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Impaired Negative Selection in CD28-Deficient Mice
CELLULAR IMMUNOLOGY ARTICLE NO.
187, 131–138 (1998)
CI981332
Impaired Negative Selection in CD28-Deficient Mice Patricia J. Noel,* Maria-Luisa Alegre,*,† Steven L. Reiner,*,‡ and Craig B. Thompson*,†,‡,1 *Gwen Knapp Center for Lupus and Immunology Research, †Howard Hughes Medical Institute, and ‡Department of Medicine, University of Chicago, Chicago, Illinois 60637 Received May 20, 1998; accepted May 22, 1998
T cell antigen receptors (TCR) expressed on developing T cells can react with self-peptides presented by proteins encoded by the major histocompatibility complex (MHC). Depending on the relative strength of these interactions, thymocytes are either negatively selected as potentially autoreactive and deleted or positively selected to become mature T cells. Developmental selection may also be regulated by signals in addition to those mediated through the TCR. In peripheral T cells, the CD28 receptor plays an important role in enhancing the survival and expansion of T cells activated by TCR engagement. Therefore, we have investigated the role of CD28 in regulating the selection of thymocytes using CD28-deficient mice. Surprisingly, we found a 50% increase in cell number in the thymi of CD28-deficient compared to wildtype mice, suggesting that CD28 might play a role in negative selection. Negative selection of doublepositive thymocytes was found to be significantly reduced in response to either antigen or antibody crosslinking of the TCR complex in CD28-deficient animals. This was not due to a generalized defect in thymocyte survival as thymocytes from CD28-deficient and wildtype mice displayed similar sensitivity to apoptosis initiated by either g-irradiation or dexamethasone. In contrast to its role in T cell activation and survival in the peripheral immune system, the CD28 receptor appears to participate in the intracellular signaling events that result in negative selection in the thymus. © 1998 Academic Press
INTRODUCTION In the thymus, immature T cells that have successfully rearranged their TCR genes undergo further developmental selection. This results in the survival of T cells expressing TCRs with the capability of reacting with antigens expressed in the context of self-MHC 1
To whom correspondence should be addressed at Gwen Knapp Center for Lupus and Immunology Research, University of Chicago, 924 E. 57th Street, R413A, Chicago, IL 60637-5420. Fax: (773) 7021576.
(positive selection) and the deletion of potentially autoreactive T cells (negative selection). These processes are thought to be largely regulated by TCR engagement of self-peptide/MHC complexes. In addition to the intrinsic binding affinity of a single TCR for a particular peptide–MHC complex, both the density of TCRs on thymocytes and the density of MHC–peptide on the presenting cells contribute to the overall avidity of the interaction between the thymocytes and the selecting cells (1–3). Thymocytes that react below a certain threshold die by neglect, while positive selection occurs when cells react with intermediate strength or avidity. Finally, cells that react too strongly with peptide–MHC are negatively selected and die by apoptosis. Signaling through the TCR alone may not be sufficient to induce negative selection (4, 5). For example, the thymic epithelium can mediate positive selection, while cells of hematopoietic origin generally found in the cortex are principally responsible for negative selection (6 –9). It has become clear that additional cell surface molecules play a role in T cell development. The binding of the CD4 and CD8 coreceptors to their respective MHC ligands, MHC class II and class I, can increase the affinity of the TCR for peptide–MHC during early maturation (10, 11). Adhesion molecules can also promote TCR–peptide–MHC interactions. Blockade of LFA-1 and ICAM-1 has been shown to inhibit negative selection (12). Thus, it is clear that a variety of receptors can influence a thymocyte’s decision to undergo positive or negative selection. One class of T cell receptors whose role in T cell development has remained controversial is that of costimulatory receptors. These receptors have traditionally been identified through their ability to influence the outcome of T cell receptor engagement in the periphery. In particular, signaling through the T cell surface receptor, CD28, is critical for the proliferation and survival of activated T cells in the periphery (13–15). CD28 is expressed at high levels on double-positive (DP) thymocytes (16), while its counterreceptors, B7-1 and B7-2, are found on thymic epithelial and dendritic cells within the corticomedullary and medullary regions of the thymus (17, 18). Considering this pattern of expression, and
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the critical role that CD28/B7 interactions play in the periphery, it has been speculated that CD28 may also be involved in T cell development. A number of studies have attempted to identify a role for CD28 in T cell development. Blockade of CD28/B7 interactions via the addition of CTLA4Ig to fetal thymic organ cultures (FTOC) was found to have little effect on the deletion of potentially autoreactive thymocytes in a class II-specific transgenic model (5). There have been conflicting results obtained in studies where B7 engagement has been prevented by the addition of blocking antibodies. Several investigators found no effect on clonal deletion under these conditions (5, 19, 20). In contrast, one recent study showed reduced negative selection in both suspension and FTOC cultures (21). Finally, one study has demonstrated that cross-linking of CD28 on thymocytes is capable of augmenting anti-CD3 induced deletion in vitro (4), whereas another has implicated CD28 and/or CTLA4/B7 interactions in the rescue of thymocytes from glucocorticoid-induced cell death (22). Initial studies that characterized CD28-deficient mice suggested that CD28 signal transduction is not absolutely required for either positive or negative selection (23–25). However these studies did not examine the possibility that CD28 signal transduction might contribute to determining the fate of a thymocyte. To explore this issue, we have studied thymic selection in CD28-deficient mice in greater detail. Our studies suggest that CD28/B7 interactions can contribute to signaling information used to establish a threshold for negative selection in a developing thymocyte. MATERIALS AND METHODS Mice C57BL/6J (B6) and BALB/c mice were purchased from the Jackson Laboratory (Bar Harbor, ME). The derivation of the CD28-deficient mice has been described elsewhere (24). CD28-deficient BALB/c mice carrying a TCR transgene specific for ovalbumin (DO11.10) were produced by appropriate breeding. Breeding and maintenance of the mice were carried out at the University of Chicago (Chicago, IL) in a specificpathogen-free animal facility. All work was performed in accordance with University of Chicago guidelines for animal use and care. In Vivo Depletion of Thymocytes Seven-week-old B6, CD28-deficient and age-matched wildtype controls were injected intraperitoneally (ip) with either 1–100 mg of the anti-CD3 mAb 145-2C11 in 100 ml of PBS or with 100 ml of PBS as a control. Mice were sacrificed 48 h later and single cell suspensions were prepared from thymus and spleen by passage through nylon mesh. Erythrocytes were depleted from
