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The Journal of Immunology, 2003, 170: 3002-3006.
Copyright © 2003 by The American Association of Immunologists

CD28 Signal Enhances Apoptosis of CD8 T Cells After Strong TCR Ligation 1

Xue-Zhong Yu2,*, Paul J. Martin*,{dagger} and Claudio Anasetti*,{dagger}

* Human Immunogenetics Program, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109; and {dagger} Department of Medicine, University of Washington, Seattle, WA 98195


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
High avidity ligation of the TCR induces negative selection in the thymus and can also induce apoptosis of peripheral T cells. Costimulation through CD28 enhances T cell activation and facilitates negative selection in the thymus, but the role of CD28 in peripheral T cell deletional tolerance has not been investigated. We used 2C CD28 wild-type and 2C CD28-deficient strains to assess the effects of CD28 and TCR avidity on peripheral T cell expansion and apoptosis. We compared the activation, division, expansion, and apoptosis of CD28+/+ and CD28-/- 2C cells in response to self-Ag (Kb), alloantigens with intermediate (Kbm3), high (Ld), or very high (Ld + QL9 peptide) avidity. With intermediate avidity alloantigen, the CD28 signal enhanced T cell activation and expansion. However, when T cells encountered high avidity alloantigen, the CD28 signal reduced T cell expansion and increased apoptosis. These results indicate that the CD28 signal can down-regulate peripheral T cell responses by increasing apoptosis when TCR ligation exceeds a critical threshold.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The strength of TCR avidity for antigenic peptides strictly regulates T cell fate in the thymus (1, 2). T cell precursors with TCRs that bind self-peptide/MHC complexes with low avidity are selected to survive and further mature. Conversely, T cell precursors with TCRs that recognize self-Ag with high avidity are eliminated through apoptosis. Apoptosis can also be induced in peripheral T cells when Ag is continuously or repeatedly encountered. The Ag-driven apoptosis of activated T cells is important in regulating the peripheral T cell pool (3, 4). TCR signaling has been shown to induce peripheral T cell tolerance by deletion in a number of model systems. Superantigen binding to a family of TCR molecules that express the same V{beta} can activate T cells and induce peripheral T cell tolerance by deletion (5). High dose Ag can paradoxically suppress immune responses in adult animals. In vivo administration of high dose or high avidity myelin basic protein can deplete Ag-specific T cells and abrogate the clinical and pathological signs of experimental autoimmune encephalomyelitis in mice (6, 7). Therefore, it has been clearly shown that strong TCR engagement can induce T cell apoptosis in both the thymus and the periphery.

Several lines of evidence indicate that costimulatory molecules can contribute to the negative selection of T cells. Negative selection of double-positive thymocytes was significantly reduced in response to either Ag or anti-TCR/CD3 Ab in CD28-deficient mice (8). CD28 is also involved in thymocyte negative selection mediated by superantigen both in vitro (9, 10) and in vivo (11). Furthermore, it has been recently reported that prenatal blockade of B7-1 and B7-2 substantially inhibits clonal deletion of T cells in the thymus and leads to an accumulation of autoreactive T cells in the periphery (12). A largely unexplored issue is whether the CD28 signal regulates negative selection of peripheral T cells. While CD28 provides costimulatory signals and enhances T cell activation (13), it is possible that the CD28 signal facilitates T cell negative selection through Ag-activated apoptosis in peripheral T cells.

