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A Role for the B7-1/B7-2:CD28/CTLA-4 Pathway During Negative Selection¹

Janet E. Buhlmann^*, Sheryl Krevsky Elkin and Arlene H. Sharpe^2,*

^* Departments of Pathology, Immunology Research Division, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115; and Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, MA 02115

	Abstract

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Although costimulation plays an important role in activating naive T cells, its role in negative selection is controversial. By following thymocyte deletion induced by endogenous superantigens in mice lacking B7-1 and/or B7-2, we have identified a role for both B7-1 and B7-2 in negative selection. Studies using CD28-deficient and CD28/CTLA-4-double-deficient mice have revealed that either CD28 or another as yet undefined coreceptor can mediate these B7-dependent signals that promote negative selection. Finally, CTLA-4 delivers signals that inhibit selection, suggesting that CTLA-4 and CD28 have opposing functions in thymic development. Combined, the data demonstrate that B7-1/B7-2-dependent signals help shape the T cell repertoire.

	Introduction

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Negative selection is the process by which autoreactive T cells are eliminated from the T cell repertoire during maturation in the thymus (1). T cells with high affinity/avidity for self MHC-peptide complexes are deleted. This is a critical central mechanism for eliminating autoreactive T cells, and it is important to understand how negative selection is regulated. In vitro studies have shown that engagement of the TCR alone is insufficient to trigger apoptosis (2), and studies are now focusing on what additional signals are required for negative selection.

The B7-1/B7-2:CD28/CTLA-4 pathway is an important, but complex pathway due in part to the dual specificity of B7-1 and B7-2 for two coreceptors, CD28 and CTLA-4, and the opposing outcomes of CD28 and CTLA-4 signaling. Signaling through CD28 promotes T cell activation, whereas signaling through CTLA-4 down-regulates T cell responses. Due to the importance of B7-CD28 interactions for the activation of naive T cells, the role of CD28 costimulation in negative selection has been studied, but is controversial. Several groups, using both in vitro and in vivo models of selection, have shown that CD28 costimulation is critical for the proper elimination of autoreactive T cells (2, 3, 4, 5, 6, 7). However, conflicting studies indicate that CD28 is not required for negative selection (8, 9, 10, 11). Similar conflicting results were seen when the role of CTLA-4 in negative selection was examined. Studies using either a transgenic model or an endogenous superantigen (SAg)³ model of deletion on a CTLA-4-deficient background showed no alteration of negative selection (12, 13). However, when an Ab to CTLA-4 was administered in vivo during CD3-induced deletion, inhibition of negative selection was observed (14). Interpretation of these results is complicated by whether the Ab blocked CTLA-4 engagement or stimulated CTLA-4 by mimicking ligand engagement. Other studies have investigated B7-1 and B7-2 in thymic selection and suggested their participation in the development of central tolerance (15, 16, 17, 18, 19). Providing additional support for B7 involvement, Foy et al. (20) demonstrated decreased expression of B7-2 in the medullary region of the thymus in the absence of CD40 signals. The authors postulated that reduced B7-2 expression disrupted the costimulatory signals necessary for negative selection.

To investigate the role of the B7-1/B7-2:CD28/CTLA-4 pathway in thymic selection, we measured the deletion of SAg-reactive T cells in mice lacking either the ligands or receptors. A unique property of SAg is their ability to interact with MHC II molecules, in a nonprocessed, nonpeptide-dependent manner, and cause the deletion of T cells bearing reactive V TCRs (21). All murine genomes contain endogenous SAg as a result of the random integration of mouse mammary tumor viruses (Mtv) (22). The presence of these Mtv causes the deletion of T cells containing reactive TCRs. Although different from TCR recognition of MHC-peptide complexes, these T cells are potentially autoreactive cells. Therefore, this is a useful model system of negative selection. BALB/c mice are positive for Mtv-6, Mtv-8, and Mtv-9 (23). As a result, V3⁺ (Mtv-6)-, V11⁺ (Mtv-8 and Mtv-9)-, and V12⁺ (Mtv-8 and Mtv-9)-containing T cells are deleted.