spleen samples by hypotonic lysis in NH4ClK buffer. Triplicate samples were counted and stained as previously described (26) with FITC-CD4, PE-CD8, FITCCD4 1 PE-CD8, or appropriate nonbinding isotype controls (PharMingen, La Jolla, CA). Thymocyte subpopulations were analyzed using flow cytometry (FACScalibur, Becton Dickinson, Mountain View, CA) and Lysis II or CellQuest software. Proliferation Assays Bulk lymph node cells were isolated from CD28-expressing and CD28-deficient BALB/c mice carrying the DO11.10 transgene and 2 3 105 cells/well were plated in 96-well flat-bottomed microtiter plates (Costar Corp., Cambridge, MA) in DMEM (GIBCO-BRL) supplemented with 10% fetal calf serum, penicillin (100 U/ml), streptomycin (100 mg/ml), 10 mM Hepes, 50 mM b-ME, and 0.1 mM nonessential amino acids. The I-Ad-restricted OVA peptide 323–339 (ISQAVHAAHAEINEAGR) specific for the DO11.10 receptor or the I-Ad-restricted ovalbumin (OVA) control peptide 324–334 (SQAVHAAHAEI) was added at the indicated concentrations at the start of the culture period. The plates were incubated at 37°C, 7% CO2 and pulsed with 1 mCi tritiated thymidine ([3H]TdR, ICN Biochemicals) per well for the final 8 h of a 48-h culture period. Plates were harvested utilizing a Tomtec Mach II cell harvester and counted on a 1205 Betaplate liquid scintillation counter (Wallac Inc., Gaithersburg, MD). All results are expressed as the mean 6 SD of triplicate cultures. In Vitro Depletion of Thymocytes Cells were isolated from the thymi of 5- to 7-week-old CD28-deficient or CD28-expressing BALB/c mice carrying the DO11.10 transgene. Single cell suspensions were prepared and cells were plated at 1 3 106/ml in 24-well plates (Costar Corp., Cambridge, MA) in medium and OVA323-339 (10 and 100 mM) or OVA control peptide (100 mM). The plates were incubated at 37°C, 7% CO2. Forty-eight and 72 h later, triplicate samples were harvested and thymocyte subpopulations were analyzed by flow cytometry. Cells were simultaneously stained using anti-CD4-PE (PharMingen), anti-CD8Tricolor (Caltag Laboratories, Burlingame, CA) and the clonotypic antibody KJ126-FITC, generously provided by J. Auger and J. Bluestone (University of Chicago). A total of 25000 live cells was collected per sample and the percentage of specific deletion of clonotypic DP thymocytes was calculated as follows: (Clonotype1 DPcontrol peptide 2 Clonotype1 DPseleting peptide)/ Clonotype1 DPcontrol peptide. Single cell suspensions were prepared from the thymi of 6-week-old B6 wildtype and CD28-deficient mice. Half of the thymocytes were incubated in complete medium for 24 h, in the presence of the indicated concentrations of dexamethasone. The other half of the cells were g-irradi-
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ated for different time periods corresponding to 0.4, 2, 10, and 20 Gy, and cultured for 24 h in complete medium. All cells were harvested at 24 h, stained with propidium iodide, anti-CD4-FITC, and anti-CD8-tricolor, and analyzed by flow cytometry to assess for viability of the different subsets of thymocytes. DNA Fragmentation Analysis Thymoycte single cell suspensions were isolated from age-matched B6 wildtype and CD28-deficient mice and subjected to 5 Gy g-irradiation or cultured in the presence and absence of 1 mM dexamethasone at 1 3 106/ml in 6-well plates as indicated above. At the indicated time points, cells were harvested and genomic DNA was isolated as previously described (26). Samples were treated with RNase A (0.5 mg/ml) and then subjected to electrophoresis through 1.5% SeaKem agarose (FMC Bioproducts, Rockland, ME) gels. RESULTS The Thymus Is Enlarged in CD28-Deficient Mice Gross observation suggested that CD28-deficient thymi were larger than those of age- and sex-matched mice expressing CD28. In order to assess whether this difference was significant, we determined the overall number of thymocytes in CD281/1 and CD282/2 mice. Figure 1A shows that the total number of thymocytes is elevated in the CD28-deficient mice compared to CD28-expressing animals. Typically, CD28-deficient thymi displayed a 50% increase in cell number over that of wildtype thymi. This is in contrast to other lymphoid organs in which total cell numbers are not significantly different between CD281/1 and CD282/2 mice [(24) and data not shown]. Although there was significant enlargement of the CD28-deficient thymi, the percentage distribution of T cell subpopulations, as defined by CD4 and CD8 expression, was similar to that in CD28-expressing animals (data not shown). T-Cell-Receptor-Mediated Deletion of DP Thymocytes One possible explanation for an increase in thymocyte number in CD28-deficient mice could be that costimulation alters the threshold for negative selection. To investigate this possibility, we studied thymocyte deletion induced via stimulation of the TCR complex in wildtype and CD28-deficient mice. Mice were injected with increasing concentrations of anti-CD3 and thymi were analyzed 48 h later by determining total cell counts and T cell subpopulations by flow cytometry. Figure 1B shows that following a 3-mg dose of antiCD3, there was approximately a 15% reduction in the size of the CD28-deficient thymi. In contrast, wildtype thymi showed a reduction of approximately 60% at this dose of anti-CD3. Increasing the dose of anti-CD3 re-
FIG. 1. Thymus size in wildtype and CD28-deficient mice. (A) Thymi were removed from wildtype and CD28-deficient mice and single cell suspensions were prepared. Thymocyte samples were counted in triplicate and total cell number for each thymus was determined. The mean and standard deviation was calculated for each group of thymi (n 5 15) and the statistical significance determined by performing a Student t test (P , 0.001). (B) Mice were injected ip with either PBS or anti-CD3 at the indicated doses; 48 h later mice were sacrificed and thymi were removed. Three mice were analyzed in each treatment group. Thymocyte samples were counted in triplicate and the total cell number of each thymus calculated. The data are representative of at least three independent experiments.
sulted in the induction of more efficient deletion in CD28-deficient thymi, while wildtype thymi achieved essentially maximal deletion at the lower dose. Further increases in the dose of anti-CD3 by as much as 10-fold more were able to induce further deletion in both wildtype and CD28-deficient thymi; however, deletion in CD28-deficient thymi never occurred as efficiently as in wildtype thymi. To determine whether CD28 deficiency causes a selective defect in thymocyte deletion following TCR engagement, surviving T cells were analyzed by staining for the expression of CD4 and CD8 (Fig. 2). Using this assay, the survival of more immature double-positive cells (CD41, CD81) can be assessed relative to that of more mature single-positive cells (CD41, CD82 or CD42, CD81). The results indicate that anti-CD3
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FIG. 2. In vivo deletion of double-positive thymocytes. Thymocyte samples from mice injected ip with PBS or 1 or 100 mg of anti-CD3 were stained for CD4 and CD8 as described under Materials and Methods and analyzed by flow cytometry. The percentage of DP cells in each sample is indicated in the figure. The data are representative of at least three independent experiments.
