CTLA-4 Engagement Acts as a Brake on CD4+ T Cell Proliferation and Cytokine Production but Is Not Required for Tuning T Cell Reactivity in Adaptive Tolerance

Manabu Inobe and Ronald H. Schwartz
J Immunol December 15, 2004, 173 (12) 7239-7248; DOI: https://doi.org/10.4049/jimmunol.173.12.7239

Abstract

Adaptive tolerance is the physiologic down-regulation of T cell responsiveness in the face of persistent antigenic stimulation. In this study, we examined the role of CTLA-4 in this process using CTLA-4-deficient and wild-type TCR transgenic, Rag2−/−, CD4+ T cells transferred into a T cell-deficient, Ag-expressing host. Surprisingly, we found that the tuning process of adoptively transferred T cells could be induced and the hyporesponsive state maintained in the absence of CTLA-4. Furthermore, movement to a deeper state of anergy following restimulation in vivo in a second Ag-bearing host was also unaffected. In contrast, CTLA-4 profoundly inhibited late T cell expansion in vivo following both primary and secondary transfers, and curtailed IL-2 and IFN-γ production. Removal of this braking function in CTLA-4-deficient mice following Ag stimulation may explain their lymphoproliferative dysregulation.

Self-tolerance is initially established by elimination of self-reactive T cells through thymocyte negative selection. Although this is the major mechanism of self-tolerance, its imperfection has been repeatedly demonstrated in many different models (1, 2, 3, 4). A substantial fraction of either superantigen (superAg)-reactive T cells (1) or TCR transgenic T cells (2, 3, 4) escapes from death in the thymus, even when T cells develop under circumstances of abundant Ag expression. Thus, peripheral mechanisms that deal with such escaped self-reactive T cells are required to ensure the integrity of the host. One way this can occur is for mature CD4+ T cells to adapt to persistent Ag stimulation by down-modulating (tuning) their sensitivity to specific Ag restimulation (5). This CD4+ T cell response is called adaptive tolerance (6) or in vivo anergy (7), and is thought to function also in the case of self Ag (4, 8). This mechanism, along with activation-induced cell death (9) and regulatory T cells (10, 11), constitutes a set of peripheral tolerance mechanisms that keep self-reactive T cells harmless in the periphery.

Adaptive tolerance was first described in a population of T cells left following expansion and subsequent death induced by superAg stimulation (12, 13, 14). Later, Ag-specific models were developed, in which a monoclonal T cell population derived from a TCR transgenic mouse is adoptively transferred into either an Ag-bearing recipient (15, 16) or an Ag-free syngeneic recipient, followed by peptide Ag challenge (17). From these models, many specific characteristics regarding the induction and maintenance of adaptive tolerance have been addressed. First, a defect in Ag-induced proliferation and IL-2 production is the most important hallmark of the state, although these responses are not completely disabled. Second, a general down-regulation has been seen for a variety of effector cytokines, such as IFN-γ and IL-4 production, distinguishing adaptive tolerance from differentiation of naive T cells toward the Th1 or Th2 phenotype. Third, the process is a cell-autonomous mechanism involving TCR desensitization. Fourth, this state is reversible upon removal of Ag, implying the requirement of Ag persistence for its maintenance. Finally, we have recently found that adaptive tolerance is also characterized by a changeable activation threshold (5) and that a deeper state of tolerance can be induced upon retransfer of the tolerant T cells into a second Ag-bearing recipient, expressing the same level of Ag (18).

CTLA-4 is a well-known regulator of T cell activation (19). Although originally cloned from a CD8 CTL library, it was subsequently shown to negatively regulate CD4+ T cell expansion by recruitment of phosphatases to the TCR signaling complex. Because of this, the requirement of CTLA-4 for adaptive tolerance has been examined in a number of experiments (7, 20, 21, 22, 23, 24, 25, 26) and postulated from some of these to play a key role. The most striking fact that has reinforced this hypothesis is the finding that the CTLA-4-deficient mouse develops a lymphoproliferative disease and dies at an early age (27, 28). Because thymocyte development in CTLA-4-deficient mice is normal (29, 30), it was speculated that the deficiency results in a breakdown in peripheral tolerance. Despite an extensive effort, however, the role of CTLA-4 in adaptive tolerance is still controversial, because both affirmative (7, 20, 21, 22) and negative (23, 24, 25, 26) results have been published. Initial attempts to address this question used a blocking Ab against CTLA-4 (7, 20, 21, 23, 24, 25). This approach, however, might have given an incomplete blockade, particularly because we now know that CTLA-4 is largely sequestered intracellularly (31, 32, 33) until TCR engagement mobilizes it to the cell surface (34, 35). More recently, CTLA-4-deficient mice were generated on a TCR transgenic, Rag−/− background (22, 26). These mice do not manifest a lymphoproliferative disease, suggesting that the dysregulation is a postenvironmental Ag-induced activation event. Unfortunately, when those mice were used to look at the role of CTLA-4 in in vivo tolerance induction, again conflicting data were reported. Because one set of experiments was conducted with CD8+ T cells and the other with CD4+ T cells, we decided to re-examine this refractory question in our well-characterized CD4+ adaptive tolerance model (5, 18). In this model, nonpathogenic, TCR transgenic, naive T cells are allowed to respond to a persistent antigenic challenge, making it possible for us to follow the long-term fate of the transferred T cells.

For this purpose, naive CD4+ T cells from pigeon cytochrome c (PCC)2-specific, TCR transgenic mice on a Rag2−/− background, with or without a homozygous CTLA-4 deficiency, were adoptively transferred into a T cell-deficient mouse that was persistently expressing PCC under the control of an MHC class I promoter and an Ig enhancer. CTLA-4 deficiency resulted in an overexpansion of naive CD4+ T cells following Ag stimulation and a persistence of IL-2 and IFN-γ production. Nonetheless, CTLA-4-deficient CD4+ T cells eventually entered the same tolerant state as did wild-type (WT) T cells. This was evidenced by hypoproliferation in vivo, down-regulation of cytokine production in vitro, entry into a deeper tolerant state following retransfer into a second Ag-bearing host, and reversal of the state following retransfer into an Ag-free host. These observations suggest that CTLA-4 is not necessary for the tuning mechanisms involved in adaptive tolerance.