Our studies indicate that B7-1 and B7-2 molecules are required for deletion mediated by endogenous SAg, but not exogenous SAg. B7-1 and B7-2 have overlapping functions for the deletion of V11⁺ and V12⁺ T cells. Similarly, in the absence of CD28 there is a defect in V11⁺ and V12⁺ selection, suggesting CD28 transmits the B7-1/B7-2-dependent signals. However, the requirement for CD28-mediated V11⁺ and V12⁺ deletion is eliminated in the absence of both CD28 and CTLA-4, suggesting that CTLA-4 inhibits negative selection. In contrast to V11 and V12, B7-1 appears to be more important than B7-2 for the deletion of V3⁺-bearing thymocytes. Surprisingly, deletion of V3⁺ T cells occurs independent of both CD28 and CTLA-4. These data suggest the presence of a third receptor that mediates B7-1/B7-2-dependent negative selection. Thus, B7-1/B7-2 costimulation delivers signals critical for achieving central tolerance.

	Materials and Methods

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Animals

All animals were housed and maintained in the animal facility of Brigham and Women’s Hospital under specific pathogen-free conditions in accordance with institutional and Institutional Animal Care and Use Committee standards. Wild-type (WT) BALB/c mice were purchased from Taconic Farms (Germantown, NY), BALB/c CD28-deficient mice and BALB/c CD40-deficient mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and bred at Brigham and Women’s Hospital. B7-1-deficient, B7-2-deficient, and B7-1/B7-2-deficient mice, on the BALB/c background, were generated, as previously described (24), and bred at Brigham and Women’s Hospital. These mice were all backcrossed at least 10 generations onto the BALB/c background. Because the integration site of Mtv-6 (responsible for the deletion of V3⁺ thymocytes) is between B7-1 and B7-2 on chromosome 16, the presence of Mtv-6 in B7-deficient mice was confirmed by PCR. CD28/CTLA-4-deficient mice were generated, as previously described (25), and bred at Brigham and Women’s Hospital and backcrossed at least eight generations onto the BALB/c background.

Flow cytometry

All Abs, including isotype controls, were purchased from BD PharMingen (San Diego, CA): V8.1/8.2 (MR5-2), V11 (RR3-15), V12 (MR11-1), V3 (KJ25), CD4 (RM4-5), CD8 (53.6-7), hamster Ig (A19-4), rat IgG2a (R35-95), and rat IgG2b (A95-1). All Abs were diluted in supernatant containing the Fc-blocking Ab 2.4G2 before addition to the cells. Thymocytes or splenocytes were incubated on ice for 20 min in the dark and then washed twice with FACS buffer (PBS with 2% BSA and 0.01% sodium azide). Cells were either analyzed directly or fixed in PBS containing 1% formaldehyde. Fixed cells were stored at 4°C in the dark until analysis. Cell analysis was done on a FACSCalibur cytometer using CellQuest software (BD Biosciences, San Jose, CA).

Staphylococcal enterotoxin B (SEB) studies

A previously described protocol was adapted for these experiments (26). Briefly, SEB from Staphylococcus aureus (Sigma-Aldrich, St. Louis, MO) was reconstituted at 400 µg/ml in sterile PBS. Starting at 1 wk of age, pups were administered 20 µg of SEB i.p. three times per week for 4 wk. At the end of the injection schedule, mice were sacrificed and the presence of V8.1/8.2⁺ T cells was determined, both in the thymus and spleen, by flow cytometry, as described above.

	Results

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Involvement of B7-1 and B7-2 in negative selection

Endogenous SAg are produced from the open reading frame within the long terminal repeat of mouse Mtv that have randomly integrated within the murine genome (22). BALB/c mice express SAg that cause the deletion of V3⁺, V11⁺, and V12⁺ T cells, but not V8.1/8.2⁺ T cells (23). To determine whether B7s were required for negative selection, we isolated thymocytes from either WT or B7-1^-/-/B7-2^-/- (B7 double knockout (DKO)) mice and examined V expression. To analyze CD4⁺ single-positive (SP) thymocytes, cells were gated on the absence of CD8. WT mice had a large population of CD4⁺, V8.1/8.2⁺ T cells present within the thymus, but very low levels (less than 1%) of CD4⁺ T cells expressing either V3-, V11-, or V12-containing TCRs, as expected. Although the expression of V8⁺ T cells is not significantly different from WT mice, B7 DKO mice express increased numbers of V3-, V11-, or V12-bearing T cells (Fig. 1). B7 DKO CD4 SP thymocytes are 2.3% V3⁺, 2.7% V11⁺, and 2.2% V12⁺. There is a modest decrease in the percentage of V8.1/8.2⁺ CD4 SP thymocytes (WT, 17.2%; B7 DKO, 15.7%), which may represent compensation for the increased number of V3-, V11-, or V12-positive thymocytes. When splenocytes from WT or B7 DKO mice were examined, V3⁺, V11⁺, and V12⁺ (Fig. 2) (data not shown) were present at significantly higher levels than in WT mice, indicating that the thymocytes matured and emigrated out of the thymus to become part of the peripheral repertoire.