treatment results in the selective cell death of DP cells in wildtype thymi. In contrast, CD28-deficient DP thymocytes were highly resistant to cell death upon treatment with anti-CD3. At a dose of 100 mg of anti-CD3, there were greater than 10-fold more double-positive thymocytes surviving in CD28-deficient compared to wildtype animals (Fig. 2). These data suggest that the enlargement observed in CD28-deficient thymi is due to the decreased efficiency with which deletion occurs in the absence of CD28. In Vitro Clonal Deletion of Thymocytes In order to extend our observations and to control for the systemic effects of anti-CD3, we also analyzed peptide-mediated events in mice expressing the transgenic TCR from the DO11.10 T cell hybridoma. This TCR recognizes chicken ovalbumin (OVA) peptide in the context of I-Ad (27). It has been established that OVA peptide fragment 323–339 delivers a deletional signal to developing thymocytes and also provides a strong activation signal to mature OVA-specific CD41 cells in the periphery (27). In contrast, OVA fragment 324 –334 does not induce responses from transgenic T cells. We first tested both OVA peptides for their ability to activate mature T cells in a standard proliferation assay as measured by [3H]thymidine uptake. As expected,
OVA324 –334 did not elicit any response from either wildtype or CD28-deficient lymphocytes. In contrast, OVA323–339 was able to activate both types of T cells in a dose-dependent manner although the response of the CD28-deficient cells was three- to fourfold lower than the wildtype response (Fig. 3). We then assessed the ability of OVA peptide to deliver a deletional signal when added to thymocyte cultures. The OVA323–339 was able to induce deletion in a concentrationdependent manner in the clonotypic OVA-reactive DP population of TCR transgenic, CD281/1 mice (Fig. 4A). In contrast, little deletion was observed in CD28deficient DP transgenic thymocytes incubated with 10 mM of OVA323–339. Although more significant antigen-specific deletion of clonotypic CD28-deficient DP thymocytes occurred after 3 days of incubation with the highest dose of antigenic peptide, deletion was still approximately three fold lower than that observed in CD281/1 cells. A representative flow cytometry profile of clonotype-positive thymocyte subsets 72 h after incubation with control or deleting peptide is shown in Fig. 4B: a subset of clonotype-positive DP thymocytes was deleted following exposure to the stimulating but not the control peptide. Addition of exogenous APC to in vitro thymocyte cultures increased the level of deletion of clonotypic DP cells exposed to the selecting
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FIG. 3. Proliferation of peripheral T cells bearing a TCR transgene. Bulk lymph node cells were isolated from DO11.10 wildtype and CD28-deficient mice and cultured in medium and 10 or 100 mM OVA323-339 or 100 mM of a control peptide for 48 h. Proliferation was measured as described under Materials and Methods. The data are representative of at least three independent experiments.
thymocytes were equally sensitive to dexamethasoneand g-irradiation-induced death (Fig. 5). All thymocytes subsets (DP, DN, and SP cells) were affected equally by these treatments (data not shown). As an assay of apoptosis, genomic DNA was prepared and analyzed to determine the extent of DNA degradation that had occurred. Figure 6 shows that genomic DNA prepared either from freshly isolated thymocytes or cells that had been cultured in medium without treatment showed no nucleosomal laddering in samples from wildtype and CD28-deficient animals. The slightly smaller, more defined band in the samples that had been in culture for 24 h indicates a slightly higher percentage of cell death in these cells as opposed to the DNA from freshly isolated thymocytes. However, there was no significant apoptosis as indicated by the absence of laddering. In contrast, DNA from both wildtype and CD28-deficient thymocytes exhibited a high degree of laddering in cultures that contained dexamethasone or had been exposed to g-irradiation, indicating that a significant portion of the cells were undergoing apoptosis. Thus, it appears that CD28 does not protect thymocytes from glucocorticoid or g-irradiation induced cell death in thymocytes. Taken together, the above experiments indicate that CD28/B7 interactions play a specific role in enhancing cell death in response to signaling events mediated through the TCR, but do not contribute to thymocyte death in response to glucocorticoids or g-irradiation. DISCUSSION
peptide in both wildtype and CD28-deficient cells (data not shown). However, the difference between CD28deficient and wildtype cells was maintained because CD28-deficient transgenic DP thymocytes underwent significantly less deletion than CD28-expressing cells. Taken together with the experiments using anti-CD3 mAb, these results demonstrate that lack of CD28 results in less efficient deletion of DP thymocytes when TCR signaling is initiated either through crosslinking with antibody or interaction with peptide/MHC. Thymocyte Apoptosis Induced by Glucocorticoids or g-Irradiation We have shown that CD28 plays a role in the efficient induction of deletion in DP thymocytes when the induction is mediated by signaling events through the TCR. Exposure of thymocytes to glucocorticoids and g-irradiation are also well-known methods for induction of cell death in thymocytes (28). In order to determine whether CD28/B7 interactions influenced these mechanisms of cell death, the survival of wildtype and CD28-deficient thymocytes was examined following exposure to different doses of dexamethasone or g-irradiation. Viability of the thymocytes subsets was assessed by flow cytometry. Wildtype and CD28-deficient
This study suggests that the CD28 receptor contributes information that determines whether the threshold for negative selection has been exceeded in a developing thymocyte. In the absence of the CD28 receptor, thymocytes require significantly greater signaling through the TCR to undergo negative selection. CD28 does not appear to be absolutely required in vivo since deletion of thymocytes can be initiated in CD28deficient animals in response to high levels of TCR crosslinking or high doses of antigenic peptide. It is likely that CD28 aids the negative selection process by helping to increase the avidity of the interactions between thymocytes and APC, as a result of increased adherence acting to prolong TCR–MHC engagement. Avidity is thought to be a combination of the density of molecules interacting on thymocytes and APC and the intrinsic affinity of specific TCR for peptide–MHC. Thus, in the absence of CD28 there are fewer total interactions taking place possibly resulting in reduced total avidity and less efficient negative selection. In addition to the reduced density of surface interactions in the absence of CD28, costimulatory signals important for the induction of negative selection may also be lost. Hogquist et al. (2) define efficacy in selection as the overall signaling properties of particular
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FIG. 4. Peptide-induced clonal deletion of thymocytes bearing a TCR transgene. Thymocytes were isolated from DO11.10 wildtype and CD28-deficient mice and cultured in medium, 10 or 100 mM of OVA323–339, or 100 mM of a control peptide for 48 and 72 h. Thymocytes were simultaneously stained with anti-CD4-PE, anti-CD8-tricolor, and anti-KJ126-FITC. A total of 25000 live cells was collected per sample and the percentage of clonotype-expressing thymocytes was determined. The percentage of clonotype-positive DP thymocytes was similar in untreated CD281/1 and CD282/2 thymocytes. For A, percentage specific deletion of clonotype-positive DP cells was calculated as described under Materials and Methods. Bars represent the mean 6 standard deviation from triplicate samples and are representative of four independent experiments. The statistical significance between deletion induced by 100 mM of selecting peptide in CD281/1 and CD282/2 clonotype-expressing DP thymocytes was determined by a Student t test and was P , 0.001. (B) A representative flow cytometry profile of KJ1261 thymocyte subsets 72 h after incubation with 100 mM of control or deleting ovalbumin peptides.
ligands including TCR interactions, peptide concentration, and contributions from accessory molecules. As in the avidity model, high efficacy results in negative selection. The reduced negative selection of DP thymocytes observed in CD28-deficient mice after crosslinking of CD3 suggests that CD28 has intrinsic signaling properties that contribute to the efficient induction of negative selection, and is not simply a result of de-
creased adhesion. Therefore, the less efficient negative selection observed in the absence of CD28 can be explained by lowered avidity and/or efficacy resulting from a combination of reduced density of surface receptors, resultant lower-affinity TCR–peptide interactions, and a loss of CD28 signaling. In contrast to the results obtained when deletion was induced using anti-CD3 antibody or peptide antigen,
FIG. 5. Susceptibility of CD281/1 and CD282/2 thymocytes to dexamethasone- and g-irradiation-induced cell death. Thymocytes from wildtype and CD28-deficient mice were incubated for 24 h in the presence of different concentrations of dexamethasone or exposed to different amounts of g-irradiation. Cells were stained at 24 h with anti-CD4-FITC, anti-CD8-tricolor, and propidium iodide, and analyzed by flow cytometry. 10,000 events/sample were collected and the percentage deletion was calculated based on the number of live cells present among the 10,000 events. Results are expressed as the mean 6 standard deviation from triplicate samples and are representative of two independent experiments.