Materials and Methods

Mice

All mice carrying various transgenic and mutant alleles were backcrossed onto a B10.A/SgSnAi (B10.A) background. They were bred at the National Institute of Allergy and Infectious Diseases contract facility at Taconic Farms (Germantown, NY) and housed in our National Institutes of Health animal facility before use. Both facilities are accredited by the American Association for Accreditation of Laboratory Animal Care. All procedures were approved by the Animal Care and Use Committee in National Institute of Allergy and Infectious Diseases, and all their guidelines were followed. The 5C.C7 TCR transgenic mouse on a Rag2-deficient background, whose T cells recognize the PCC peptide 81–104 in the context of I-Ek (referred to as WT), has been described previously (18). In this study, CTLA-4-deficient mice with the same TCR specificity (referred to as CTLA-4 knockout (CTLA-4KO)) were generated by backcrossing five times the CTLA-4KO mouse developed in the laboratory of J. Allison (University of California, Berkeley, CA) (29) to the B10.A 5C.C7 TCR transgenic, Rag2−/− mouse. B10.A/CD3ε−/− mice as well as mPCC-CD3ε−/− mice, which express a membrane-targeted form of PCC (mPCC) under the control of an MHC class I promoter and an Ig enhancer, have also been described (18).

Adoptive transfer

Naive T cells were isolated from lymph nodes (LNs) (cervical, axillary, brachial, inguinal, mesenteric, periaortic, and pancreatic) of WT or CTLA-4KO mice and pooled (≥90% CD4+ T cells) before adoptive transfer. A total of 3 × 106 cells was injected i.v. into the mPCC-CD3ε−/− mouse. In some cases, T cells were purified, as described below, from the first recipient’s LNs, and 3 × 106 cells were transferred i.v. into a second fresh recipient, either an mPCC-CD3ε−/− or an Ag-free CD3ε−/− mouse.

In vitro T cell culture and cytokine assay

To evaluate their cytokine-making ability, T cells were recovered and purified from the recipient LNs. In brief, LN cells were first treated with Dynabeads (Dynal Biotech, Great Neck, NY) coupled with a sheep anti-mouse IgG and depleted of Ig-positive cells. The remaining cell population was then incubated with an Ab mixture against B220 (RA3-6B2), CD11b (M1/70), and I-Ek (14-4-4S). All Abs were purchased from BD Biosciences (San Jose, CA). The cells were then treated with Dynabeads coupled with anti-mouse IgG and anti-rat IgG and positive cells again depleted. Thus, T cells were negatively purified, and the yield was typically ≥80% CD4+ T cells. When T cells were purified 1–3 days after in vivo transfer, the recipient spleen (instead of LNs) was used as a source for the cells, because a majority of the T cells (≥90%, data not shown) are recovered from the spleen up to 2 days after transfer. After enrichment, 10,000 purified T cells (≤12,500 total cells) were cultured with various concentrations of the synthetic peptide PCC 81–104 (Bachem, King of Prussia, PA) in the presence of 3000 rad irradiated splenocytes from B10.A (5 × 105) or CD3ε−/− (3 × 105) mice in 200 μl of medium placed in a 96-well flat-bottom plate (Corning Glass, Corning, NY) for 48 h. The culture medium was a 1:1 mixture of RPMI 1640 and Eagles Hanks Amino Acids (EHAA; BioSource International, Rockville, MD) plus 10% FCS, 4 mM glutamine, 200 μg/ml penicillin, 200 μg/ml streptomycin, 25 μg/ml gentamicin, and 50 μM 2-ME. IL-2, IFN-γ, and IL-4 were assayed in the 48-h supernatant by using Quantikine M kit (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions. Naive T cells were subjected to the same assay. In this case, the LN cells from WT or CTLA-4KO mice were used without purification.

Flow cytometry

Because all recipient mice were CD3ε−/−, T cells were routinely stained with a PE-labeled anti-Vβ3 Ab (KJ25) and a CyChrome-labeled anti-CD4 Ab (RM4-5). Fc receptor blocking with the 2.4G2 mAb was performed before Vβ3/CD4 staining. In some cases, T cells were further stained with a FITC-labeled Ab against CD69 (H1.2F3) or CD25 (PC61) to evaluate for activation of the T cells. For intracellular staining of CTLA-4, T cells were first reacted with FITC-labeled anti-Vα11 (RR8-1) and CyChrome-labeled anti-CD4 (RM4-5) before fixation/permeabilization using a Cytofix/Cytoperm kit (BD Biosciences), according to the manufacturer’s instructions. PE-labeled Ab against CTLA-4 (4F10) and a hamster IgG isotype control (G235-2356) were used in this assay. All Abs were purchased from BD Biosciences.

CFSE analysis

T cells were labeled with the CFSE dye before transfer to evaluate in vivo proliferation. T cells were incubated with 2 μM CFSE (Molecular Probes, Eugene, OR) in PBS plus 0.5% FCS for 15 min at 37°C. After three washes with 10 ml of PBS containing 5% FCS, 3 × 106 naive T cells or 1 × 106 LN T cells from the first recipient were injected i.v. into an mPCC-CD3ε−/− mouse. The splenocytes were then isolated at the indicated time points, and the T cell CFSE profile was analyzed by gating on the Vβ3+CD4+ population using a FACSCalibur machine. The area under each division peak was quantified and calculated as a percent contribution of the initial cohort (5). The division profile of naive T cells was fitted to a Gaussian distribution by Prism software to estimate a mean division number (5). The naive T cells proliferated up to 60 h after transfer with nearly a constant rate once they began dividing, allowing us to estimate a lag time to first division and an early division rate from the X-value at Y = 1 and the slope of the linear regression analysis, respectively. In contrast, CFSE-labeled T cells in the second transfer were quantified only as the sum of the division number of each peak multiplied by the percent contribution estimated as described above, because of a poor fit of the data to a Gaussian distribution. This is partly due to cell heterogeneity acquired by the T cell population following the first in vivo transfer. This was discerned in a second form of analysis, the rate of disappearance of the undivided peak at various times after the second transfer. This second analysis revealed that the undivided population of T cells disappears in a biphasic manner (5). Because there are at least two populations distinguished by their rate of cell division, the division profile poorly fits a Gaussian distribution function. The consequence for the mean division number analysis for the second transfer is that it predominantly represents the population with the higher division rate, and thus obscures the cell heterogeneity.