View larger version (33K): [in this window] [in a new window]   FIGURE 1. Negative selection is defective in B7-1/B7-2 DKO mice. Thymocytes were isolated from mice of the indicated genotype on the BALB/c background. Thymocytes from mice ranging in age from 4 to 12 wk old were stained for the presence of various V-bearing TCRs. Thymocytes were incubated with CD8 PE, CD4 CyChromeC, and either V8 FITC, V11 FITC, or V12 FITC. For detection of V3+, T cells were stained with CD8 FITC, V3 PE, and CD4 CyC. Dot plots represent thymocytes based upon forward light scatter vs side light scatter profile and the absence of CD8. Left column, V expression pattern of CD4 SP WT thymocytes; right column, that of B7-1/B7-2 DKO thymocytes. The x-axis represents the indicated V staining, while the y-axis is CD4 staining. The numbers in the upper right corner indicate the percentage of V+CD4+ thymocytes present.

View larger version (14K): [in this window] [in a new window]   FIGURE 2. Cells that escape deletion in the thymus can be found in the periphery. Splenocytes from BALB/c mice of the indicated genotype were isolated and stained for expression of specific V-bearing T cells. Surface staining and data analysis were performed, as described in Fig. 1. The data represent the expression of either V11 or V3 on CD4+ splenocytes. Each point represents an individual mouse. The bar indicates the mean value of each group. **, Indicates a p value <0.005; *, indicates a p value <0.05.

View larger version (17K): [in this window] [in a new window]   FIGURE 3. B7-1 KO and B7-2 KO mice do not display identical defects in negative selection as compared with B7-1/B7-2 double KO mice. Thymocytes were isolated from the indicated strain of mouse (WT, n = 16; B7 DKO, B7-1/B7-2 double KO, n = 16; B7-2, B7-2 single KO, n = 16; and B7-1, B7-1 single KO, n = 12). Cells were stained and analyzed, as described in Fig. 1. Data are presented as the percentage of CD4+ CD8- thymocytes expressing the indicated V chain. Each point represents an individual mouse, and the bars indicate the mean. **, Indicates a p value <0.005.

View larger version (13K): [in this window] [in a new window]   FIGURE 4. Deletion mediated by exogenous SAg is normal in the absence of B7-1 and B7-2. One-week-old mice were injected i.p. with 20 µg of SEB, three times per week, for 4 wk. After 4 wk, mice were sacrificed and thymocytes were isolated for staining and FACS analysis. Cells were stained with V8.1/8.2 FITC, CD8 PE, and CD4 CyC, and analyzed, as described in Fig. 1. Control mice were untreated mice of the same age and the indicated genotype (n = 8). SEB-treated animals were pooled from three different experiments (n = 16). Each point represents an individual mouse, and the bars indicate the mean.

Given the defects seen in B7 DKO mice, we investigated which receptor transmits the critical B7-1/B7-2-dependent signals because B7-1 and B7-2 can signal through CD28 and CTLA-4. Surprisingly, in CD28 knockout (KO) mice there was no significant defect in V3 thymocyte deletion compared with WT mice. However, there was a significant defect in the deletion of V11⁺ and V12⁺ thymocytes (Fig. 5) (data not shown) in the absence of CD28. These data suggest that CD28 is critical for the deletion of V11⁺ and V12⁺ T cells, but is not required for the deletion of V3⁺ T cells.