To assess how CD28 and TCR avidity influence clonal expansion and depletion in peripheral T cells, we have used 2C TCR transgenic mice with or without CD28 molecules, since the specificity and avidity of the 2C TCR for Ag are well defined (14, 15). 2C T cells are MHC class I restricted and are positively selected in the thymus by Kb molecules. 2C TCR recognizes Ld, Kbm3 and Kbm11 alloantigens with high, intermediate, and low avidity, respectively. The specificity of 2C cells for Ld is directed to a naturally occurring peptide, termed p2Ca. This 8-mer peptide, when presented by Ld, displays high avidity for 2C TCR molecules. A 9-mer variant of p2Ca, termed QL9, has 100-fold higher binding avidity for Ld, and the Ld/QL9 complex has 10-fold higher avidity for 2C TCR molecules than the Ld/p2Ca complex (16). Here we present evidence that the CD28 signal reduces T cell expansion and facilitates apoptosis both in vitro and vivo after strong TCR ligation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

C57BL/6 (B6), B6.C-H2bm3 (bm3), and (BALB/c x B6)F1 (CB6F1) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). (B6 x bm3)F1 mice were bred at the Fred Hutchinson Cancer Research Center (Seattle, WA). Founders for the 2C strain were provided by Dr. D. Y. Loh (Nippon Roche Research Center, Kamakura-shi, Japan). B6 CD28-/- mice were a gift from Dr. C. Thompson (17). 2C CD28-/- mice were generated by intercrossing 2C and B6 CD28-/- mice. All the mice used in this study were housed in microisolator cages. Experimental procedures were reviewed and approved by the institutional animal care and use committee of the Fred Hutchinson Cancer Research Center.

T cell purification and proliferation

CD8+ T cells were purified by positive selection with a magnetic cell separation system (Miltenyi Biotech, Auburn, CA) as described previously (18, 19). The purity of CD8+ cells used in this study ranged from 95 to 99%. Cells were cultured in RPMI 1640 medium containing 10% FBS, 2 mM glutamine, 25 mM HEPES, 1 mM sodium pyruvate, 5 x 105 M 2-ME, 100 U/ml penicillin, and 100 µg/ml streptomycin. To measure proliferative responses, purified CD8+ 2C cells were cultured in 96-well, flat-bottom plates at 2.5 x 104/well together with 2.5 x 105/well irradiated (21 cGy) stimulators (splenocytes). In some cultures, antigenic peptide QL9 (QLSPFPFDL) (synthesized by United Biochemical Research, Seattle, WA) was added at 10 µM in addition to BALB/c stimulators. Eight hours before harvest, cells were pulse-labeled with [3H]TdR (1 µCi/well, 1 µCi = 37 GBq). DNA was harvested onto glass-fiber filters, and thymidine incorporation was measured as counts per minute in a Topcount liquid scintillation counter (Packard, Meridian, CT).

Transplantation

CD28+/+ and CD28-/- 2C TCR transgenic mice were designated donors, and B6, (B6 x bm3)F1, and CB6F1 mice were designated recipients. Lymphocytes from lymph nodes and spleens or purified CD8+ cells were isolated from responder mice, labeled with CFSE (Molecular Probes, Eugene, OR) as previously described (20), and injected via the tail vein into recipients. Recipient mice were exposed to a total body irradiation, so that donor T cells could be easily visualized after adoptive transfer. The number of responder cells injected varied among experiments (8–15 x 106 purified T cells or 30–40 x 106 mixed spleen and lymph node cells), but equal numbers of cells were transplanted into each recipient in any given experiment.

Immunofluorescence analysis

Cell staining was performed to measure the expression of surface or intracellular molecules according to standard techniques. Abs used for staining include FITC-labeled anti-CD4, CyChrome-labeled anti-CD8, allophycocyanin-labeled anti-CD8{alpha}, PE-labeled annexin V, biotin-labeled anti-IL-2, biotin-labeled anti-H2Kb, streptavidin-PE, and streptavidin-CyChrome (all from BD PharMingen, San Diego, CA). 1B2, the clonotypic mAb, was prepared and labeled with FITC or biotin in our laboratory. Analysis was performed using a FACScan or FACSCalibur and CellQuest software (BD Biosciences, San Jose, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The CD28 signal enhances T cell apoptosis during initiation of an immune response to high avidity Ag in vitro