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FIG. 6. DNA fragmentation in thymocytes treated with g-irradiation or dexamethasone. Thymocytes from wildtype and CD28-deficient mice were cultured in the presence and absence of dexamethasone or exposed to 5 Gy g-irradiation. Genomic DNA was prepared from freshly isolated cells and from treated samples at 24 h and subjected to electrophoresis through agarose gels.
when thymocytes were treated with steroids or g-irradiation, similar amounts of cell death were observed in wildtype and CD28-deficient thymocytes. Previous studies have shown that the thymic microenvironment produces glucocorticoids which are capable of inducing apoptosis in thymocytes, and are thought to be involved in a “death by neglect” mechanism of thymocytes not selected to survive (22). Our results suggest that CD28 signaling is not involved in rescuing thymocytes from glucocorticoids-induced cell death, whereas it plays a role in decreasing the threshold for negative selection. There have been several attempts to examine the significance of the expression of the CD28 counterreceptors, B7-1 and B7-2, on thymic stromal cells. While some studies have found that negative selection can occur under conditions of B7 blockade (5, 19, 20), others have suggested that blocking B7-1 and B7-2 on thymic stromal cells could enhance thymocyte cell survival in response to TCR crosslinking in vitro (21, 29). These experiments have been difficult to interpret because B7-1 and B7-2 share the ability to bind to two separate receptors, CD28 and CTLA-4. Both of these receptors are expressed on TCR-activated T cells but are thought to have opposing functions. In contrast to the ability of CD28 to contribute to mature T cell survival and proliferation, CTLA-4 signal transduction inhibits T cell proliferation and, under some circumstances, can promote cell death (30 –34). B7 expression on thymic stromal cells could promote positive or negative selection by engaging CTLA-4. Alternatively, B7-1 and B7-2 could promote negative selection through CD28 signal transduction. Such signal transduction might boost the signal initiated through the
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TCR to levels that promote a thymocyte’s autonomous decision to undergo apoptosis. The role of CD28 in the promotion of thymocyte negative selection contrasts with its ability to promote survival in peripheral T cells. However, these seemingly contradictory functions may be consistent with the known ability of CD28 to synergize with the TCR to activate intracellular signal transduction. For example, crosslinking of the TCR and CD28 has been shown to synergize in the activation of Jun N-terminal kinase (JNK) (35). Several recent studies have suggested that in immature cells JNK activation can promote apoptosis (36, 37). Thus, one possible mechanism by which CD28 can promote negative selection of thymocytes is by cooperating with TCR signaling to activate the JNK pathway thus resulting in apoptosis. Since negative selection takes place in CD28-deficient mice, additional costimulatory receptors may exist that play a role in negative selection. One other molecule suggested to be involved in negative selection is gp39. Noelle et al. have demonstrated a requirement for gp39 –CD40 interactions in negative selection when antigen was endogenously expressed both by blockade of gp39 and in gp39-deficient mice (38). Interestingly, the same study also demonstrated that B7-2 expression in the thymus is greatly reduced in the absence of gp39, suggesting that gp39 may indirectly affect the role of CD28 in negative selection by altering its ability to interact with its ligand. Another molecule potentially involved in costimulation during T cell development is CD30. CD30-deficient mice have also been demonstrated to have a defect in negative selection with a phenotype similar to that of CD28-deficient mice, i.e., hypercellularity of the thymus with normal percentage distribution of thymic subsets, and a lack of any autoimmune disease (39). As a member of the tumor necrosis factor receptor family, it is possible that CD30 is involved in mediating death signals during negative selection. However, CD30 has been shown to have costimulatory function in mature cells (40, 41) and is just as likely to mediate its function through the increased avidity/efficacy interactions we are proposing for CD28. Finally, Fas has been shown recently also to play a role in negative selection of immature SP thymocytes in vivo when high doses of antigen are used, but not at low doses of antigen (42). To this end, CD28 may help determine the signaling threshold for negative selection that depends on the quantity of antigen expressed, as well as on the ligation of other surface molecules, such as Fas, gp39, and CD30. Alternatively, CD28 signaling may influence the type of thymocyte deletion occurring after TCR ligation, i.e., Fas-dependent or -independent. The data presented here argue for a strength-ofsignal model for thymocyte negative selection. Such a model would explain why CD28, while not absolutely essential for thymocyte selection, plays an important
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role in the determination of developmental fate. CD28 costimulation synergizes with TCR signaling to increase the avidity and/or efficacy of T cell–stromal cell interactions. However, the outcome of these interactions depends on the developmental stage of the T cell. In the periphery, CD28 costimulation promotes survival and expansion of mature T cells, while in the thymus signaling through CD28 contributes to more efficient induction of negative selection.
19. 20. 21. 22. 23. 24.
ACKNOWLEDGMENTS 25. We thank Carol Sampson for secretarial support and Mandel Davis for expert technical assistance. We also thank A. Kelekar, R. Gedrich, R. Arch, E. Chuang, and M. Gilfillan for critical review of the manuscript. S. L. Reiner is supported by the Burroughs Wellcome Fund and the National Institutes of Health (AI-01309). P. J. Noel is supported by a postdoctoral fellowship from the Cancer Research Institute. This work is supported in part by the National Institutes of Health (AI-35294).