BrdU incorporation

Eight-tenths of a milligram of BrdU (Sigma-Aldrich, St. Louis, MO) was injected i.p. twice per day into mPCC-CD3ε−/− recipients that had received naive T cells either 5 or 21 days earlier. One mouse was sacrificed each day for 5 days. LNs and spleens were isolated, and the cells were combined. The incorporation of BrdU by the Vβ3+CD4+ T cell population was analyzed with a BrdU Flow kit (BD Biosciences), according to the manufacturer’s instructions.

Statistics

Statistical tests were performed using Student’s independent t test. The results were considered significant at p < 0.05.

Results

Naive CTLA-4KO CD4+ T cells overexpand in response to persistent antigenic stimulation in vivo

When WT T cells were transferred into mPCC-CD3ε−/− recipients, a robust T cell expansion was seen, as previously reported (5, 18). After a ∼100-fold expansion of the initial population, approximately one-half of the T cells consistently disappeared from lymphoid organs (Fig. 1⇓) (5, 18). Thereafter, the population size stabilized. The complexity of this T cell response suggests that more than a single event is taking place. One is clearly naive T cell activation, which gives rise to a robust expansion phase. The others appear to be an apoptotic phase accompanied by a desensitization phase, both of which allow the cells to adapt to the persistent antigenic stimulation. CTLA-4KO T cells with the same TCR were transferred into an identical Ag-bearing recipient, and the effect of the KO on the kinetics of the response was examined (Fig. 1⇓). CTLA-4KO T cells began their expansion just like WT T cells, but did not stop as early. They continued dividing for 1 more day and reached a plateau population size that was 5-fold greater than that of WT cells (1-2 × 108 cells per mouse). There was no obvious death phase, and the population size remained at this higher level for as long as 49 days. Thus, the most striking effect from CTLA-4 deficiency is the increase in burst size of the T cell expansion.

FIGURE 1.
FIGURE 1.

Naive CTLA-4KO transgenic T cells overexpand in response to persistent Ag stimulation in vivo. A total of 3 × 106 naive transgenic LN T cells from WT (□) or CTLA-4KO (•) donor mice was injected i.v. into mPCC-CD3ε−/− recipients. The donor T cell numbers were measured in peripheral LNs and spleen as the Vβ3+CD4+ population at the indicated time points after transfer. The data are an average of three independent experiments ± SEM. As a control, the same number of LN T cells from WT (○) or CTLA-4KO (▪) donor mice was transferred into Ag-free CD3ε−/− recipients, and the T cell numbers were determined as well.

In contrast to what was observed for Ag-driven proliferation, CTLA-4 had no effect on the homeostatic expansion induced by the lymphopenic environment of an Ag-free CD3ε−/− recipient (Fig. 1⇑) (18). WT T cells underwent a ∼5-fold expansion over 50 days. CTLA-4-deficient T cells exhibited similar behavior as measured by both an increase in cell number and CFSE dilution (data not shown).

CTLA-4 deficiency does not affect the initiation of cell division nor the early expansion rate, despite effects on the t1/2 of activation markers by 48 h

To investigate the mechanism responsible for the overexpansion of CTLA-4KO T cells, early T cell division was quantitatively evaluated by a CFSE dilution assay up to 60 h after transfer (Fig. 2⇓, A and B). The estimated lag time to the first division for the WT and CTLA-4-deficient T cells was the same, 34 h, and all the cells in both populations had divided at least once by 42 h. There was also a similar rate of expansion over the entire 60-h observation period. The average doubling time for the WT population was 6.1 h, and for the CTLA-4-deficient population, 6.3 h. This was somewhat surprising because CTLA-4 expression, detected by intracellular staining, showed that nearly 100% of the WT transgenic T cells were positive by 48 h after transfer (Fig. 2⇓C). The CTLA-4 was functioning at that time point, as shown by the effect it had on the maintenance of T cell activation markers (Fig. 3⇓). Both CD69 and CD25 were expressed with identical kinetics and amount (mean fluorescence intensity (MFI)) up to 36 h after transfer of WT or CTLA-4-deficient T cells. By 48 h, when CTLA-4 expression was maximal, both activation markers had begun to decline. At this point, the rate of disappearance (percentage positive) of CD69 and CD25 was faster for WT T cells than CTLA-4-deficient T cells. The CD25+ MFI also fell off more quickly in WT T cells; this was not the case for CD69. These differences were also observed at 60 h. Thus, we conclude that CTLA-4 does not affect the division rate of the T cells out to 60 h, despite its functional engagement before 48 h (presumably by B7). Because the T cell expansion is 5-fold greater for cells from CTLA-4-deficient mice, CTLA-4 must exert (or delay) its effect on burst size (until) after 60 h.

FIGURE 2.
FIGURE 2.

WT and CTLA-4KO naive transgenic T cells proliferate with the same kinetics in the early phase of expansion, despite maximal CTLA-4 expression by 2 days. A total of 3 × 106 naive transgenic LN T cells from WT (□) or CTLA-4KO (•) donor mice was injected i.v. into mPCC-CD3ε−/− recipients with (A and B) or without (C) CFSE labeling. Splenocytes were isolated at the indicated times after T cell transfer. A, The CFSE profiles of WT (left) or CTLA-4KO (right) T cells at various times after transfer. B, The data in A were analyzed and fitted to a Gaussian distribution to estimate the mean division number, as described in Materials and Methods. The mean division numbers are plotted vs time. Two values, the lag time to first division and the doubling time, estimated by a linear regression analysis, are indicated in the box. C, CTLA-4 expression (percentage positive) in WT 5C.C7 transgenic T cells was analyzed by intracellular staining at different times after transfer. The figures are one of two similar experiments performed using a single mouse for each point.

FIGURE 3.
FIGURE 3.