To test the effects of CD28 and TCR signals on proliferation, clonal expansion, and apoptosis of peripheral T cells in vitro, we compared the response of T cells from 2C TCR transgenic mice with or without CD28 after stimulation by Ags with distinct strength. CD8+ cells from 2C CD28+/+ or 2C CD28-/- mice were incubated with irradiated B6 (Kb), bm3 (Kbm3), or BALB/c (Ld) stimulators alone or with BALB/c stimulators plus exogenous QL9 peptide. The complex of exogenous QL9 peptide and Ld provides a very high avidity ligation to 2C TCRs (16). CD28 costimulation enhanced T cell proliferation in response to Kbm3 or Ld alloantigens, but inhibited T cell proliferation in response to Ld plus QL9 peptide on days 2 and 3 (Fig. 1A). The absolute number of live cells correlated with the level of proliferation (data not shown). These results suggest that CD28 costimulation can inhibit T cell proliferation and expansion during the initial response to very high avidity TCR ligation.



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  		FIGURE 1. The effect of TCR avidity and CD28 on T cell activation, expansion, and apoptosis in vitro. Purified CD8+ cells from CD28+/+ or CD28-/- 2C mice were incubated with irradiated splenocytes from B6, (B6 x bm3)F1, or BALB/c mice or with BALB/c splenocytes plus exogenous QL9 peptide. After 2, 3, and 4 days, cells were harvested for the following measurements. A, T cell proliferation was measured with [3H]TdR incorporation, and results are shown as the mean of triplicate determinations. B, T cell apoptosis was measured by annexin V staining, and the data shown are the percentage of annexin V+ cells on gated 1B2+/CD8+ 2C responder cells. The results represent one of two replicate experiments.

 
To test whether the inhibitory effect of CD28 costimulation on T cell proliferation was due to increased apoptosis, cells were harvested on days 2, 3, and 4 and stained with annexin V (Fig. 1B). In response to self (Kb) Ag, T cells showed a high level of apoptosis regardless of CD28 expression. The level of apoptosis was lower for 2C CD28+/+ than for 2C CD28-/- cells in response to intermediate avidity alloantigen (Kbm3), indicating that engagement with Kbm3 can generate enough signals to maintain T cell survival in the presence of CD28 costimulation. Under a higher level of engagement with alloantigen (Ld), T cells had a higher level of proliferation and a lower level of apoptosis, and CD28 costimulation enhanced T cell proliferation. At the highest level of TCR engagement (Ld + QL9), 2C CD28+/+ cells showed a significantly higher level of apoptosis (p < 0.001) than 2C CD28-/- cells on days 2 and 3, suggesting that the inhibitory effect of CD28 costimulation on T cell proliferation was due to increased apoptosis.

In response to Ld + QL9, the level of apoptosis on 2C CD28+/+ cells was reduced on day 4 compared with that on day 3, and the level of proliferation was higher for 2C CD28+/+ cells than for 2C CD28-/- cells on day 4 (Fig. 1A). We hypothesized that TCR engagement with high avidity ligands might induce TCR internalization, thereby preventing T cells from receiving signals that lead to apoptosis. Under these conditions, high levels of IL-2 induced by strong TCR ligation and CD28 costimulation might be able to drive subsequent proliferation at a higher rate for the T cells with internalized TCRs. To test these hypotheses, TCR expression and IL-2 production by 2C CD28+/+ and CD28-/- cells were measured after stimulation with Kb, Ld alone, or Ld + QL9. Ld + QL9 induced maximal TCR down-regulation (Fig. 2, A and B). Ld + QL9 induced high levels of IL-2 production by 2C CD28+/+ cells, but not by 2C CD28-/- cells (Fig. 2C). A restimulation assay showed that 2C cells incubated with Ld + QL9 for 4 days were no longer able to respond to TCR stimulation, but remained responsive to IL-2 stimulation (Fig. 2D). These data suggest that TCR down-regulation is an effective mechanism to prevent apoptosis of peripheral T cells for at least some time after stimulation with high avidity Ag and CD28.



