CD40 Ligand Functions Non-Cell Autonomously to Promote Deletion of Self-Reactive Thymocytes

Joy A. Williams, Susan O. Sharrow, Anthony J. Adams and Richard J. Hodes
J Immunol March 15, 2002, 168 (6) 2759-2765; DOI: https://doi.org/10.4049/jimmunol.168.6.2759

Abstract

CD40 ligand (CD40L)-deficient mice have been shown to have a defect in negative selection of self-reactive T cells during thymic development. However, the mechanism by which CD40L promotes deletion of autoreactive thymocytes has not yet been elucidated. We have studied negative selection in response to endogenous superantigens in CD40L-deficient mice and, consistent with previous reports, have found a defect in negative selection in these mice. To test the requirement for expression of CD40L on T cells undergoing negative selection, we have generated chimeric mice in which CD40L wild-type and CD40L-deficient thymocytes coexist. We find that both CD40L wild-type and CD40L-deficient thymocytes undergo equivalent and efficient negative selection when these populations coexist in chimeric mice. These results indicate that CD40L can function in a non-cell-autonomous manner during negative selection. Deletion of superantigen-reactive thymocytes was normal in B7-1/B7-2 double-knockout mice, indicating that CD40-CD40L-dependent negative selection is not solely mediated by B7 up-regulation and facilitation of B7-dependent T cell signaling. Finally, although the absence of CD40-CD40L interactions impairs negative selection of autoreactive CD4+ and CD8+ cells during thymic development, we find that self-reactive T cells are deleted in the mature CD4+ population through a CD40L-independent pathway.

Negative selection of autoreactive T cells during development in the thymus is a critical step in creating a T cell repertoire tolerant of self. Integral to this process is the removal of thymocytes expressing TCRs with high affinity for self-Ags. Signaling through the TCR is a requisite step in the negative selection process, but a number of in vitro studies have determined that TCR signals alone may not be sufficient to induce death of immature thymocytes (1, 2). Although in vitro manipulation of thymocytes has clearly implicated costimulatory signals in the process of TCR-triggered thymocyte death, the role of costimulatory molecules during in vivo negative selection is less well established.

A critical role for costimulation in negative selection has been reported in studies that demonstrated a defect in negative selection in CD40 ligand (CD40L)2-deficient mice (3, 4, 5). However, the mechanism by which CD40L functions in negative selection has not yet been elucidated. Stimulation of CD40 on APCs by engagement of CD40L expressed on T cells results in increased levels of cell surface molecules including B7-1, B7-2, MHC-II, and LFA-1, as well as increased production of a number of cytokines such as IL-12 and TNF-α (reviewed in Ref. 6). Recently, however, it has been shown that signaling during CD40-CD40L interactions may be bidirectional (7). Signaling through CD40L on T cells has been implicated in promoting cytokine expression and short term T cell effector responses (8). Blair et al. (9) have found that activation of peripheral CD4+ T cells through engagement of CD3 and CD40L results in significantly more apoptotic death than is induced by anti-CD3/CD28 treatment. Thus, signaling through CD40L appears to facilitate activation (and death) of mature T cells. This led us to consider the possibility that direct signaling through CD40L might also play a role in thymic negative selection.

To address the question of whether CD40L functions in a cell-autonomous manner during negative selection, we have used bone marrow chimeras in which developing thymocytes either do or do not express CD40L. We conclude that CD40L knockout (KO) thymocytes are negatively selected as efficiently as CD40L wild-type (WT) thymocytes when these populations coexist in a chimeric environment. Thus, CD40-CD40L interactions can function in negative selection through a non-cell-autonomous mechanism that does not require signaling through CD40L specifically on those T cells undergoing negative selection. Finally, although the absence of CD40-CD40L interactions clearly impairs negative selection of autoreactive CD4+ and CD8+ cells during thymic development, we find that self-reactive CD4+ T cells but not CD8+ T cells are eliminated at a late stage of development through a CD40L-independent pathway, resulting in diminished levels of these cells in the mature CD4+ T cell population.

Materials and Methods

Antibodies

Anti-CD4, -CD8, -CD45.1 (Ly5.2), and -TCR Vβ Abs were purchased from PharMingen (San Diego, CA). TCR Vβ Abs used were as follows: KJ25, anti-murine Vβ3; MR9-4, anti-murine Vβ5.1, Vβ5.2; RR4-7, anti-murine Vβ6; MR5-2, anti-murine Vβ8.1, Vβ8.2; V21.5, anti-murine Vβ10, RR3-15, anti-murine Vβ11; MR11-1, anti-murine Vβ12; and H57-597, anti-murine TCR β-chain.