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Jones, L. A., Izon, D. J., Nieland, J. D., Linsley, P. S., and Kruisbeek, A. M., Int. Immunol. 5, 503, 1993. Tan, R., The, S.-J., Ledbetter, J. A., Linsley, P. S., and Teh, H.-S., J. Immunol. 149, 3217, 1992. Amsen, D., and Kruisbeek, A. M., Int. Immunol. 8, 1927, 1996. Wagner, D. H., Jr., Hagman, J., Linsley, P. S., Hodsdon, W., Freed, J. H., and Newell, M. K., J. Exp. Med. 184, 1631, 1996. Lucas, P. J., Negishi, I., Nakayama, K., Fields, L. E., and Loh, D. Y., J. Immunol. 154, 5757, 1995. Shahinian, A., Pfeffer, K., Lee, K. P., Kundig, T. M., Kishihara, K., Wakeham, A., Kawai, K., Ohashi, P. S., Thompson, C. B., and Mak, T. W., Science 261, 609, 1993. Walunas, T. L., Sperling, A. I., Khattri, R., Thompson, C. B., and Bluestone, J. A., J. Immunol. 156, 1006, 1996. Noel, P. J., Boise, L. H., Green, J. M., and Thompson, C. B., J. Immunol. 157, 636, 1996. Murphy, K. M., Heimberger, A. B., and Loh, D. Y., Nature 250, 1720, 1990. Wyllie, A. H., Nature 284, 555, 1980. Kishimoto, H., Cai, Z., Brunmark, A., Jackson, M. R., Peterson, P. A., and Sprent, J., J. Exp. Med. 184, 531, 1996. Walunas, T. L., Lenschow, D. J., Bakker, C. Y., Linsley, P. S., Freeman, G. J., Green, J. M., Thompson, C. B., and Bluestone, J. A., Immunity 1, 405, 1994. Krummel, M. F., and Allison, J. P., J. Exp. Med. 182, 459, 1995. Waterhouse, P., Penninger, J. M., Timms, E., Wakeham, A., Shahinian, A., Lee, K. P., Thompson, C. B., Griesser, H., and Mak, T. W., Science 270, 985, 1995. Tivol, E. A., Borriello, F., Schweitzer, A. N., Lynch, W. P., Bluestone, J. A., and Sharpe, A. H., Immunity 3, 541, 1995. Gribben, J. G., Freeman, G. J., Boussiotis, V. A., Rennert, P., Jellis, C. L., Greenfield, E., Barber, M., Restivo, V. A., Ke, X., Gray, G. S., and Nadler, L. M., Proc. Natl. Acad. Sci. USA 92, 811, 1995. Su, B., Jacinto, E., Hibi, M., Kallunki, T., Karin, M., and BenNeriah, Y., Cell 77, 727, 1994. Verheij, M., Bose, R., Lin, X. H., Yao, B., Jarvis, W. D., Grant, S., Birrer, M. J., Szabo, E., Zon, L. I., Kyriakis, J. M., Haimovitz-Friedman, A., Fuks, Z., and Kolesnick, R. N., Nature 380, 75, 1996. Xia, Z., Dickens, M., Raingeaud, J., Davis, R. J., and Greenberg, M. E., Science 270, 1326, 1995. Foy, T. M., Page, D. M., Waldschmidt, T. J., Schoneveld, A., Laman, J. D., Masters, S. R., Tygrett, L., Ledbetter, J. A., Aruffo, A., Claassen, E., Xu, J. C., Flavell, R. A., Oehen, S., Hedrick, S. M., and Noelle, R. J., J. Exp. Med. 182, 1377, 1995. Amakawa, R., Hakem, A., Kundig, T. M., Matsuyama, T., Simard, J. J. L., Timms, E., Wakeham, A., Mittruecker, H.-W., Griesser, H., Takimoto, H., Schmits, R., Shahinian, A., Ohashi, P. S., Penninger, J. M., and Mak, T. W., Cell 84, 551, 1996. Gruss, H.-J., Boiani, N., Williams, D. E., Armitage, R. J., Smith, C. A., and Goodwin, R. G., Blood 83, 2045, 1994. Gilfillan, M. C., Noel, P. J., Podack, E. R., Reiner, S. L., and Thompson, C. B., J. Immunol. 160, 2181, 1998. Kishimoto, H., Surh, C. D., and Sprent, J., J. Exp. Med. 187, 1427, 1998.
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Impaired Negative Selection in CD28-Deficient Mice Patricia J. Noel,* Maria-Luisa Alegre,*,† Steven L. Reiner,*,‡ and Craig B. Thompson*,†,‡,1 *Gwen Knapp Center for Lupus and Immunology Research, †Howard Hughes Medical Institute, and ‡Department of Medicine, University of Chicago, Chicago, Illinois 60637 Received May 20, 1998; accepted May 22, 1998
T cell antigen receptors (TCR) expressed on developing T cells can react with self-peptides presented by proteins encoded by the major histocompatibility complex (MHC). Depending on the relative strength of these interactions, thymocytes are either negatively selected as potentially autoreactive and deleted or positively selected to become mature T cells. Developmental selection may also be regulated by signals in addition to those mediated through the TCR. In peripheral T cells, the CD28 receptor plays an important role in enhancing the survival and expansion of T cells activated by TCR engagement. Therefore, we have investigated the role of CD28 in regulating the selection of thymocytes using CD28-deficient mice. Surprisingly, we found a 50% increase in cell number in the thymi of CD28-deficient compared to wildtype mice, suggesting that CD28 might play a role in negative selection. Negative selection of doublepositive thymocytes was found to be significantly reduced in response to either antigen or antibody crosslinking of the TCR complex in CD28-deficient animals. This was not due to a generalized defect in thymocyte survival as thymocytes from CD28-deficient and wildtype mice displayed similar sensitivity to apoptosis initiated by either g-irradiation or dexamethasone. In contrast to its role in T cell activation and survival in the peripheral immune system, the CD28 receptor appears to participate in the intracellular signaling events that result in negative selection in the thymus. © 1998 Academic Press
INTRODUCTION In the thymus, immature T cells that have successfully rearranged their TCR genes undergo further developmental selection. This results in the survival of T cells expressing TCRs with the capability of reacting with antigens expressed in the context of self-MHC 1
To whom correspondence should be addressed at Gwen Knapp Center for Lupus and Immunology Research, University of Chicago, 924 E. 57th Street, R413A, Chicago, IL 60637-5420. Fax: (773) 7021576.
(positive selection) and the deletion of potentially autoreactive T cells (negative selection). These processes are thought to be largely regulated by TCR engagement of self-peptide/MHC complexes. In addition to the intrinsic binding affinity of a single TCR for a particular peptide–MHC complex, both the density of TCRs on thymocytes and the density of MHC–peptide on the presenting cells contribute to the overall avidity of the interaction between the thymocytes and the selecting cells (1–3). Thymocytes that react below a certain threshold die by neglect, while positive selection occurs when cells react with intermediate strength or avidity. Finally, cells that react too strongly with peptide–MHC are negatively selected and die by apoptosis. Signaling through the TCR alone may not be sufficient to induce negative selection (4, 5). For example, the thymic epithelium can mediate positive selection, while cells of hematopoietic origin generally found in the cortex are principally responsible for negative selection (6 –9). It has become clear that additional cell surface molecules play a role in T cell development. The binding of the CD4 and CD8 coreceptors to their respective MHC ligands, MHC class II and class I, can increase the affinity of the TCR for peptide–MHC during early maturation (10, 11). Adhesion molecules can also promote TCR–peptide–MHC interactions. Blockade of LFA-1 and ICAM-1 has been shown to inhibit negative selection (12). Thus, it is clear that a variety of receptors can influence a thymocyte’s decision to undergo positive or negative selection. One class of T cell receptors whose role in T cell development has remained controversial is that of costimulatory receptors. These receptors have traditionally been identified through their ability to influence the outcome of T cell receptor engagement in the periphery. In particular, signaling through the T cell surface receptor, CD28, is critical for the proliferation and survival of activated T cells in the periphery (13–15). CD28 is expressed at high levels on double-positive (DP) thymocytes (16), while its counterreceptors, B7-1 and B7-2, are found on thymic epithelial and dendritic cells within the corticomedullary and medullary regions of the thymus (17, 18). Considering this pattern of expression, and