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  		FIGURE 2. TCR modulation and IL-2 production in vitro. Cell cultures were established as indicated in Fig. 1. Four days after culture, cells were harvested for the following tests. A and B, TCR expression was tested by staining with anti-TCR mAb on 2C cells after stimulation with Kb, Ld, or Ld + QL9. C, IL-2 production in culture supernatant was measured by ELISA assay. D, 2C CD28+/+ cells were stimulated with Ld or Ld + QL9 in the primary culture for 4 days. Viable cells were isolated and restimulated with medium, IL-2, Ld + QL9, or both IL-2 and Ld + QL9 for an additional 2 days. T cell proliferation was measured by [3H]TdR incorporation, and results are shown as the mean of triplicate tests. The results represent one of two replicate experiments.

 
The CD28 signal enhances apoptosis of T cells in response to high avidity Ag in vivo

To test whether the inhibitory effect of CD28 also applies in vivo, lymphocytes from 2C CD28+/+ or 2C CD28-/- mice were labeled with CFSE and injected into B6, (B6 x bm3)F1, or CB6F1 recipients, which express a self-Ag (Kb), intermediate (Kbm3), or high (Ld) avidity alloantigen, respectively. We measured the expansion, activation, division, and apoptosis of CD28+/+ and CD28-/- 2C cells in recipient spleens 84 h after adoptive transfer. The absolute numbers of 2C CD28+/+ cells were, on the average, 0.3, 2.5, and 0.7 x 105/spleen (n = 3), and those of 2C CD28-/- cells were 0.5, 1.9, and 3.5 x 105/spleen (n = 3) in B6, (B6 x bm3)F1, and CB6F1 recipients, respectively.

In syngeneic recipients (Kb), CD28+/+ and CD28-/- 2C T cells represented 3–4% of recipient splenocytes (Fig. 3A) and did not produce IL-2 regardless of CD28 expression (Fig. 3B). Similar fractions of 2C CD28+/+ and CD28-/- cells divided once or twice, but the annexin V+ subpopulation was larger among 2C CD28-/- cells than among 2C CD28+/+ cells (Fig. 3C). These data indicate that the CD28 signal has no effect on homeostatic proliferation, but may facilitate T cell survival when the TCR interacts with self-peptide/MHC complexes.



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  		FIGURE 3. The effect of TCR avidity and CD28 on T cell clonal expansion, activation, division, and apoptosis in vivo. Spleen and lymph node cells isolated from CD28+/+ or CD28-/- 2C TCR transgenic donors were labeled with CFSE and transferred into irradiated B6, (B6 x bm3)F1, or CB6F1 mice. Eighty-four hours after the transplant, splenocytes were pooled from three mice in each group. A, Splenocytes were stained for expression of 2C TCR and CD8. The numbers represent the percentage of 1B2+/CD8+ 2C donor cells in recipient spleen. B, Intracellular IL-2 expression was analyzed on gated 1B2+/CD8+ 2C cells, and the numbers represent the mean fluorescence intensity of individual curves. C, Annexin V and CFSE profiles were analyzed on gated 1B2+/CD8+ 2C donor cells, and the numbers indicate the percentage of annexin V+ cells. The results represent one of three replicate experiments.

 
In recipients expressing intermediate avidity alloantigen (Kbm3), the number of CD28+/+ cells expanded 8-fold compared with that in syngeneic recipients (Fig. 3A) and produced low levels of IL-2 (Fig. 3B). 2C CD28-/- cells expanded 4-fold (Fig. 3A) and did not produce IL-2 (Fig. 3B). The size of the annexin V+ subpopulation was similar with 2C CD28-/- cells and 2C CD28+/+ cells (Fig. 3C). These results demonstrate that the CD28 signal enhanced T cell activation and expansion when the TCR interacts with intermediate avidity alloantigen in vivo.

