines as
standards. Intracellular IFN- levels were measured by flow cytometry
on permeabilized cells, as described (24).
Protein biochemistry
Naive or primed 2C/RAG2-/- T cells were stimulated for 48 h with P815.B71 cells, as described above, and purified by Ficoll-Hypaque centrifugation. Cells (20 x 106) were lysed in a buffer consisting of 0.5% Triton X-100, 50 mM Tris (pH 7.6), 50 mM NaCl, 5 mM EDTA, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 25 µM p-nitrophenyl p-guanidinobenzoate, and 1 mM PMSF. Lysates remained on ice for 20 min and were then centrifuged for 10 min at 14,000 x g. For analysis of whole-cell lysates, supernatants were immediately denatured in reducing sample buffer. For immunoprecipitation of CTLA-4, supernatants were incubated at 4°C for 1 h with protein G-agarose beads that had previously been coated with 4F10 (5 µg/ml). Immunoprecipitating beads were washed with lysis buffer, and the proteins were eluted by boiling in reducing sample buffer. Samples were electrophoresed on 14% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Blots were blocked with 5% BSA, incubated with either a combination of phospho-specific CTLA-4 Abs (1 µg/ml each in 5% milk buffer) (12) or a goat anti-CTLA-4 antiserum (Santa Cruz Biotechnology, Santa Cruz, CA; 1:1000 in TBST), and developed using the appropriate HRP-conjugated second step Ab and ECL substrate (Amersham Pharmacia Biotech, Piscataway, NJ). SHP-2 was detected using a specific rabbit antiserum (Upstate Biotechnology, Lake Placid, NY). All Western blots were exposed to film for varying lengths of time, and films generating subsaturating levels of intensity were selected for imaging.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Purified naive 2C/RAG2-/- T cells respond
well to P815.B71 cells in vitro, resulting in production of IL-2 and
IFN-, proliferation, and differentiation into effector CTL
(22). Use of the RAG2-deficient background extinguishes
expression of endogenous TCR genes and ensures a naive T cell surface
phenotype (22). 2C/RAG2-/- mice
also vigorously reject allogeneic P815 tumors in vivo, resulting in
priming of the majority of the T cells in the mouse based on surface
expression of CD44 and CD62L, as well as induction of cytolytic
activity (21, 25). Thus, immunization via tumor rejection
provides a convenient means by which to prime T cells from these
mice.
T cells were isolated from naive 2C/RAG2-/-/CTLA-4+/+, 2C/RAG2-/-/CTLA-4+/-, and 2C/RAG2-/-/CTLA-4-/- mice, and flow cytometric analysis was performed to determine expression of the TCR, CD28, CD8, and CTLA-4, as well as of the activation markers CD44 and CD62L. As expected, surface expression of the TCR, CD28, and CD8 were comparable on T cells from each of these mice, and the cells were reproducibly 95–99% CD44low and CD62Lhigh. The naive T cell phenotype in 2C/RAG2-/-/CTLA-4-/- mice is in keeping with the lack of overt autoimmunity and normal mouse life span observed (data not shown). In addition, total cellular CTLA-4 expression analyzed on permeabilized cells following stimulation for 48 h was detected on the CTLA-4+/+ T cells but not on the CTLA-4-/- T cells, with the 2C/RAG2-/-/CTLA-4+/- T cells showing an intermediate level of expression (data not shown).
Purified naive CD8+ T cells from
2C/RAG2-/-/CTLA-4+/+ or
CTLA-4-/- mice were stimulated with P815.B71
cells in vitro, and thymidine incorporation was measured over time.
Contrary to expectations, the CTLA-4-deficient cells did not exhibit an
augmented proliferative response compared with CTLA-4-expressing cells
(Fig. 1
A). In fact, thymidine
incorporation by the CTLA-4-/- cells was
reproducibly less than that of the CTLA-4+/+
cells, with a 5–30% reduction observed in various experiments.
Production of IL-2 by T cells from CTLA-4+/+ and
CTLA-4-/- mice also was comparable (Fig. 1B), as was IFN- secretion (data not shown). Thus, CTLA-4
does not appear to be a negative regulator during the initial
activation of naive CD8+ T cells in vitro.