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the critical role that CD28/B7 interactions play in the periphery, it has been speculated that CD28 may also be involved in T cell development. A number of studies have attempted to identify a role for CD28 in T cell development. Blockade of CD28/B7 interactions via the addition of CTLA4Ig to fetal thymic organ cultures (FTOC) was found to have little effect on the deletion of potentially autoreactive thymocytes in a class II-specific transgenic model (5). There have been conflicting results obtained in studies where B7 engagement has been prevented by the addition of blocking antibodies. Several investigators found no effect on clonal deletion under these conditions (5, 19, 20). In contrast, one recent study showed reduced negative selection in both suspension and FTOC cultures (21). Finally, one study has demonstrated that cross-linking of CD28 on thymocytes is capable of augmenting anti-CD3 induced deletion in vitro (4), whereas another has implicated CD28 and/or CTLA4/B7 interactions in the rescue of thymocytes from glucocorticoid-induced cell death (22). Initial studies that characterized CD28-deficient mice suggested that CD28 signal transduction is not absolutely required for either positive or negative selection (23–25). However these studies did not examine the possibility that CD28 signal transduction might contribute to determining the fate of a thymocyte. To explore this issue, we have studied thymic selection in CD28-deficient mice in greater detail. Our studies suggest that CD28/B7 interactions can contribute to signaling information used to establish a threshold for negative selection in a developing thymocyte. MATERIALS AND METHODS Mice C57BL/6J (B6) and BALB/c mice were purchased from the Jackson Laboratory (Bar Harbor, ME). The derivation of the CD28-deficient mice has been described elsewhere (24). CD28-deficient BALB/c mice carrying a TCR transgene specific for ovalbumin (DO11.10) were produced by appropriate breeding. Breeding and maintenance of the mice were carried out at the University of Chicago (Chicago, IL) in a specificpathogen-free animal facility. All work was performed in accordance with University of Chicago guidelines for animal use and care. In Vivo Depletion of Thymocytes Seven-week-old B6, CD28-deficient and age-matched wildtype controls were injected intraperitoneally (ip) with either 1–100 mg of the anti-CD3 mAb 145-2C11 in 100 ml of PBS or with 100 ml of PBS as a control. Mice were sacrificed 48 h later and single cell suspensions were prepared from thymus and spleen by passage through nylon mesh. Erythrocytes were depleted from
spleen samples by hypotonic lysis in NH4ClK buffer. Triplicate samples were counted and stained as previously described (26) with FITC-CD4, PE-CD8, FITCCD4 1 PE-CD8, or appropriate nonbinding isotype controls (PharMingen, La Jolla, CA). Thymocyte subpopulations were analyzed using flow cytometry (FACScalibur, Becton Dickinson, Mountain View, CA) and Lysis II or CellQuest software. Proliferation Assays Bulk lymph node cells were isolated from CD28-expressing and CD28-deficient BALB/c mice carrying the DO11.10 transgene and 2 3 105 cells/well were plated in 96-well flat-bottomed microtiter plates (Costar Corp., Cambridge, MA) in DMEM (GIBCO-BRL) supplemented with 10% fetal calf serum, penicillin (100 U/ml), streptomycin (100 mg/ml), 10 mM Hepes, 50 mM b-ME, and 0.1 mM nonessential amino acids. The I-Ad-restricted OVA peptide 323–339 (ISQAVHAAHAEINEAGR) specific for the DO11.10 receptor or the I-Ad-restricted ovalbumin (OVA) control peptide 324–334 (SQAVHAAHAEI) was added at the indicated concentrations at the start of the culture period. The plates were incubated at 37°C, 7% CO2 and pulsed with 1 mCi tritiated thymidine ([3H]TdR, ICN Biochemicals) per well for the final 8 h of a 48-h culture period. Plates were harvested utilizing a Tomtec Mach II cell harvester and counted on a 1205 Betaplate liquid scintillation counter (Wallac Inc., Gaithersburg, MD). All results are expressed as the mean 6 SD of triplicate cultures. In Vitro Depletion of Thymocytes Cells were isolated from the thymi of 5- to 7-week-old CD28-deficient or CD28-expressing BALB/c mice carrying the DO11.10 transgene. Single cell suspensions were prepared and cells were plated at 1 3 106/ml in 24-well plates (Costar Corp., Cambridge, MA) in medium and OVA323-339 (10 and 100 mM) or OVA control peptide (100 mM). The plates were incubated at 37°C, 7% CO2. Forty-eight and 72 h later, triplicate samples were harvested and thymocyte subpopulations were analyzed by flow cytometry. Cells were simultaneously stained using anti-CD4-PE (PharMingen), anti-CD8Tricolor (Caltag Laboratories, Burlingame, CA) and the clonotypic antibody KJ126-FITC, generously provided by J. Auger and J. Bluestone (University of Chicago). A total of 25000 live cells was collected per sample and the percentage of specific deletion of clonotypic DP thymocytes was calculated as follows: (Clonotype1 DPcontrol peptide 2 Clonotype1 DPseleting peptide)/ Clonotype1 DPcontrol peptide. Single cell suspensions were prepared from the thymi of 6-week-old B6 wildtype and CD28-deficient mice. Half of the thymocytes were incubated in complete medium for 24 h, in the presence of the indicated concentrations of dexamethasone. The other half of the cells were g-irradi-
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ated for different time periods corresponding to 0.4, 2, 10, and 20 Gy, and cultured for 24 h in complete medium. All cells were harvested at 24 h, stained with propidium iodide, anti-CD4-FITC, and anti-CD8-tricolor, and analyzed by flow cytometry to assess for viability of the different subsets of thymocytes. DNA Fragmentation Analysis Thymoycte single cell suspensions were isolated from age-matched B6 wildtype and CD28-deficient mice and subjected to 5 Gy g-irradiation or cultured in the presence and absence of 1 mM dexamethasone at 1 3 106/ml in 6-well plates as indicated above. At the indicated time points, cells were harvested and genomic DNA was isolated as previously described (26). Samples were treated with RNase A (0.5 mg/ml) and then subjected to electrophoresis through 1.5% SeaKem agarose (FMC Bioproducts, Rockland, ME) gels. RESULTS The Thymus Is Enlarged in CD28-Deficient Mice Gross observation suggested that CD28-deficient thymi were larger than those of age- and sex-matched mice expressing CD28. In order to assess whether this difference was significant, we determined the overall number of thymocytes in CD281/1 and CD282/2 mice. Figure 1A shows that the total number of thymocytes is elevated in the CD28-deficient mice compared to CD28-expressing animals. Typically, CD28-deficient thymi displayed a 50% increase in cell number over that of wildtype thymi. This is in contrast to other lymphoid organs in which total cell numbers are not significantly different between CD281/1 and CD282/2 mice [(24) and data not shown]. Although there was significant enlargement of the CD28-deficient thymi, the percentage distribution of T cell subpopulations, as defined by CD4 and CD8 expression, was similar to that in CD28-expressing animals (data not shown). T-Cell-Receptor-Mediated Deletion of DP Thymocytes One possible explanation for an increase in thymocyte number in CD28-deficient mice could be that costimulation alters the threshold for negative selection. To investigate this possibility, we studied thymocyte deletion induced via stimulation of the TCR complex in wildtype and CD28-deficient mice. Mice were injected with increasing concentrations of anti-CD3 and thymi were analyzed 48 h later by determining total cell counts and T cell subpopulations by flow cytometry. Figure 1B shows that following a 3-mg dose of antiCD3, there was approximately a 15% reduction in the size of the CD28-deficient thymi. In contrast, wildtype thymi showed a reduction of approximately 60% at this dose of anti-CD3. Increasing the dose of anti-CD3 re-
FIG. 1. Thymus size in wildtype and CD28-deficient mice. (A) Thymi were removed from wildtype and CD28-deficient mice and single cell suspensions were prepared. Thymocyte samples were counted in triplicate and total cell number for each thymus was determined. The mean and standard deviation was calculated for each group of thymi (n 5 15) and the statistical significance determined by performing a Student t test (P , 0.001). (B) Mice were injected ip with either PBS or anti-CD3 at the indicated doses; 48 h later mice were sacrificed and thymi were removed. Three mice were analyzed in each treatment group. Thymocyte samples were counted in triplicate and the total cell number of each thymus calculated. The data are representative of at least three independent experiments.