Mice

BALB/c (BALB), C57BL/6 (B6), and BALB/c × C57BL/6 F1 (CB6F1) mice were obtained from Frederick Cancer Research Facility (Frederick, MD) and were maintained at Bioqual (Rockville, MD). To generate I-E+ CD40L WT and CD40L KO mice expressing the desired superantigens (SAgs), female CD40L KO mice on a B6 background (mammary tumor virus (Mtv)-8+, -9+, and -I-E−) were crossed with CBA/J males (Mtv-6+, -7+, -8+, -9+, -17+, and -I-E+). The CD40L gene is carried on the X chromosome, and male progeny from this cross are therefore B6 × CBA/J CD40L KO. To generate CD40L WT male mice, CBA/J (CD40L WT) females were crossed with B6 (CD40L WT) males. B6 CD40L KO, B6 CD28 KO, BALB CD28 KO, and BALB CD40 KO mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and were maintained at Bioqual. BALB mice deficient in both B7-1-deficient and B7-2 (BALB B7 double-KO (DKO)) were a generous gift from A. Sharpe (10). CB6F1 CD28+/− and CB6F1 CD28−/− mice were generated by crossing B6 CD28+/+ × B6 CD28−/− mice and then crossing B6 CD28+/− progeny to BALB CD28−/− mice.

Flow cytometric analysis

Single-cell suspensions were prepared from thymus and spleen. Anti-FcR mAb 24G2 (blocks FcγII and FcγIII) was added to prevent FcR-mediated binding of mAb to cells, and cells were then incubated with FITC-labeled mAb, PE-labeled mAb, biotinylated mAb, and Cy5 conjugate (PharMingen) sequentially. Viable cells were analyzed by FACScan (BD Biosciences, San Jose, CA) using CellQuest software. When necessary, four-color staining was performed using FITC-labeled mAb, PE-labeled mAb, Cy-5-labeled mAb biotinylated-mAb, and streptavidin-Alexa 594 conjugate (Molecular Probes, Eugene, OR). Viable cells, as determined by propidium iodide exclusion, were then analyzed on a dual-laser FACStarPlus (BD Biosciences). The percent of cells expressing a particular Vβ was determined as follows: (% Vβ+ − % isotype control+)/(% H57 (TCRβ)+ − % isotype control+).

Mixed bone marrow chimeras

Radiation bone marrow chimeras were prepared as described previously (11). Recipient mice were lethally irradiated with 1000 rad and reconstituted with 107 T cell-depleted bone marrow cells. CD40L WT/CD40L KO mixed chimeras were generated by combining equal numbers of bone marrow cells from B6.Ly5.1 × CBA/J (CD40L WT) and from B6.Ly5.2 × CBA/J (CD40L KO) males and reconstituting lethally irradiated B6.Ly5.1 × CBA/J (CD40L WT) host mice. CD40L KO-only chimeras were generated by reconstituting B6.Ly5.1 × CBA/J (CD40L KO) host mice with bone marrow from B6.Ly5.2 × CBA/J (CD40L KO) males. Analysis of chimeras was performed 6–10 wk after reconstitution.

Statistical analyses

Statistical differences were established by Student t test. Values given were obtained by two-tailed paired t tests.

Results

The requirement for CD40L during negative selection is non-cell autonomous

We have examined the role of CD40L in a SAg-mediated model of negative selection in which thymocytes expressing TCRs that recognize endogenous retroviral Ags associated with permissive MHC class II (I-E) molecules are deleted during thymic development (reviewed in Ref. 12). Female B6 CD40L WT or CD40L KO mice were crossed with male CBA/J mice to generate male B6 × CBA/J (Mtv-6+, -7+, -8+, -9+, -17+, and -I-E+) offspring that were CD40L WT or CD40L KO, respectively. Negative selection in response to Mtv-7, -8, and -9 was then examined in these mice. As illustrated in Fig. 1⇓, CD4+ single-positive (SP) thymocytes expressing Vβ5, Vβ6, Vβ11, and Vβ12 were almost undetectable in the B6 × CBA/J CD40L WT mice but were clearly present in the B6 × CBA/J CD40L KO mice, consistent with earlier reports that negative selection of self-reactive T cells is impaired in CD40L-deficient mice (3, 4, 5). A similar pattern of defective negative selection was observed in the CD8+ SP population of B6 × CBA/J CD40 KO mice (data not shown). The frequency of Vβ10-expressing thymocytes was measured as a control for the specificity of deletion, given that Vβ10-containing TCRs do not recognize endogenous Mtv products. In fact, a compensatory increase in the proportion of Vβ10-expressing cells was observed in CD40L WT relative to CD40L KO mice.