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We reasoned that T cell priming might be necessary to reveal a
potential inhibitory effect of CTLA-4. To this end,
2C/RAG2-/-/CTLA-4+/+,
CTLA-4+/-, and CTLA-4-/-
mice were immunized with living P1.HTR tumor cells in vivo. Splenic
CD8+ T cells were isolated 10 days later and
analyzed by flow cytometry. As shown in Fig. 2, the majority of cells from
2C/RAG2-/-/CTLA-4+/+ and
CTLA-4-/- mice were primed as assessed by
increased CD44 expression, and comparable levels of clonotypic TCR also
were maintained. Similar results were observed for the
CTLA-4+/- T cells (data not shown). Each of
these cell types was then stimulated with P815 or P815.B71 cells, and
thymidine incorporation was measured over time. As shown in Fig. 3, A and B,
substantially greater proliferation of the
CTLA-4-/- cells compared with the
CTLA-4+/- or CTLA-4+/+
cells was observed following priming by immunization in vivo. In
addition, the difference between cell types was most striking when B7-1
was expressed on the stimulator cells. Thus, B7-1 costimulation
resulted in greater augmentation of proliferation of CTLA-4-deficient
than of CTLA-4-expressing T cells.
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C) and of IFN- (Fig. 3D) by
2C/RAG2-/-/CTLA-4-/- T
cells was augmented 
2-fold compared with that seen with the
CTLA-4+/- or CTLA-4+/+
cells. This difference was again seen when P815.B71, but not when P815,
cells were used as stimulators. Thus, the increased proliferation of
the CTLA-4-deficient T cells can be attributed to greater production of
the autocrine growth factor IL-2. Similar results were also seen when
the various T cell populations were primed with P815.B71 cells in vitro
(data not shown). Finally, it was conceivable that differences in proliferation between primed CTLA-4+/+ and CTLA-4-/- cells were due to less death in the CTLA-4-deficient population. However, this was not the case, as the number of live cells, dead cells, and total cells was increased proportionally in the CTLA-4-/- cell population (data not shown).
Single-cell analysis upon activation of primed CTLA-4+/+ and CTLA-4-/- cells
Increased responsiveness of the primed
CTLA-4-/- T cell population could result either
from a greater number of T cells being activated or from an increased
magnitude of response by individual cells. To address these
possibilities, three types of single-cell analyses were performed by
flow cytometry. One of the early measurable events that occurs when T
cells are stimulated is an increase in cell size. Examination of cell
size by forward light scatter following stimulation with P815.B71 cells
revealed that a greater proportion of primed
2C/RAG2-/-/CTLA-4-/- T
cells was undergoing blastogenesis at 24 h compared with primed
CTLA-4+/+ cells (73 vs 30%; Fig. 4A). Even 48 h after
activation, a fraction of CTLA-4+/+ T cells had
not been triggered to increase in size. In contrast to primed cells,
naive CTLA-4-/- T cells did not show this more
rapid and uniform cell size increase compared with naive
CTLA-4+/+ cells (data not shown).
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B, the rate of cell division for those cells that actually
divided was comparable between primed CTLA-4+/+
and CTLA-4-/- T cells, as reflected by the
number of cell divisions achieved by 48 h. However, a significant
fraction (16%) of the CTLA-4+/+ cells maintained
high CFSE staining, reflecting a failure to divide. Gating on this
nondividing population revealed that it corresponded to the fraction of
cells that had not increased in size by day 2 (data not shown). In
contrast to primed cells, naive CTLA-4-/- T
cells did not show this increased proportion of cells dividing when
compared with naive CTLA-4+/+ cells and, in fact,
lagged slightly behind (data not shown), consistent with the modest
reduction in thymidine incorporation observed (Fig. 1). Taken together,
these results suggest that in the absence of CTLA-4, a greater
proportion of the primed T cell population undergoes blastogenesis, DNA
synthesis (as measured by thymidine uptake), and cell division, with
more rapid kinetics.