CTLA-4KO transgenic T cells express CD69 and CD25 with the same kinetics as WT transgenic T cells, but maintain expression longer. WT and CTLA-4KO transgenic LN T cells were transferred into mPCC-CD3ε−/− recipients. The splenocytes were isolated at various times after transfer, and CD69 (A) and CD25 (B) expression were evaluated by FACS. The percentage of positive T cells is plotted vs time in the upper panels. The MFI of the positive cells is shown in the lower panels. WT (□), CTLA-4KO (•). The figures are one of two similar experiments performed using a single mouse for each point. For statistical analysis, the same experiment was repeated one more time for the 48-h time point, and the average ± SEM is shown (total n = 3). ∗, p < 0.05 (WT vs CTLA-4KO).

CTLA-4KO T cells down-regulate cytokine production following exposure to persistent antigenic stimulation

In this adoptive transfer model, we have previously shown that an adaptive tolerant state is induced following naive T cell activation (5, 18). During this process, T cells modulate their sensitivity to Ag stimulation and reduce their potential to make cytokines on restimulation in vitro. To examine whether this occurred for CTLA-4-deficient T cells, the cells were taken at various times after transfer and stimulated in vitro with the PCC peptide for 48 h, and three cytokines secreted into the medium were measured by ELISA. First, we examined IL-2, IFN-γ, and IL-4 produced by naive T cells (Fig. 4⇓). WT T cells produced both IL-2 and small amounts of IFN-γ in a dose-dependent manner; IL-4, however, was not detected (data not shown). Cytokine production reached a plateau at 10 μM PCC stimulation. IL-2 production was not significantly different for naive CTLA-4KO T cells. IFN-γ production by these T cells was also the same. The lack of an effect possibly relates to the need to induce the CTLA-4 molecule (Figs. 2⇑C and 3⇑).

FIGURE 4.
FIGURE 4.

Cytokine production by CTLA-4KO naive transgenic T cells is not significantly different from WT naive transgenic T cells upon in vitro stimulation. WT (□) and CTLA-4KO (•) transgenic T cells were isolated from naive TCR transgenic mouse LNs and stimulated with various doses of PCC peptide in the presence of irradiated APC. The supernatant was collected after 48 h of culture, and IL-2 (A) or IFN-γ (B) was assayed by ELISA. The data are an average of three independent experiments ± SEM.

Next, we isolated T cells present in the Ag-bearing recipient for 1, 2, or 21 days and stimulated them in vitro for 48 h to see whether cytokine production was down-regulated between the early and late time points (Fig. 5⇓). As previously described (5, 18), WT T cells isolated from a day 21 recipient showed reduced potential to make IL-2 in comparison with activated T cells from a day 1 recipient (Fig. 5⇓A). In three experiments, this decrease was 84%. Surprisingly, by day 21, CTLA-4-deficient T cells had also undergone a down-regulation in IL-2 production similar to that of WT T cells. For three experiments, this decrease was 93%. IFN-γ and IL-4 production by day 21 T cells from both mice were also down-regulated when compared with that of activated T cells isolated from recipients at day 2 (Fig. 5⇓, B and C). At day 2, WT T cells produced 610-fold more IFN-γ than naive T cells following in vitro stimulation, while CTLA-4-deficient T cells produced 730-fold more (n = 3). By 21 days, however, both populations had dampened down their IFN-γ production by 97% (n = 3). A small number of T cells also differentiated toward IL-4 producers after 2 days. Both WT and CTLA-4-deficient T cells produced similar amounts of IL-4 in vitro (1800 vs 1500 pg/ml, n = 3). By day 21, again both T cells down-regulated IL-4 production to the same level. In three experiments, this decrease was 97% for the WT and 95% for the CTLA-4-deficient T cells. Thus, the adaptive tolerant state appeared to occur normally in the absence of CTLA-4.

FIGURE 5.
FIGURE 5.

Cytokine production is down-regulated by persistent Ag stimulation in vivo in both WT and CTLA-4KO transgenic T cells. T cells were purified from LNs of the recipient at 21 days after transfer (filled symbols). To compare the cytokine production between tolerant T cells and activated T cells that are potent cytokine producers, T cells were also purified from the spleen of a recipient at either 1 day (for IL-2) or 2 days (for IFN-γ and IL-4) after transfer (open symbols). A total of 1 × 104 purified WT (left panels) or CTLA-4KO (right panels) T cells was stimulated by various doses of PCC peptide in the presence of irradiated APC. The supernatant was collected after 48 h of culture, and IL-2 (A), IFN-γ (B), and IL-4 (C) were assayed by ELISA. The data are an average of three independent experiments ± SEM.

Because the lack of CTLA-4 resulted in a prolongation of the proliferative expansion phase, we investigated whether the kinetics of adaptive tolerance induction was also delayed in the CTLA-4-deficient T cells. To test this possibility, a more detailed time course of the modulation of cytokine production was undertaken (Fig. 6⇓). T cells were purified from recipients at days 1, 2, 3, 4, 7, 14, 21, and 35 after transfer and stimulated with 10 μM PCC in vitro, and IL-2, IFN-γ, and IL-4 production was assayed after 48 h of culture. Day 1 WT T cells, which showed a full activation phenotype, as judged by CD69 and CD25 expression (Fig. 3⇑), did not alter IL-2 production from that of naive cells. CTLA-4-deficient T cells produced a slightly higher amount of IL-2 at day 1, but this was not significantly different from WT T cells. By day 2, IL-2 production by WT T cells was clearly diminished, while CTLA-4-deficient T cells maintained naive levels of production. By day 3, however, IL-2 production was also down in the CTLA-4-deficient T cells. Thus, CTLA-4 limits the duration of IL-2 production potential as well as T cell expansion, although at different time points. In contrast to IL-2, IFN-γ and IL-4 required 2 days to be maximally induced, and the level of induction was nearly the same for both WT and CTLA-4-deficient T cells. By day 4, WT T cells had fully down-regulated all three cytokines and maintained those low levels out to day 35. The same was true for IL-4 production by CTLA-4-deficient T cells. However, the onset of IFN-γ down-regulation by CTLA-4-deficient T cells was delayed. Day 3 levels were as high as day 2, and it took >7 days to be lowered to the level observed for WT T cells (Fig. 6⇓B). The T cells then maintained low levels of IFN-γ production for at least 35 days. Thus, the presence of CTLA-4 results in a more rapid down-regulation of both IFN-γ and IL-2 production potential.