sulted in the induction of more efficient deletion in CD28-deficient thymi, while wildtype thymi achieved essentially maximal deletion at the lower dose. Further increases in the dose of anti-CD3 by as much as 10-fold more were able to induce further deletion in both wildtype and CD28-deficient thymi; however, deletion in CD28-deficient thymi never occurred as efficiently as in wildtype thymi. To determine whether CD28 deficiency causes a selective defect in thymocyte deletion following TCR engagement, surviving T cells were analyzed by staining for the expression of CD4 and CD8 (Fig. 2). Using this assay, the survival of more immature double-positive cells (CD41, CD81) can be assessed relative to that of more mature single-positive cells (CD41, CD82 or CD42, CD81). The results indicate that anti-CD3
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FIG. 2. In vivo deletion of double-positive thymocytes. Thymocyte samples from mice injected ip with PBS or 1 or 100 mg of anti-CD3 were stained for CD4 and CD8 as described under Materials and Methods and analyzed by flow cytometry. The percentage of DP cells in each sample is indicated in the figure. The data are representative of at least three independent experiments.
treatment results in the selective cell death of DP cells in wildtype thymi. In contrast, CD28-deficient DP thymocytes were highly resistant to cell death upon treatment with anti-CD3. At a dose of 100 mg of anti-CD3, there were greater than 10-fold more double-positive thymocytes surviving in CD28-deficient compared to wildtype animals (Fig. 2). These data suggest that the enlargement observed in CD28-deficient thymi is due to the decreased efficiency with which deletion occurs in the absence of CD28. In Vitro Clonal Deletion of Thymocytes In order to extend our observations and to control for the systemic effects of anti-CD3, we also analyzed peptide-mediated events in mice expressing the transgenic TCR from the DO11.10 T cell hybridoma. This TCR recognizes chicken ovalbumin (OVA) peptide in the context of I-Ad (27). It has been established that OVA peptide fragment 323–339 delivers a deletional signal to developing thymocytes and also provides a strong activation signal to mature OVA-specific CD41 cells in the periphery (27). In contrast, OVA fragment 324 –334 does not induce responses from transgenic T cells. We first tested both OVA peptides for their ability to activate mature T cells in a standard proliferation assay as measured by [3H]thymidine uptake. As expected,
OVA324 –334 did not elicit any response from either wildtype or CD28-deficient lymphocytes. In contrast, OVA323–339 was able to activate both types of T cells in a dose-dependent manner although the response of the CD28-deficient cells was three- to fourfold lower than the wildtype response (Fig. 3). We then assessed the ability of OVA peptide to deliver a deletional signal when added to thymocyte cultures. The OVA323–339 was able to induce deletion in a concentrationdependent manner in the clonotypic OVA-reactive DP population of TCR transgenic, CD281/1 mice (Fig. 4A). In contrast, little deletion was observed in CD28deficient DP transgenic thymocytes incubated with 10 mM of OVA323–339. Although more significant antigen-specific deletion of clonotypic CD28-deficient DP thymocytes occurred after 3 days of incubation with the highest dose of antigenic peptide, deletion was still approximately three fold lower than that observed in CD281/1 cells. A representative flow cytometry profile of clonotype-positive thymocyte subsets 72 h after incubation with control or deleting peptide is shown in Fig. 4B: a subset of clonotype-positive DP thymocytes was deleted following exposure to the stimulating but not the control peptide. Addition of exogenous APC to in vitro thymocyte cultures increased the level of deletion of clonotypic DP cells exposed to the selecting
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FIG. 3. Proliferation of peripheral T cells bearing a TCR transgene. Bulk lymph node cells were isolated from DO11.10 wildtype and CD28-deficient mice and cultured in medium and 10 or 100 mM OVA323-339 or 100 mM of a control peptide for 48 h. Proliferation was measured as described under Materials and Methods. The data are representative of at least three independent experiments.
thymocytes were equally sensitive to dexamethasoneand g-irradiation-induced death (Fig. 5). All thymocytes subsets (DP, DN, and SP cells) were affected equally by these treatments (data not shown). As an assay of apoptosis, genomic DNA was prepared and analyzed to determine the extent of DNA degradation that had occurred. Figure 6 shows that genomic DNA prepared either from freshly isolated thymocytes or cells that had been cultured in medium without treatment showed no nucleosomal laddering in samples from wildtype and CD28-deficient animals. The slightly smaller, more defined band in the samples that had been in culture for 24 h indicates a slightly higher percentage of cell death in these cells as opposed to the DNA from freshly isolated thymocytes. However, there was no significant apoptosis as indicated by the absence of laddering. In contrast, DNA from both wildtype and CD28-deficient thymocytes exhibited a high degree of laddering in cultures that contained dexamethasone or had been exposed to g-irradiation, indicating that a significant portion of the cells were undergoing apoptosis. Thus, it appears that CD28 does not protect thymocytes from glucocorticoid or g-irradiation induced cell death in thymocytes. Taken together, the above experiments indicate that CD28/B7 interactions play a specific role in enhancing cell death in response to signaling events mediated through the TCR, but do not contribute to thymocyte death in response to glucocorticoids or g-irradiation. DISCUSSION
peptide in both wildtype and CD28-deficient cells (data not shown). However, the difference between CD28deficient and wildtype cells was maintained because CD28-deficient transgenic DP thymocytes underwent significantly less deletion than CD28-expressing cells. Taken together with the experiments using anti-CD3 mAb, these results demonstrate that lack of CD28 results in less efficient deletion of DP thymocytes when TCR signaling is initiated either through crosslinking with antibody or interaction with peptide/MHC. Thymocyte Apoptosis Induced by Glucocorticoids or g-Irradiation We have shown that CD28 plays a role in the efficient induction of deletion in DP thymocytes when the induction is mediated by signaling events through the TCR. Exposure of thymocytes to glucocorticoids and g-irradiation are also well-known methods for induction of cell death in thymocytes (28). In order to determine whether CD28/B7 interactions influenced these mechanisms of cell death, the survival of wildtype and CD28-deficient thymocytes was examined following exposure to different doses of dexamethasone or g-irradiation. Viability of the thymocytes subsets was assessed by flow cytometry. Wildtype and CD28-deficient
This study suggests that the CD28 receptor contributes information that determines whether the threshold for negative selection has been exceeded in a developing thymocyte. In the absence of the CD28 receptor, thymocytes require significantly greater signaling through the TCR to undergo negative selection. CD28 does not appear to be absolutely required in vivo since deletion of thymocytes can be initiated in CD28deficient animals in response to high levels of TCR crosslinking or high doses of antigenic peptide. It is likely that CD28 aids the negative selection process by helping to increase the avidity of the interactions between thymocytes and APC, as a result of increased adherence acting to prolong TCR–MHC engagement. Avidity is thought to be a combination of the density of molecules interacting on thymocytes and APC and the intrinsic affinity of specific TCR for peptide–MHC. Thus, in the absence of CD28 there are fewer total interactions taking place possibly resulting in reduced total avidity and less efficient negative selection. In addition to the reduced density of surface interactions in the absence of CD28, costimulatory signals important for the induction of negative selection may also be lost. Hogquist et al. (2) define efficacy in selection as the overall signaling properties of particular
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FIG. 4. Peptide-induced clonal deletion of thymocytes bearing a TCR transgene. Thymocytes were isolated from DO11.10 wildtype and CD28-deficient mice and cultured in medium, 10 or 100 mM of OVA323–339, or 100 mM of a control peptide for 48 and 72 h. Thymocytes were simultaneously stained with anti-CD4-PE, anti-CD8-tricolor, and anti-KJ126-FITC. A total of 25000 live cells was collected per sample and the percentage of clonotype-expressing thymocytes was determined. The percentage of clonotype-positive DP thymocytes was similar in untreated CD281/1 and CD282/2 thymocytes. For A, percentage specific deletion of clonotype-positive DP cells was calculated as described under Materials and Methods. Bars represent the mean 6 standard deviation from triplicate samples and are representative of four independent experiments. The statistical significance between deletion induced by 100 mM of selecting peptide in CD281/1 and CD282/2 clonotype-expressing DP thymocytes was determined by a Student t test and was P , 0.001. (B) A representative flow cytometry profile of KJ1261 thymocyte subsets 72 h after incubation with 100 mM of control or deleting ovalbumin peptides.