In addition to recruiting a higher proportion of T cells to become
activated, it was possible that the absence of CTLA-4 also increased
the magnitude of response on an individual cell level. This hypothesis
was assessed by performing intracellular staining for IFN-
production by flow cytometry at various time points following
restimulation of primed T cells in vitro. As shown in Fig. 5A, there was no detectable
IFN- in T cells isolated from immunized
2C/RAG2-/-/CTLA-4+/+ or
CTLA-4-/- mice at baseline (time 0).
Interestingly, at each of the early time points tested, a greater
fraction of CTLA-4-deficient cells was producing IFN- compared with
the CTLA-4+/+ cells (0.9 vs 0.6% at 2 h,
7.2 vs 0.8% at 4 h, and 11.9 vs 2.9% at 7 h). In contrast,
the mean fluorescence intensity of the IFN--positive cell fraction
was similar between the CTLA-4+/+ and the
CTLA-4-/- cells at each of the various time
points (ranging from 29–36 fluorescence units). Thus, the 2-fold
increase in IFN- detected in the supernatants of the primed
CTLA-4-/- T cell population upon restimulation
in vitro (Fig. 3) can be accounted for by an increased number of cells
secreting IFN- at early time points after activation, rather than by
an increased quantity of IFN- being produced per cell.
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B, a stimulator:responder ratio of 0.1 was
required to begin to detect significant numbers (6.6%) of
IFN--positive cells in the CTLA-4+/+
population. In contrast, 10-fold fewer stimulator cells (at a ratio
of 0.01) were required to elicit IFN- staining from a significant
fraction (5.8%) of the CTLA-4-/- population.
Similar results were observed for intracellular IL-2 staining (data not
shown). Thus, absence of CTLA-4 appears to lower the activation
threshold of primed CD8+ T cells. In addition,
because the increased responsiveness of primed
2C/RAG2-/-/CTLA-4-/-
cells was observed only when B7-1 was expressed by the stimulator cells
(Fig. 3), this shift in activation threshold is not intrinsic to TCR
signaling but appears to depend upon B7-1 ligation of CTLA-4.
Proliferation in response to graded numbers of P815 vs P815.B71 cells
was also examined. As shown on Fig. 6, primed
2C/RAG2-/-/CTLA-4+/+ and
CTLA-4-/- cells proliferated comparably in
response to P815 cells (lacking B7 expression), again supporting the
notion that TCR signaling was comparable between these T cell
populations. However, the CTLA-4-/- T cells
demonstrated augmented proliferation in response to P815.B71 stimulator
cells, with the dose-response curve shifted substantially to the left.
In various experiments, 5- to 100-fold greater P815.B71 cells were
necessary to elicit a level of proliferation by
CTLA-4+/+ T cells that was comparable to that
seen by CTLA-4-/- cells. Thus, a shift in
activation threshold for proliferation was also observed in the absence
of CTLA-4.
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It was conceivable that a negative regulatory role for CTLA-4 was
manifest only with primed cells because of higher expression of CTLA-4
induced upon activation of primed T cells compared with that induced on
naive T cells. However, flow cytometric analysis on permeabilized cells
following stimulation in vitro with P815.B71 cells revealed comparable
expression of CTLA-4 on both the naive and primed populations, with a
similar biphasic kinetics of expression peaking between days 2 and 3
(Fig. 7).