FIGURE 6.
FIGURE 6.

Kinetics of cytokine down-regulation. WT (squares) and CTLA-4KO (circles) transgenic T cells were purified from recipient mice at various time points after in vivo transfer. A total of 1 × 104 purified T cells was stimulated with 10 μM PCC peptide in the presence of irradiated APC. The supernatant was collected after 48 h of culture, and IL-2 (A), IFN-γ (B), and IL-4 (C) were assayed by ELISA. The data are an average of three independent experiments ± SEM, except for day 35, which was only measured once. Cytokine production by naive WT and CTLA-4KO T cells from Fig. 4⇑ is plotted for the day 0 time point. ∗, p < 0.05 (WT vs CTLA-4KO).

Interestingly, CTLA-4-deficient T cells overshot the WT down-regulation level for IL-2 production potential (Fig. 6⇑A), and by day 7, it was 5-fold lower (p = 0.003, n = 3). This overshoot was slowly reversed, and by day 21, the IL-2 production of the CTLA-4-deficient T cells was similar to that of the WT T cells. The day 7 result shows that even the depth of tolerance induction is curtailed by CTLA-4 in a manner similar to its effect on the proliferative burst size (Fig. 1⇑) and IFN-γ production potential (Fig. 6⇑B). In addition, the unique kinetics clearly indicate that T cells can sense the depth of down-regulation induced for IL-2 production potential and adjust it back up without the participation of CTLA-4.

Hypoproliferation of adaptive tolerant T cells upon in vivo retransfer is the same for CTLA-4KO and WT T cells

T cells in the adaptive tolerant state can proliferate in response to Ag stimulation, if the concentration is high enough. This is also manifest in vivo upon retransfer of the T cells into a second T cell-depleted recipient (5, 18). In both cases, the tolerant population is hyporesponsive compared with naive T cells. Adaptively tolerant CTLA-4KO T cells were next examined to see whether their proliferative response was similarly impaired on retransfer (Fig. 7⇓). T cells were recovered from the first recipient at day 7 or 21, CFSE labeled, and transferred into new mPCC-CD3ε−/− recipients. WT T cells retrieved from the first recipient at day 21 were indeed hypoproliferative by both a mean division number analysis (Fig. 7⇓A) and measurement of the disappearance of cells from the undivided peak (Fig. 7⇓B) (see Materials and Methods). Furthermore, CTLA-4 deficiency did not make any difference in this assay, suggesting that CTLA-4KO T cells were in a similar hyporesponsive state. We also analyzed the two T cell populations at day 7 (Fig. 7⇓, C and D), when IL-2 production by the CTLA-4KO showed a deeper anergic state. The in vivo proliferation data, however, were similar to day 21, with both the CTLA-4KO and WT T cells showing a comparable hypoproliferative state using either method of analysis. Nonetheless, the data show for a second parameter of the adaptive tolerant state that there is no effect of deleting CTLA-4 on the maintenance of this state.

FIGURE 7.
FIGURE 7.

Day 7 and day 21 CTLA-4KO transgenic T cells are impaired the same as WT transgenic T cells in an in vivo proliferation assay. WT (□) and CTLA-4KO (•) transgenic T cells were recovered from recipient LNs at 7 (C and D) or 21 (A and B) days after transfer. Those T cells were retransferred into fresh mPCC-CD3ε−/− recipients after CFSE labeling. The splenocytes were isolated at the indicated times after second transfer, and the CFSE profile was analyzed. The mean division number (A and C) and percentage of T cells remaining undivided (B and D) are plotted against time. The decay of the undivided population of naive WT (○) and CTLA-4KO (▪) transgenic T cells was obtained from the experiment shown in Fig. 2⇑ and is plotted in B and D. The data for the last three points are an average of three independent experiments ± SEM. The data for the first three points are single determinations.

Slow turnover of adaptive tolerant cells in the presence of persistent Ag stimulation is the same for both CTLA-4KO and WT T cells

Adaptively tolerant T cells also undergo a slow turnover in vivo in the first Ag transgenic recipient (∼5% per day) presumably in response to persistent Ag stimulation (5). CTLA-4KO T cells keep their population size stable after the robust expansion, just as WT T cells do (Fig. 1⇑). It was possible, however, that a different balance was being kept between proliferation and disappearance from the lymphoid organs. Therefore, we determined whether the adaptively tolerant CTLA-4KO T cells had the same turnover rate in vivo (Fig. 8⇓). T cells were labeled with BrdU from day 21 to 26, and the incorporation into dividing cells was determined each day by FACS (Fig. 8⇓A). Both WT and CTLA-4KO T cells divided over this time period at the same rate (5.7 vs 5.0% per day). Thus, both T cells were maintained with similar turnover rates, despite the 5-fold difference in their pool sizes. Because IL-2 production was more strongly impaired in CTLA-4-deficient T cells 7 days after transfer (Fig. 6⇑A), we also examined the in vivo turnover from day 5 to 10, starting right after T cells begin curtailing their expansion (Fig. 8⇓B). WT T cells incorporated BrdU slowly with a rate (3.9% per day) similar to that of T cells at day 21. Importantly, CTLA-4-deficient T cells also showed a similar rate (4.8% per day), despite their higher initial labeling after the first day’s pulse. The latter presumably reflects the residual dividing cells culminating from the initial T cell expansion. These results suggest that although CTLA-4 deficiency prolonged T cell expansion for an extra day (Fig. 1⇑), the stable turnover of adaptive tolerance is rapidly established thereafter, and does not overshoot the way IL-2 production potential does (Fig. 6⇑A).

FIGURE 8.
FIGURE 8.