ligands including TCR interactions, peptide concentration, and contributions from accessory molecules. As in the avidity model, high efficacy results in negative selection. The reduced negative selection of DP thymocytes observed in CD28-deficient mice after crosslinking of CD3 suggests that CD28 has intrinsic signaling properties that contribute to the efficient induction of negative selection, and is not simply a result of de-
creased adhesion. Therefore, the less efficient negative selection observed in the absence of CD28 can be explained by lowered avidity and/or efficacy resulting from a combination of reduced density of surface receptors, resultant lower-affinity TCR–peptide interactions, and a loss of CD28 signaling. In contrast to the results obtained when deletion was induced using anti-CD3 antibody or peptide antigen,
FIG. 5. Susceptibility of CD281/1 and CD282/2 thymocytes to dexamethasone- and g-irradiation-induced cell death. Thymocytes from wildtype and CD28-deficient mice were incubated for 24 h in the presence of different concentrations of dexamethasone or exposed to different amounts of g-irradiation. Cells were stained at 24 h with anti-CD4-FITC, anti-CD8-tricolor, and propidium iodide, and analyzed by flow cytometry. 10,000 events/sample were collected and the percentage deletion was calculated based on the number of live cells present among the 10,000 events. Results are expressed as the mean 6 standard deviation from triplicate samples and are representative of two independent experiments.
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FIG. 6. DNA fragmentation in thymocytes treated with g-irradiation or dexamethasone. Thymocytes from wildtype and CD28-deficient mice were cultured in the presence and absence of dexamethasone or exposed to 5 Gy g-irradiation. Genomic DNA was prepared from freshly isolated cells and from treated samples at 24 h and subjected to electrophoresis through agarose gels.
when thymocytes were treated with steroids or g-irradiation, similar amounts of cell death were observed in wildtype and CD28-deficient thymocytes. Previous studies have shown that the thymic microenvironment produces glucocorticoids which are capable of inducing apoptosis in thymocytes, and are thought to be involved in a “death by neglect” mechanism of thymocytes not selected to survive (22). Our results suggest that CD28 signaling is not involved in rescuing thymocytes from glucocorticoids-induced cell death, whereas it plays a role in decreasing the threshold for negative selection. There have been several attempts to examine the significance of the expression of the CD28 counterreceptors, B7-1 and B7-2, on thymic stromal cells. While some studies have found that negative selection can occur under conditions of B7 blockade (5, 19, 20), others have suggested that blocking B7-1 and B7-2 on thymic stromal cells could enhance thymocyte cell survival in response to TCR crosslinking in vitro (21, 29). These experiments have been difficult to interpret because B7-1 and B7-2 share the ability to bind to two separate receptors, CD28 and CTLA-4. Both of these receptors are expressed on TCR-activated T cells but are thought to have opposing functions. In contrast to the ability of CD28 to contribute to mature T cell survival and proliferation, CTLA-4 signal transduction inhibits T cell proliferation and, under some circumstances, can promote cell death (30 –34). B7 expression on thymic stromal cells could promote positive or negative selection by engaging CTLA-4. Alternatively, B7-1 and B7-2 could promote negative selection through CD28 signal transduction. Such signal transduction might boost the signal initiated through the
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TCR to levels that promote a thymocyte’s autonomous decision to undergo apoptosis. The role of CD28 in the promotion of thymocyte negative selection contrasts with its ability to promote survival in peripheral T cells. However, these seemingly contradictory functions may be consistent with the known ability of CD28 to synergize with the TCR to activate intracellular signal transduction. For example, crosslinking of the TCR and CD28 has been shown to synergize in the activation of Jun N-terminal kinase (JNK) (35). Several recent studies have suggested that in immature cells JNK activation can promote apoptosis (36, 37). Thus, one possible mechanism by which CD28 can promote negative selection of thymocytes is by cooperating with TCR signaling to activate the JNK pathway thus resulting in apoptosis. Since negative selection takes place in CD28-deficient mice, additional costimulatory receptors may exist that play a role in negative selection. One other molecule suggested to be involved in negative selection is gp39. Noelle et al. have demonstrated a requirement for gp39 –CD40 interactions in negative selection when antigen was endogenously expressed both by blockade of gp39 and in gp39-deficient mice (38). Interestingly, the same study also demonstrated that B7-2 expression in the thymus is greatly reduced in the absence of gp39, suggesting that gp39 may indirectly affect the role of CD28 in negative selection by altering its ability to interact with its ligand. Another molecule potentially involved in costimulation during T cell development is CD30. CD30-deficient mice have also been demonstrated to have a defect in negative selection with a phenotype similar to that of CD28-deficient mice, i.e., hypercellularity of the thymus with normal percentage distribution of thymic subsets, and a lack of any autoimmune disease (39). As a member of the tumor necrosis factor receptor family, it is possible that CD30 is involved in mediating death signals during negative selection. However, CD30 has been shown to have costimulatory function in mature cells (40, 41) and is just as likely to mediate its function through the increased avidity/efficacy interactions we are proposing for CD28. Finally, Fas has been shown recently also to play a role in negative selection of immature SP thymocytes in vivo when high doses of antigen are used, but not at low doses of antigen (42). To this end, CD28 may help determine the signaling threshold for negative selection that depends on the quantity of antigen expressed, as well as on the ligation of other surface molecules, such as Fas, gp39, and CD30. Alternatively, CD28 signaling may influence the type of thymocyte deletion occurring after TCR ligation, i.e., Fas-dependent or -independent. The data presented here argue for a strength-ofsignal model for thymocyte negative selection. Such a model would explain why CD28, while not absolutely essential for thymocyte selection, plays an important
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role in the determination of developmental fate. CD28 costimulation synergizes with TCR signaling to increase the avidity and/or efficacy of T cell–stromal cell interactions. However, the outcome of these interactions depends on the developmental stage of the T cell. In the periphery, CD28 costimulation promotes survival and expansion of mature T cells, while in the thymus signaling through CD28 contributes to more efficient induction of negative selection.
19. 20. 21. 22. 23. 24.
ACKNOWLEDGMENTS 25. We thank Carol Sampson for secretarial support and Mandel Davis for expert technical assistance. We also thank A. Kelekar, R. Gedrich, R. Arch, E. Chuang, and M. Gilfillan for critical review of the manuscript. S. L. Reiner is supported by the Burroughs Wellcome Fund and the National Institutes of Health (AI-01309). P. J. Noel is supported by a postdoctoral fellowship from the Cancer Research Institute. This work is supported in part by the National Institutes of Health (AI-35294).
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