In recipients expressing high avidity alloantigen (Ld), 2C CD28+/+ cells expanded 2-fold (Fig. 3A) and produced high levels of IL-2 (Fig. 3B), while 2C CD28-/- cells expanded 7-fold (Fig. 3A) and produced intermediate levels of IL-2 (Fig. 3B). The reduced expansion of CD28+/+ cells was probably due to the increased rate of apoptosis, since the annexin V+ subpopulation was significantly larger among CD28+/+ cells than among CD28-/- cells in CB6F1 recipients (p < 0.05) and was significantly larger in CB6F1 recipients than in (B6 x bm3)F1 recipients (p < 0.01; Fig. 3C). These results indicate that the CD28 signal enhances T cell activation, but inhibits T cell expansion and increases T cell apoptosis when the TCR interacts with the high avidity alloantigen in vivo.

TCR avidity and CD28 determine T cell fate

To determine whether the inhibitory effect of high TCR avidity or CD28 on the initial T cell response is relevant to long term T cell fate, we analyzed the effect of TCR avidity on T cell fate and pathogenicity. Purified CD28+/+ 2C T cells were transferred into irradiated (B6 x bm3)F1 or CB6F1 mice. Irradiated (B6 x bm3)F1 or CB6F1 mice without T cell transfer were used as controls. We monitored the number of 2C cells in the peripheral blood of the recipients over time and found that more 2C cells persisted in (B6 x bm3)F1 than in CB6F1 mice at late time points after transplantation (Fig. 4A). On day 98, the persistence of 2C cells in the recipient spleen was measured to evaluate the fate of 2C donor cells (Fig. 4, B and C). The spleen contained an average of 18.5 x 105/spleen (n = 3) 2C cells in (B6 x bm3)F1 recipients, compared with 3.8 x 105/spleen (n = 5) in CB6F1 recipients, suggesting that more 2C cells could persist in (B6 x bm3)F1 recipients than in CB6F1 recipients. TCR expression on 2C cells was lower in CB6F1 than (B6 x bm3)F1 in at all time points tested (Fig. 4C and data not shown), indicating that engagement with Ld alloantigen caused continuous TCR modulation in vivo.



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  		FIGURE 4. The effect of TCR avidity and CD28 on T cell fate in vivo. Purified CD8+ cells from 2C CD28+/+ donors were injected into irradiated (B6 x bm3)F1 or CB6F1 recipients. Irradiated mice were injected with PBS alone as controls without graft-vs-host disease. A, On days 13, 41, and 62 after cell transfer, blood samples were collected and stained for the expression of CD8 and 2C TCR. The absolute numbers of 2C donor cells in the blood are shown. {blacksquare}, CB6F1 recipients; •, (B6 x bm3)F1 recipients; {square} and {circ}, irradiation controls. B and C, Pooled splenocytes from three to five mice in each group were stained for the expression of CD8 and1B2. The percentage and absolute number of 2C cells in recipient spleen are shown. D, Pooled thymocytes from three to five mice in each group were stained for the expression of CD4 and CD8. The numbers shown are the percentage of CD4+/CD8+ cells among total thymocytes. Splenocytes and thymocytes were analyzed 98 days after the transplant.

 
Since recipient thymocytes are sensitive targets of 2C cells (19), the total numbers of thymocytes and their phenotype were measured to evaluate the pathogenic activity of 2C cells. The total number of thymocytes averaged 1.4 x 106 in (B6 x bm3)F1 recipients (n = 3) and 10.2 x 106 in CB6F1 (n = 5) recipients, in contrast to 35.2 x 106 and 38.4 x 106 in irradiation controls of each strain (n = 3), respectively. The phenotype showed that CD4+/CD8+ double-positive thymocytes were essentially eliminated in (B6 x bm3)F1, but not in CB6F1, recipients (Fig. 4D). These results indicate that 2C cells sustain a graft vs host reaction in response to alloantigen with intermediate avidity, but not with high avidity.