Both WT and CTLA-4KO transgenic T cells have the same turnover rate in vivo. WT (□) and CTLA-4KO (•) transgenic T cells were transferred into mPCC-CD3ε−/− recipients, and the T cells were labeled with BrdU by twice daily i.p. injections from day 21 to day 26 (A) or from day 5 to day 10 (B), as described in Materials and Methods. BrdU incorporation was assayed daily by FACS, and is indicated as percentage of positive cells. The slope was calculated by fitting points to a linear regression curve. A, One of two experiments. B, Displays the results from a single experiment. All experiments were performed using a single mouse for each point.

CTLA-4KO T cells in the adaptive tolerant state were as tunable as WT T cells following transfer into a second Ag-bearing recipient, even though their burst size was again greater

One of the unique characteristics of adaptive tolerance is its plasticity, which can be seen in the modulation of cytokine production, especially IL-2. Ag persistence is required to maintain low production, and its removal leads to an increase in IL-2 production (18). Furthermore, if the T cells are transferred to a second Ag-bearing recipient (mPCC-CD3ε−/−), they slowly enter a deeper tolerant state in which they make even lower amounts of cytokines following reactivation in vitro. Fig. 9⇓ shows that CTLA-4KO T cells have the same plasticity as WT T cells. On transfer of day 21 adaptively tolerant T cells into an Ag-free recipient (CD3ε−/−), IL-2 production increased substantially by 7 days, and this was maintained out to 47 days (Fig. 9⇓A). In contrast, transfer of either tolerant T cell population into an Ag-bearing recipient (mPCC-CD3ε−/−) led to a further 10-fold decrease in IL-2 production potential. Interestingly, this second down-regulation is a much slower process than that observed with naive T cells, requiring ∼21 days to be completed. IFN-γ production was also decreased with a similar kinetic (Fig. 9⇓B). This tunable phenotype of T cell cytokine regulation was confirmed with T cells isolated from another first recipient, this time at day 35 (Fig. 9⇓, C and D). Both WT and CTLA-4-deficient tolerant T cells were able to tune cytokines in opposite directions depending on whether or not Ag was present in the second adoptive host. Moreover, there was no difference between WT and CTLA-4-deficient T cells in either the kinetics or quantitative amounts of the adaptive changes. These results reinforce the conclusion that CTLA-4 is not required for the tuning process involved in adaptive tolerance.

FIGURE 9.
FIGURE 9.

Both CTLA-4KO and WT tolerant transgenic T cells are able to regain the ability to make IL-2 when transferred into an Ag-free recipient, whereas further down-regulation of cytokine production is observed following transfer into an Ag-bearing recipient. WT (open symbols) and CTLA-4KO (filled symbols) transgenic T cells were isolated from recipient LNs at day 21 (Expt. 1) or day 35 (Expt. 2) and purified. A total of 3 × 106 T cells was retransferred into fresh recipients with (□ and •) or without (○ and ▪) Ag expression. T cells were again purified from the LNs at various times after second transfer, and the ability to produce IL-2 (A and C) and IFN-γ (B and D) was assayed in vitro, as described in Fig. 6⇑. Cytokine production by naive WT (▴) and naive CTLA-4KO (⋄) transgenic T cells from Fig. 4⇑ is plotted for the day 0 time point. All experiments were performed using a single mouse for each time point for the Ag-expressing recipients. Two mice were combined for each time point for the Ag-free recipients.

Nonetheless, CTLA-4 did play a role in the expansion of the adaptive tolerant T cells in the second Ag-bearing recipient (Fig. 10⇓, A and B). In contrast to naive T cells, the expansion on retransfer was much slower, peaking at ∼day 21 instead of day 5, but still the general pattern was the same. The initial expansion was equivalent for WT and CTLA-4KO T cells (Fig. 10⇓B). Beyond 4 days, however, there began an overexpansion in the CTLA-4KO population that culminated in a 3- to 5-fold greater expansion by day 21. This biphasic curve could correspond to the heterogeneity seen in the CFSE dilution profiles (see Fig. 7⇑ and Materials and Methods). The initial phase of the expansion over 3 days appears to come solely from the first dividing population, and interestingly, it was not affected by CTLA-4. In contrast, the second phase of proliferation from day 5 to 21 resulted in the 3- to 5-fold greater expansion in the absence of CTLA-4. Expression of CTLA-4 was examined by intracellular staining in the WT transgenic T cells after retransfer into the Ag-bearing recipient (Fig. 10⇓C). Small increases were detected at days 1 and 5. The former occurred as a sharp burst; the latter was more gradual and extended to at least day 7. These expression kinetics alone cannot explain why CTLA-4 has a functional effect in the second phase of the expansion and not in the first phase. Nevertheless, these observations show that CTLA-4 continues to have a functional effect during the second transfer, even though it again has no influence on the adaptive tolerance process (Fig. 9⇑).

FIGURE 10.
FIGURE 10.

Tolerant CTLA-4KO transgenic T cells slowly overexpand in a second Ag-bearing recipient. WT (open symbols) and CTLA-4KO (filled symbols) transgenic T cells were isolated from recipient LNs at day 21 (Expt. 1) or day 35 (Expt. 2) and purified. A total of 3 × 106 T cells was retransferred into fresh recipients, with (□ and •) or without (○ and ▪) Ag expression, as described in Fig. 9⇑. T cells in LNs and spleen were quantified by FACS, as described in Fig. 1⇑, and the total numbers were plotted against time (A and B). In experiment 2, CTLA-4 expression was evaluated by intracellular staining using WT transgenic T cells retransferred into Ag-bearing recipients, and shown as percentage positive (C). All experiments were performed using a single mouse for each time point for the Ag-expressing recipients. Two mice were combined for each time point for the Ag-free recipients.

Finally, Fig. 10⇑, A and B, shows two experiments in which adaptive tolerant T cells were transferred into an Ag-free host to examine reversal and expansion under lymphopenic condition. These cells showed a ∼6-fold increase in T cell number over 50 days, similar to what was observed for naive T cells. This expansion was also unaffected by the presence of CTLA-4. Thus, adaptive tolerance is reversible in a lymphopenic host in the absence of Ag, and this reversal is not influenced by CTLA-4 (Figs. 9⇑ and 10⇑).