View larger version (17K): [in this window] [in a new window]   FIGURE 5. CD28 costimulation mediates B7-dependent deletion of V11+ thymocytes; however, the absence of CTLA-4 overcomes the CD28 requirement. Neither CD28 nor CTLA-4 mediates B7-dependent deletion of V3+ thymocytes. Thymocytes were isolated from the indicated strain of mouse (WT, n = 16; B7, B7-1/B7-2 double KO, n = 16; CD28, CD28 single KO, n = 11; and 4/28, CTLA-4/CD28 double KO, n = 12). Cells were stained and analyzed, as described in Fig. 1. Data are presented at the percentage of CD4+ CD8- thymocytes expressing the indicated V chain. Each point represents an individual mouse, and the bars indicate the mean. **, Indicates a p value <0.005.

The role of B7-1/B7-2 vs CD40

Previous work by Foy et al. (20) suggested defective negative selection after blockade of CD40 may be the result of dysregulated B7-2 expression. We compared the negative selection in mice deficient in CD40 or both B7-1 and B7-2. Although both strains showed defects in negative selection of V3- and V11-bearing thymocytes, CD40 KO showed a more pronounced defect in the selection of V11⁺ T cells than seen in the B7 DKO mice (Fig. 6A). B7 DKO mice had a slightly more pronounced defect in V3 deletion than CD40 KO mice, suggesting that CD40 and B7 may not induce the same signals in developing thymocytes. When the T cells in the periphery of CD40 KO and B7 DKO mice were compared, a striking difference was seen with V3⁺ T cells. As discussed earlier, V3⁺ T cells are present in the periphery of B7 DKO mice; however, these cells are absent in CD40 KO mice (Fig. 6B). Together, these data suggest that while both CD40 and B7-1/B7-2 providecritical signals for negative selection, they are not equivalent, and that there may be different requirements for CD40 and B7s in peripheral selection.

View larger version (30K): [in this window] [in a new window]   FIGURE 6. CD40 KO and B7 DKO mice have distinct patterns of negative selection defects in the thymus, and V3+ T cells are not found in the periphery of CD40 KO mice. A, Thymocytes from mice of the indicated genotype were stained, as described in Fig. 1 (WT, n = 17; B7 DKO, B7-1/B7-2 double KO, n = 16; CD40 KO, CD40 KO, n = 16). Cells were analyzed, as described in Fig. 1, and each point represents an individual mouse. Data are expressed as the percentage of V-positive cells in the CD4+ population. **, Indicates a p value of less than 0.005, and the bar indicates the mean value for each group. B, Splenocytes from mice of the indicated genotype were stained and analyzed, as described in Fig. 1. Each point represents an individual mouse. Data are expressed as the percentage of V-positive cells in the CD4+ population. **, Indicates a p value <0.005; *, indicates a p value <0.05. The bar indicates the mean value for each group.

	Discussion

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Previous work has yielded contradictory results regarding the involvement of CD28 during thymic selection (2, 3, 4, 5, 6, 7, 8, 9, 10, 11). We found that CD28 costimulation is necessary for the deletion of V11⁺ and V12⁺ thymocytes, but not V3⁺ thymocytes. Distinct effects on different V-bearing T cells may reflect differences in avidity between the TCR and the SAg. SAg can delete multiple V-reactive TCRs, and a hierarchy of deletion has been seen (30). Although the molecular basis for the hierarchy is not known, one explanation could be differences in avidity between the TCR and the SAg. If the SAg responsible for deleting V11- and V3-bearing thymocytes have different affinities for their respective TCRs, they may have different costimulation requirements. Thus, it is possible that the V11-SAg interaction is of a lower avidity and requires CD28 costimulation. The normal selection of V3⁺ thymocytes in the CD28-deficient strains would suggest a different type of interaction, possibly a high avidity interaction that is CD28 independent.

Additional support for the interplay of TCR avidity and the requirements of costimulation was suggested by studies investigating Ca²⁺ flux patterns of thymocytes in response to high and low avidity engagement of CD3 and CD4 (31). Low avidity TCR interactions evoked a biphasic calcium signal that was associated with survival and positive selection. In contrast, high avidity interactions, which promoted deletion of reactive thymocytes, resulted in spikes and an elevation in the mean level of calcium. Interestingly, when CD28 was coligated in the presence of a low avidity TCR interaction, the biphasic calcium signal was converted to the spiking pattern associated with a high avidity interaction. These data suggest that under the appropriate conditions, CD28 costimulation can convert a low avidity signal into a high avidity signal, and thus may account for the variable involvement of CD28 during negative selection.