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-chain of the CD3 complex
(13). It was conceivable that naive and primed
2C/RAG2-/- T cells differed in the degree of
CTLA-4 phosphorylation after stimulation or in expression of (or
association with) SHP-2. To examine tyrosine phosphorylation of CTLA-4
with precision, a rabbit antiserum was generated that selectively
recognizes tyrosine-phosphorylated, but not nonphosphorylated, CTLA-4,
as recently described (12). The specificity of this Ab was
verified using Jurkat cells transfected with murine CTLA-4, with or
without treatment of the cells with the tyrosine phosphatase inhibitor
pervanadate to increase the phosphotyrosine content of intracellular
proteins. As shown in Fig. 8A,
pervanadate-treated, but not untreated, Jurkat transfectants contained
CTLA-4 that was recognized by this Ab following immunoprecipitation
with the anti-murine CTLA-4 mAb 4F10, whereas both conditions
yielded comparable amounts of total murine CTLA-4. Western blot
analysis using phosphorylated vs nonphosphorylated CTLA-4 peptides
confirmed the specificity of this Ab only for phosphorylated CTLA-4
(data not shown). Naive and primed 2C/RAG2-/- T
cells were then stimulated for 48 h with P815.B71 cells to induce
peak levels of CTLA-4 expression, total CTLA-4 was immunoprecipitated,
and Western blot analysis was performed using the anti-phospho
CTLA-4 Ab or a goat anti-CTLA4 antiserum. In fact, naive and primed
2C/RAG2-/- T cells showed no obvious
differences with respect to expression of phospho-CTLA-4 (Fig. 8B) or of total CTLA-4 (Fig. 8C). Western blot
analysis also was performed on whole-cell lysates to examine expression
of SHP-2. As shown in Fig. 8D, both naive and primed
2C/RAG2-/- T cells expressed comparable levels
of SHP-2. Immunoprecipitation with anti-CTLA-4 Ab followed by
Western blot analysis for SHP-2 also revealed no obvious differences
between these cell populations (Fig. 8E). Thus, the
differential regulation of naive and primed 2C T cells by CTLA-4 cannot
apparently be explained by gross differences in expression of total
CTLA-4, phosphorylated CTLA-4, or levels of expression of SHP-2.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Teleologically, one could imagine that it is more critical to control the continuous reactivation of differentiated effector cells than it is to limit the earliest stages of activation of naive T cells through CTLA-4. In light of our current results, one could propose that if primed T cells encounter Ag and B7 within an inflamed tissue, or if they return to the lymph node via lymphatics and become restimulated with Ag and B7, their cytokine production and expansion would be dampened through ligation of CTLA-4 as it becomes expressed. Like T cell anergy (28), such a scenario would limit the ability of differentiated effector T cells to continually expand in vivo.
Because the present study has used a CD8+ TCR-transgenic mouse model, it does not address directly the potential ability of CTLA-4 to affect naive vs primed CD4+ T cells. It has been reported that the lymphoproliferative syndrome seen in CTLA-4-deficient mice could be eliminated when CD4+ T cells were depleted (19), raising the possibility that, for CD4+ T cells, the naive state may be susceptible to CTLA-4 regulation. Direct examination of CD4+ TCR-transgenic T cells having a naive surface phenotype also revealed modest inhibition by CTLA-4, although primed CD4+ T cells were still inhibited to a greater extent (29). Our current results, along with another recent report (27), do demonstrate that CTLA-4 negatively regulates primed CD8+ T cells. Taken together with the observation that cytokine production by differentiated CD4+ Th1 and Th2 clones is inhibited upon CTLA-4 ligation (10), it seems likely that CTLA-4 does indeed regulate the activation of both CD4+ and CD8+ primed T cells.
T cells have been shown to be capable of expressing B7 family molecules, and it has been suggested that altered glycosylation in T cells makes these ligands unable to bind CD28 while retaining some ability to bind CTLA-4 (30, 31). However, in our current study, the differences seen between CTLA-4-positive and CTLA-4-negative T cells were most striking when CD80 was expressed on the Ag-bearing stimulator cells. Thus, B7 molecules expressed by APCs rather than by T cells are likely to be the major limiters of T cell activation via CTLA-4 under physiologic conditions. However, a minor contribution of T cell-expressed B7 family members cannot be excluded.
Although CTLA-4-deficient 2C/RAG-/- T cells displayed increased thymidine incorporation compared with CTLA-4+/+ cells, they did not expand perpetually. Rather, as they reached peak proliferative responses, they subsequently quiesced, and cell death mechanisms appeared to be intact in both CTLA-4+/+ and CTLA-4-/- populations. Because primed 2C T cells are potently lytic, it seems likely that the limited proliferative response generally observed in vitro is secondary to elimination of the APCs, thus removing the antigenic stimulus and aborting IL-2 production. That being the case, the lymphoproliferative syndrome seen with conventional CTLA-4-deficient mice in vivo may rely either upon persistence of Ag or upon a survival signal present in vivo but lacking in the in vitro culture.