T cell fate and pathogenicity were also evaluated by comparing 2C CD28+/+ and CD28-/- cells in CB6F1 recipients. We found that 2C CD28-/- cells produced a little IL-2 (Fig. 3B) and expressed a minimal amount of Bcl-xL over time and then disappeared in CB6F1 recipients before 76 days after transplantation (data not shown). Thus, it was not possible to address the relevance of CD28 on peripheral T cell deletion through Ag-activated apoptosis in the long term.

Cross-linking CD28 with its agonistic Ab results in T cell depletion through apoptosis in vivo

Given that the CD28 signal inhibits expansion and enhances apoptosis of T cells during the initial response to high avidity alloantigen, we speculated that more intense CD28 signals would further enhance T cell apoptosis. To test this hypothesis, purified CD8+ cells from 2C CD28+/+ or CD28-/- mice were transplanted into irradiated CB6F1 mice. The recipients were treated with control or anti-CD28 mAb (37.51), which has agonistic effects in vitro (21) and in vivo (our unpublished data). Four days after adoptive transfer, the 2C CD28-/- cells had greater expansion and lower levels of apoptosis than CD28+/+ cells (Fig. 5). Furthermore, cross-linking with an agonistic anti-CD28 mAb caused extensive depletion of 2C CD28+/+ cells, and a large fraction of the remaining cells were annexin V+. Anti-CD28 mAb had no effect on 2C CD28-/- cells or on host CD8+ cells (Fig. 5). These results suggest a potential strategy to eliminate Ag-specific T cells and induce tolerance by intensifying CD28 signaling with an agonistic anti-CD28 mAb.



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  		FIGURE 5. Anti-CD28 mAb depletes Ag-activated 2C cells in vivo. Purified CD8+ cells from CD28+/+ or CD28-/- 2C donors were injected into irradiated CB6F1 recipients. Recipients were divided into two groups of three mice each and treated with control or anti-CD28 mAb at 100 µg/mouse on days 0 and 2. Four days after cell transfer, pooled splenocytes were stained for the expression of 1B2, CD8, and annexin V. The numbers represent the percentage of 1B2+/CD8+ 2C donor cells in the recipient spleen (left panels) and the percentage of annexin V+ cells in gated 2C donor cells (right panels). The results represent one of three replicate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It has been long established that T cells are activated and then proliferate, differentiate, and acquire effector function after proper stimulation with specific Ag. However, it has been recently observed that high dose or high avidity TCR ligation can attenuate the T cell response by depleting a portion of newly Ag-activated T cells through apoptotic cell death (4, 22). In this study, we demonstrate that the inhibitory regulation can be achieved by high avidity alloantigens, indicating that T cell fate depends on the total TCR signal received under conditions where costimulatory signals (i.e., CD28 signal) remain constant. It has been shown that CD28 costimulation reduces the extent of TCR engagement required for effective proliferation or cytokine production, possibly by lowering the threshold of TCR signal transduction for T cell activation and promoting the formation of an immunological synapse (23, 24). This is the first study to address the influence of CD28 costimulation on the inhibitory regulation mediated by high avidity TCR ligation. The results reveal that CD28 costimulation can negatively regulate peripheral T cell responses by enhancing apoptosis when TCR ligation exceeds a critical threshold.

The data presented in this study suggest that combined signals through TCR and CD28 determine both positive and negative regulation. If the level of signal delivered from TCR binding alone is insufficient to reach the threshold required for activation, signals delivered through CD28 are mandatory for a proliferative response. If the level of signal delivered from TCR binding alone is sufficient to reach the threshold required for activation, the signals through CD28 amplify proliferation. If, however, the level of signal delivered from TCR binding alone greatly exceeds the threshold required for activation, signals through CD28 enhance Ag-activated apoptosis. Thus, the engagement of CD28 is critical for both positive and negative regulation on CD8+ T cells.