Additionally, deletion of SAg-reactive thymocytes can occur at different stages of the transition from double-positive to SP thymocyte (10). It is possible the restricted expression of B7-1, B7-2, CD28, and CTLA-4 within the thymus may influence the participation of this pathway during negative selection. B7-1 and B7-2 have very restricted expression within the thymus. Originally, B7-1 expression was detected on thymic stromal cells (32). Later studies determined that B7-1 and B7-2 expression was restricted to the medullary region of the thymus on both epithelial cells and dendritic cells (DC) (15, 33, 34, 35). Variable levels of CD28 can be detected on developing thymocytes (36). CD4^-CD8^- thymocytes express a low level of CD28 that is up-regulated as the cells mature into CD4⁺CD8⁺ thymocytes. As the thymocyte develops into a SP thymocyte, CD28 expression is down-modulated to an intermediate level. In contrast to CD28, CTLA-4 appears to have more restricted expression. Surface expression of CTLA-4 on freshly isolated murine thymocytes was undetectable; however, double-negative and CD4 SP thymocytes contain intracellular stores of CTLA-4 (14). In vivo administration of CD-3 resulted in the elevation of intracellular pools of CTLA-4 and detectable surface expression on both double-positive and SP thymocytes. When the in vivo expression of human CTLA-4 was investigated, expression was restricted to the medulla of the thymus, mainly on CD4⁺CD8^-CD1^- thymocytes (37). Our studies with administration of an exogenous SAg (SEB) also suggest that how the Ag is encountered may influence costimulation requirements. When SEB was injected over the course of 4 wk, there was no difference between WT and B7 DKO animals. Although these data suggest a difference in the presentation of endogenous and exogenous Ags, this is a less reliable model of negative selection, as the SEB also activates peripheral T cells. Thus, the timing of the Ag encounter, thymic location (cortex vs medulla), type of the APC and the level of CD28 and CTLA-4 expression on the thymocyte could influence the costimulatory requirements for selection.

Our studies show that the absence of B7-1 and B7-2 is not equivalent to the absence of CD28. The difference between the B7 DKO and CD28 KO mice was most evident in the selection of V3⁺ thymocytes, which was normal in the CD28 KO mice, but impaired in the B7 DKO mice. One potential reason for this difference is that CTLA-4 also mediates deletion of V3⁺ thymocytes. To address this issue, we examined negative selection in mice lacking both B7-1/B7-2 receptors. Mice deficient in both CTLA-4 and CD28 displayed no defects in negative selection, in contrast to defects observed in CD28-deficient mice and the B7 DKO mice. The defect of V11 deletion in the CD28 KO indicates that CD28 costimulation is required for the deletion of these thymocytes. The absence of a V11 deletion defect in the CTLA-4/CD28 DKO mice suggests that CTLA-4 provides an inhibitory signal that prevents the deletion of these SAg-reactive cells. When CTLA-4 is eliminated, the deletion of V11-bearing thymocytes indicates that CD28 costimulation is no longer required for deletion. The decreased number of V3⁺ thymocytes in the 4/28 DKO mice, as compared with WT and CD28 KO mice, further suggests that CTLA-4 delivers an inhibitory signal to developing thymocytes. In the absence of CTLA-4, V3⁺ thymocytes are more readily deleted, suggesting an inhibitory signal has been removed. CTLA-4 may influence a developing thymocyte’s fate by regulating the level of activation/stimulation required for deletion, perhaps lowering the threshold of signals required to induce apoptosis.

The impaired deletion of V3⁺ thymocytes in the B7 DKO mice compared with the normal selection in both CD28 KO and 4/28 DKO mice implies the existence of another B7 receptor. These data are consistent with studies in other model systems that suggest the presence of a third receptor. Mandelbrot et al. (25) were able to demonstrate B7-dependent costimulation in mice lacking both CD28 and CTLA-4. Studies using a cardiac allograft model demonstrated that treatment of CTLA-4/CD28 DKO recipients with either CTLA-4Ig or anti-B7-2 was able to significantly prolong the survival of fully allogeneic cardiac grafts (25, 52).