Cytokine production by T cells appears to be a quantal event, such that when a TCR-mediated activation threshold of a given T cell within a population is crossed, the response from that cell is "all or none" (32). Thus, conditions resulting in increased cytokine production by a given T cell population do so by increasing the fraction of T cells participating in the response. Ligation of CD28 appears to operate, in part, by lowering the activation threshold of T cells, allowing increased numbers of cells to respond to a given TCR stimulus (2). Our present results suggest that engagement of CTLA-4 has the opposite effect, decreasing the fraction of primed T cells among a population that respond, particularly at early time points following stimulation. This observation is consistent with the notion that CTLA-4 engagement raises the activation threshold of cells, countering indirectly the positive effect of CD28. This level of control may ensure that T cells bearing the highest affinity TCR, thus having the lowest intrinsic activation threshold, are selected for continued propagation upon repeated encounter with persistent Ag.
Because CTLA-4 can inhibit T cell activation even in the absence of CD28 (25, 33), it seems likely that CTLA-4 ligation actually antagonizes TCR-dependent signaling events. However, the biochemical basis for this inhibition is not completely understood. Cotransfection experiments have demonstrated that Src family kinases can mediate tyrosine phosphorylation of the cytoplasmic tail of CTLA-4 (12). Our present results demonstrate for the first time that CTLA-4 also becomes tyrosine-phosphorylated when normal T cells are stimulated with natural ligands on Ag-bearing APCs, supporting the physiologic relevance of this event. Phosphorylation could enable recruitment of molecules that initiate a negative signal capable of countering a positive signal delivered via the TCR complex. Alternatively, the rapid endocytosis that CTLA-4 undergoes may mediate sequestration of signaling molecules away from the TCR complex, thus limiting the extent of a positive signal.
The observation that primed T cells are preferentially inhibited through CTLA-4 suggests that biochemical differences in receptor-mediated signaling events exist between naive and previously activated T cells. It is conceivable that CTLA-4 delivers a particular signal only in primed T cells, which allows it to down-regulate T cell activation. Our present results suggest that the functional differences between naive and primed T cells are unlikely to be accounted for by differential phosphorylation of CTLA-4 or by differences in expression of or association with SHP-2. Although our biochemical experiments were not exhaustive and do not rule out the possibility of subtle contributions of these factors, several alternative hypotheses can be considered. It is possible that only a subpopulation of CTLA-4 molecules interacts functionally with the TCR complex to dephosphorylate CD3 components, and that this interaction occurs only in primed T cells. It is also possible that SHP-2 association with CTLA-4 is not sufficient to inhibit TCR signaling. Finally, it is worth considering that the difference in susceptibility to CTLA-4 inhibition between naive and primed T cells may not lie in a difference in CTLA-4 signaling, but rather in a distinction in TCR signaling such that a TCR-activated pathway that is only present in primed T cells is the principal one antagonized by CTLA-4. This possibility would be consistent with the recent observation that activation of the Jnk pathway is detectable in primed, but not naive, T cells (34). A better understanding of the biochemical events triggered by CTLA-4 and by the TCR complex in naive and primed T cells will be necessary to distinguish between these possibilities. Our results suggest that primed TCR-transgenic T cells will provide an ideal model to study signaling via CTLA-4.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Thomas F. Gajewski, Departments of Pathology and Medicine and Committee on Immunology, University of Chicago, 5841 South Maryland Avenue, MC2115, Chicago, IL 60637.
3 Abbreviations used in this paper: SHP-2; Src homology domain 2-containing protein tyrosine phosphatase; Jnk, c-Jun N-terminal kinase; RAG2, recombinase-activating gene 2.
Received for publication October 11, 2000. Accepted for publication January 1, 2001.
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and ZAP70. J. Exp. Med. 186:1645.This article has been cited by other articles:
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) 2C/RAG2-/- mice were
stimulated with mitomycin C-treated P815. B71 cells. Replicate cultures
were pulsed daily with [3H]TdR and harvested, and
incorporated radioactivity was assessed. B, The same
CD8+ T cells from A were stimulated without
(
) or with (
) P815.B71 cells, and supernatants were collected
after 24 h to assess IL-2 production. Similar results were
obtained in three independent experiments.