Our results suggest that a stronger antigenic stimulus is required for an inhibitory effect of CD28 costimulation in vitro (Ld + QL9) than in vivo (Ld alone). The ability of Kbm3 alloantigen to induce the proliferation of 2C CD28-/- cells in vivo (Fig. 3), but not in vitro (Fig. 1), may reflect the higher efficiency of productive interaction between T cells and APCs in vivo compared with in vitro. We want to emphasize that the effect of the combined signal though TCR and CD28 on the T cell response is biological relevant to long term T cell fate. On the one hand, the CD28 signal protects T cells from cell death by up-regulating growth and survival factors, such as IL-2 and Bcl-xL (25, 26), and thus maintains the T cell response over a long term. On the other hand, the CD28 signal facilitates Ag-activated T cell apoptosis when the TCR signal exceeds a certain threshold and thus curtails the T cell response in the long term. Taken together with the finding that excessive CD28 signal delivered by agonistic Ab enhances Ag-activated T cell apoptosis in vivo (Fig. 5), we propose that intermediate avidity Ag with proper CD28 costimulation provides the optimal stimulation for an effective long term T cell response.

We have observed that CD28 costimulation reduces T cell proliferation after stimulation with Ld + QL9 on days 2 and 3, but the level of proliferation of 2C CD28+/+ surpasses that of 2C CD28-/- cells on day 4. These results (Fig. 1) confirm the data reported by Cai et al. (16). Furthermore, additional data in this study (Fig. 2) suggest that the augmentation of the response to high avidity peptide on day 4 may result from the high level of IL-2-driven proliferation and the low level of Ag-activated apoptosis after TCR internalization (Fig. 2). TCRs on 2C cells was continually internalized in Ld recipients, but these cells were unable survive over a long term compared with the same cells in Kbm3 recipients (Fig. 4). Thus, the IL-2-dependent T cell replication seen on day 4 in vitro does not translate into a long term advantage for 2C cells activated with high avidity Ag in vivo.

As the inhibition of proliferation was closely associated with high level of apoptosis, we reason that the increased apoptosis may account for the decreased proliferation. The data are consistent with other reports showing that the inhibition of proliferation resulted from increased cell death (7, 27, 28, 29). As the proliferative inhibition and apoptotic death of 2C cells were driven by high avidity TCR ligation with alloantigens, we speculate this observed inhibition resembles the activation-induced cell death reported by others (6, 7, 27, 28, 30). Fas has been implicated in Ag-activated apoptosis of CD4+ cells (4, 27, 31), but it is not required for apoptosis of 2C cells (data not shown). Nevertheless, the data presented here are consistent with other reports that Ag-activated apoptosis of CD8+ cells is mediated by other death receptors in vitro, such TNF receptors, rather then Fas (29, 32). Furthermore, recent evidence has suggested that death receptors, including Fas and TNF receptors, are not required for Ag-activated apoptosis in vivo (33, 34, 35, 36).

In conclusion, the CD signal can down-regulate peripheral T cell responses by increasing apoptosis when TCR ligation exceeds a critical threshold. This finding suggests a new approach for the induction of T cell tolerance by engaging CD28 molecules (Fig. 5), which may have implications for the inhibition of deleterious T cell responses, such as graft-vs-host disease (37), graft rejection, and autoimmunity.


   Acknowledgments
 
We thank Drs. Philip Greenberg and Robert Randolph for helpful discussion of this project, and Ms. Sasha Bidwell and Lisa Rapalus for their technical assistance.


   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 This work was supported by National Institutes of Heath Grants CA84132 (to X.-Z.Y.) and CA18029 and AI51693 (to C.A.). Back

2 Address correspondence and reprint requests to Dr. Xue-Zhong Yu, Fred Hutchinson Cancer Research Center, Mail box D2-100, Fairview Avenue North, Seattle, WA 98109. E-mail address: xyu{at}fhcrc.org Back

Received for publication November 25, 2002. Accepted for publication January 7, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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