Although the identity of this third receptor is unclear, it seems unlikely that the new members of the CD28 superfamily, inducible costimulatory molecule (ICOS) and programmed cell death-1 (PD-1) (38, 39), could transmit the B7-1/B7-2-dependent signals. Studies have shown that in the absence of B7-1/B7-2 costimulation, T cells express lower levels of ICOS (40), suggesting cross-regulation of costimulatory pathways. However, Brodie et al. (41) demonstrated that ICOS does not bind to B7-1 or B7-2. This does not eliminate the potential involvement of the ICOS pathway during negative selection. Thymic expression of ICOS is primarily restricted to the medulla and the corticomedullary junction (42), and this localized expression suggests that ICOS may have a role in thymic selection. The normal numbers of T cells in ICOS-deficient mice suggest that ICOS is not required for thymic development; however, similar results were seen when CD28- or CTLA-4-deficient mice were analyzed. Thus, a more in-depth analysis of negative selection in ICOS-deficient mice or in studies using Ab treatments may reveal an unappreciated role for ICOS in negative selection. PD-1 is also a member of the CD28 superfamily, but this receptor has been postulated to have a role in positive selection (43, 44). Studies have investigated negative selection in the absence of PD-1, and it occurs normally; therefore, it is unlikely that PD-1 is transmitting B7-1/B7-2-dependent signals. Further studies are needed to identify this additional B7-1/B7-2 coreceptor.

Our studies also point to distinct roles for CD40 and B7-1/B7-2 in negative selection. Although negative selection defects are present when either the CD40 or the B7 pathway is disrupted, there are differential V expression patterns. CD40-deficient animals have a profound recovery of V11⁺ and V12⁺ thymocytes, with more modest recovery of V3⁺ thymocytes. In contrast, the most profound recovery in B7 DKO mice is seen in the V3⁺ population, with lower levels of V11⁺ and V12⁺ thymocytes, relative to CD40 KO mice. These results are consistent with previous studies that have shown CD40 to be a critical regulator of negative selection (7, 20, 45).

The ability of CD40 to activate APCs may confer a more global regulatory function for CD40 in negative selection. CD40 is expressed on both B cells and DC; CD40 stimulation has been shown to up-regulate MHC II expression and peptide presentation by DC (46, 47, 48, 49). Studies have suggested that up-regulation of MHC II synthesis is required to get surface expression of SAg (22). SAg can be expressed by both DC and B cells (50), which are required for thymic selection (51). Activation of B cells can lead to the up-regulation of SAg mRNA (22). Thus, the importance of CD40 in negative selection may be 2-fold. First, CD40 controls the expression of Ags that will shape the repertoire of TCRs expressed in the mature T cell population. Second, CD40 regulates the expression of costimulatory molecules, such as B7-1 and B7-2, which provide the signals that will determine the fate of the developing thymocyte.

This work demonstrates that there is a complex interplay of costimulatory pathways during negative selection. CD40 may be a critical regulator of the capacity of epithelial and hemopoietic cells to effectively present Ag. The contribution of B7-1 and B7-2 to negative selection may be through the relative balance of costimulation received by the developing thymocyte. Engagement of CTLA-4 inhibits deletion, while engagement of CD28 promotes negative selection under certain conditions. It also appears there may be another undefined B7 receptor, which delivers important costimulatory signals, that can trigger apoptosis. Further studies are needed to determine whether the avidity of an Ag-MHC-TCR interaction influences the need for costimulation during negative selection. TCR transgenic T cells with altered peptide ligands of known avidity will allow direct investigation of this question. Studies are currently underway to identify the other receptor transmitting B7-dependent signals and may lead to the discovery of another member in the rapidly expanding CD28/CTLA-4 family.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants AI38310 and AI40614 (to A.H.S.), and a postdoctoral fellowship from the Cancer Research Institute and National Institutes of Health Grant T32 CA72320 (to J.E.B.).

² Address correspondence and reprint requests to Dr. Arlene H. Sharpe, 221 Longwood Avenue, Boston, MA 02115. E-mail address: asharpe{at}rics.bwh.harvard.edu

³ Abbreviations used in this paper: SAg, superantigen; DC, dendritic cell; KO, knockout; Mtv, mammary tumor virus; SEB, staphylococcal enterotoxin B; SP, single positive; WT, wild type; DKO, double knockout; ICOS, inducible costimulatory molecule; PD-1, programmed cell death 1.

Received for publication December 16, 2002. Accepted for publication March 24, 2003.

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