Discussion

The adaptive tolerant state has been characterized as a hyporesponsive condition in which both in vivo proliferation and in vitro cytokine production are down-regulated. When CTLA-4-deficient CD4+ T cells were put into this state, their responses to Ag restimulation were identical with those observed for tolerant WT CD4+ T cells. These included a slow in vivo turnover rate of 4–5% per day; diminished IL-2, IL-4, and IFN-γ production in vitro; a retarded, biphasic proliferative response following transfer into an Ag-bearing recipient; a slow entry into an even deeper tolerant state following this retransfer; and a recovery of IL-2 production following transfer into a second recipient not expressing the Ag. Thus, CD4+ T cells are able to modify their activation threshold to accommodate changes in the Ag environment without CTLA-4.

The role of CTLA-4 in the induction phase of adaptive tolerance was more complex. CTLA-4 is not expressed in naive T cells, but is induced with TCR signaling and augmented by CD28 costimulation (36). Expression peaks at ∼48 h (Fig. 2⇑C), but large amounts of the molecule remain intracellular (31, 32, 33), requiring TCR re-engagement for surface display (34, 35). In our studies, we first saw functional effects of CTLA-4 between 42 and 48 h, when the down-regulation of CD25 and to a lesser extent CD69 was faster in the presence of CTLA-4 than in its absence (Fig. 3⇑). In contrast, effects on proliferation (Figs. 1⇑ and 2⇑) did not manifest themselves until beyond 3 days (72 h) when WT cells slowed their division rate, while KO T cells continued to expand at the original rate for another full day. This difference in timing of effects may simply reflect a difference in when each process is susceptible to negative feedback or how long it takes for the CTLA-4 signal to have an effect on the process. For example, cell cycle machinery, such as cyclins and kinases, could be induced to high enough levels by the initial stimulation to insure six or seven rounds of division (37, 38) even though CTLA-4 has shut down any further production of these factors by 48 h (39).

The effect of CTLA-4 on cytokine production following restimulation in vitro appeared to have its major influence during the down-regulation of the response. IL-2 showed CTLA-4-induced differences as early as day 2, while IFN-γ production differences manifested themselves from days 3–7. In both cases, the presence of CTLA-4 more quickly reduced cytokine responsiveness. The down-regulation of IL-4 production potential, however, was not significantly different. The most striking observation in the cytokine data was the apparent overshoot in the down-regulation of IL-2 production potential from days 4–7 by cells from the CTLA-4KO (Fig. 5⇑A). We interpret this as an increase in the production of an anergic factor or some other negative feedback loop in the KO. This is consistent with a general function for CTLA-4 as a mechanism for curtailing most aspects of an initial T cell immune response, both negative and positive. In contrast, the tuning process functioned independently of CTLA-4, and in this case operated slowly over the next 2 wk after the overshoot to readjust the level of IL-2 response potential back up toward a similar level to that of WT T cells. Thus, adaptation can go in either direction independent of CTLA-4.

CTLA-4 effects were also examined following retransfer of the adaptively tolerant T cells into a second lymphopenic, Ag-bearing host. The initial subdued proliferative response was the same for tolerant cells from the KO and the WT mice even though CTLA-4 levels were quickly up-regulated by this in vivo restimulation (Figs. 7⇑ and 10⇑C). CTLA-4 instead had an effect on the second wave of proliferation between days 5 and 21, resulting in a 3- to 5-fold smaller expansion (Fig. 10⇑B). This difference in expansion could be the result of increased cell death or decreased proliferation. CTLA-4 has clearly been shown to inhibit cell cycle progression (39, 40, 41). In contrast, there are reports that CTLA-4 can induce cell death during activation through either unique signaling (42, 43) or by making the cells sensitive to apoptosis (44). Nonetheless, CTLA-4 does not inhibit Bcl-x expression (45) nor Fas-mediated apoptosis (27). CTLA-4 can also inhibit CD28-induced up-regulation of the glucose transporter required for ATP generation and survival (46, 47); however, adaptively tolerant T cells are constantly stimulated to divide by persistent Ag (Fig. 8⇑), and therefore are not likely to undergo atrophic cell death (48). Thus, we think it most likely that the primary effect of CTLA-4 is an inhibition of proliferative expansion.

Once the cells expanded, they maintained the 3- to 5-fold difference in pool sizes for at least 50 days. This raises the possibility that CTLA-4 might also play a role in T cell homeostasis. It is well established that cytokines such as IL-7 and IL-15 are required to maintain memory T cell survival (49, 50). It is, therefore, conceivable that CTLA-4 might influence the level of receptors for these cytokines and thus help sustain the difference in pool sizes. One might also conjecture from the 5-fold difference in circulating cells that the number of T cells that migrate to the nonlymphoid tissues would be greater in the CTLA-4-deficient mice. In a pilot experiment, single cell suspensions were prepared from various tissues by collagenase D digestion 7 days after transfer of naive transgenic T cells into Ag-bearing hosts. Interestingly, in liver and kidney, ∼10 times as many CTLA-4-deficient T cells were detected as WT T cells (data not shown). This dichotomy was even greater than that seen in the lymphoid tissues, and raises the possibility that CTLA-4 also has a small regulatory effect on the expression of cell surface receptors and/or ligands responsible for tissue-specific migration.

Adaptive tolerance continued to operate following retransfer of the T cells into the second Ag-bearing host. Cytokine production slowly decreased another 10-fold over 21 days (Fig. 9⇑) as the cells moved into a deeper state of anergy. Importantly, however, there was no effect of CTLA-4 on this process (Fig. 9⇑). There was also no involvement of CTLA-4 in the reversal from adaptive tolerance after transfer of the cells into an Ag-free environment (Fig. 9⇑). We thus conclude that CTLA-4 is not necessary for the tuning mechanisms involved in adaptive tolerance.

The role of CTLA-4 in various other in vivo tolerance models has been examined with both CTLA-4KO mice and anti-CTLA-4 Abs. In vivo blockade of CTLA-4 enhanced the priming of responsive T cells, but failed to prevent the induction of tumor Ag-specfic tolerance (24). In vivo blockade also had no effect on intranasal-induced oral tolerance (23). In contrast, in a staphylococcal enterotoxin B superantigen model, blocking Abs administered in vivo enhanced both CD4+ T cell recovery and subsequent IL-2 production following restimulation in vitro (20). The difference from our results could relate to effects on cell death, which is not very pronounced in our model, but is in the superAg models. In addition, there is concern when using a blocking Ab in vivo that the blockade might be incomplete. Finally, the depletion by this Ab of regulatory T cells, which express CTLA-4 constitutively (51, 52), might also allow an increase in IL-2 production to occur.

These caveats are not a problem in the KO models, which examined monoclonal T cell populations from TCR transgenic, Rag-deficient mice immunized with soluble peptides. However, here again, different conclusions have been reached. Greenwald et al. (22) reported that the KO could not be induced into an unresponsive state, while Frauwirth et al. (26) reported that their KO could. One possible explanation for the discrepancy is that the former looked at a CD4+ TCR transgenic, while the latter examined a CD8+ TCR transgenic. Our model, however, involves a CD4+ TCR transgenic, and we observe, like Frauwirth et al., little effect of CTLA-4 on in vivo tolerance. Another possibility is the shift in the baseline IL-2 response that both investigators observed in the KO, i.e., their naive T cells produced 2- to 3-fold more IL-2. Thus, when anergy was induced in the KO, its remaining response might appear as a nontolerant state. In our model, this was not an issue, as both the KO and WT naive T cells produced comparable amounts of IL-2 (Fig. 4⇑). A third possibility is that the differences between our results and Greenwald et al. may relate to the nature of the Ag presentation. Our model presents naive CD4+ T cells with a constant level of Ag exposure, while the Greenwald et al. model gives a brief stimulation with soluble peptide. Because there is some evidence that the strength of a TCR signal can influence the kinetics of cytokine down-regulation (5), the weaker peptide stimulus might combine with the delay in cytokine down-regulation seen in the KO (Fig. 6⇑) to postpone the onset of anergy until beyond the 1-wk time point at which Greenwald et al. assessed their tolerant state. A fourth possibility is that no regulatory T cells are produced in our model during in vivo activation, i.e., the accumulated T cell population in the recipient mouse has no effect on the proliferation of a new cohort of naive T cells subsequently transferred into it (18). In contrast, the DO11.10 TCR transgenic T cells used by Greenwald et al. have been shown to be capable of developing into regulatory T cells (53). In addition, such regulatory functions can be induced in the periphery by peptide stimulation (54). Thus, it is possible that CTLA-4 deficiency caused a disruption in regulatory T cell development during in vivo activation of the DO11.10 T cells by peptide Ag stimulation. The absence of such regulatory cells could result in an enhanced response by the KO T cells. Finally, a major difference between the two models is that our protocol involves transferring the TCR transgenic T cells into a lymphopenic host, whereas Greenwald et al. transferred their T cells into an intact syngeneic host. Although, in general, we have found the lymphopenic host to give an augmented proliferative response and a diminished level of apoptosis, the anergy induced appears to be comparable to the original unresponsive state described by Pape et al. (17) following peptide stimulation, at least as defined by diminished cytokine production. Furthermore, we have recently transferred CD45 isotype-marked, WT transgenic, T cells into a replete mPCC-expressing host and found a similar form of anergy induction (N. Singh and R. Schwartz, unpublished observations). Thus, we feel that the conclusions drawn about CTLA-4 and adaptive tolerance from our lymphopenic model will apply equally to an intact host. At the very least, our findings should be relevant in bone marrow transplantation, in which the irradiated patient has to contend with the resurgence of chronic viral infections (55).

So, if adaptive tolerance can be established in the CTLA-4-deficient mouse, why does it develop a lymphoproliferative disease? This process is only seen when the KO allele is expressed with a polyclonal T cell population (22, 56), suggesting that the dysregulation is a post-Ag activation event involving TCRs reactive against particular self or environmental Ags. Our observations further demonstrate that the initial CD4+ T cell response, including the appearance of the activation markers CD69 and CD25, the lag time to division, and the initial division rate, as well as the amount of IL-2 and IFN-γ produced by naive T cell stimulation in vitro are all comparable in the presence or absence of CTLA-4. Similar observations have been demonstrated for naive CD8+ T cell responses in CTLA-4-deficient mice (57, 58, 59). Attempts to manipulate CTLA-4 in autoimmune models have also been consistent with this conclusion (60, 61). CTLA-4 blockade had no effect during the priming phase of diabetes induction, while the incidence and its grade were enhanced with treatment later in the time course of the disease. Walker et al. (62) also showed in a diabetes model that the magnitude of the initial T cell expansion in the spleen was not significantly affected by the absence of CTLA-4, but that the subsequent accumulation of T cells in the pancreatic LNs and the development of diabetes were greatly enhanced in the KO. Finally, Eagar et al. (63) recently published that CTLA-4 blockade solely in the ear was sufficient to allow the onset of delayed-type hypersensitivity effector function in a fixed cell, Ag presentation, tolerance model. From all these experiments, we and others (19) would conclude that CTLA-4 only acts as a brake on CD4+ TCR-induced responses, including proliferation, cytokine production, and homing. This can impact on the duration and amount of T cell effector function elicited during an immune response, and thus influence the final outcome of that response on autoimmune disease and/or immunity. In this sense then, CTLA-4 could cooperate with a separate adaptive tolerance mechanism to dampen down the immune response in the case of persistent Ag.

Acknowledgments

We thank Dr. Alfred Singer for a critical reading of the manuscript, and Dr. Nevil Singh for valuable comments and discussion as well as guidance on experimental procedures. We also thank Elizabeth Majane for generation and supply of mutant mice.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 Address correspondence and reprint requests to Dr. Ronald H. Schwartz, Laboratory of Cellular and Molecular Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 4/Room 111, 4 Center Drive, MSC-0420, Bethesda, MD 20892-0420. E-mail address: rs34r{at}nih.gov

  • ↵2 Abbreviations used in this paper: PCC, pigeon cytochrome c; KO, knockout; LN, lymph node; MFI, mean fluorescence intensity; mPCC, membrane-targeted form of PCC; WT, wild type.

  • Received June 14, 2004.
  • Accepted September 18, 2004.

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