FIGURE 1.
FIGURE 1.

SAg-mediated negative selection is defective in CD40L KO mice. Thymocytes from 8-wk-old CD40L WT and CD40L KO B6 × CBA/J mice were stained with PE-anti-CD4, FITC-anti-CD8 and biotin-conjugated anti-TCR Vβ mAbs. TCR Vβ histograms of CD4 SP gated cells were plotted. Numbers in the upper right corner indicate the percent Vβ positive in the CD4 SP gated population. Data are representative of five independent experiments.

To determine whether direct signaling through CD40L is required for deletion of self-reactive thymocytes, we established bone marrow chimeras in which donor-derived T cells consist of a mixture of cells derived from CD40L WT and CD40L KO bone marrow and in which both host and donor mice are I-E+ and express the relevant Mtv Ags 7, 8, and 9. Deletion of Vβ5, Vβ6, Vβ11, and Vβ12-expressing thymocytes was then assessed in both CD40L WT and CD40L KO thymocytes. Our expectation was that if negative selection of a thymic T cell requires signaling through CD40L expressed by that T cell, then only those thymocytes derived from the CD40L WT donor would be deleted in response to SAgs in the chimeras. If, however, the role of CD40L during negative selection is not cell autonomous, then deletion should proceed in both CD40L KO and WT donors. CD40L WT/CD40L KO chimeras were generated by combining equal numbers of bone marrow cells from B6 × CBA/J CD40L WT and B6 × CBA/J CD40L KO males and reconstituting lethally irradiated B6 × CBA/J CD40L WT host mice. The B6 × CBA/J CD40L KO donor cells were Ly5.2+ and thus could be distinguished from the B6 × CBA/J CD40L WT Ly5.2− donor and host populations. Staining for Ly5.2 on thymocytes indicated that CD40L WT and CD40L KO donor-derived cells were present in approximately equal numbers in the thymus (Ly5.2− (CD40L WT), 56 ± 3%; Ly5.2+ (CD40L KO), 44 ± 3%) and was similar in all thymic subpopulations (data not shown). Furthermore, when splenocytes from the chimeras were stimulated with PMA-ionomycin, CD40L was up-regulated on Ly5.2− but not on Ly5.2+ T cells, confirming the ability of Ly5.2 expression to distinguish CD40L-expressing and -deficient T cell populations in these chimeras (Fig. 2⇓). Analysis of Vβ expression in the CD40L WT/CD40L KO chimeras revealed that, as expected, deletion of Vβ5-, Vβ6-, Vβ11-, and Vβ12-expressing thymocytes occurred in the CD40L WT population (Fig. 3⇓). Of particular interest was the fate of Vβ5-, Vβ6-, Vβ11-, and Vβ12-expressing thymocytes in the CD40L KO population. We found that, similar to the CD40L WT population, CD4+ and CD8+ SP thymocytes expressing Vβ5, Vβ6, Vβ11, and Vβ12 were deleted in the CD40L KO population. In contrast, in CD40L KO-only chimeras, made by reconstituting CD40L KO host mice with exclusively CD40L KO bone marrow, deletion of Vβ5-, Vβ6-, Vβ11 and Vβ12-expressing thymocytes was impaired. In CD4 SP thymocytes, the levels of Vβ5-, Vβ6-, Vβ11-, and Vβ12-expressing thymocytes in the CD40L KO-only chimera were similar to those found in the I-E−, nondeleting C57BL/6 controls. In CD8 SP thymocytes, the comparability with the C57BL/6 control was less consistent. Overall, the results from the CD40L WT/CD40L KO chimeras indicate that direct cell-autonomous signaling via CD40L is not required for SAg-mediated negative selection.

FIGURE 2.
FIGURE 2.

PMA-ionomycin-stimulated CD40L expression on splenic T cells from mixed CD40L WT-CD40L KO bone marrow chimeras. Purified splenic T cells were stimulated with 50 ng/ml PMA and 1 μg/ml ionomycin for 6 h. Surface expression of CD40L was analyzed on Ly5.2− and Ly5.2+ gated CD4+ T cells. Bold line, CD40L; thin line, negative Ab control.

FIGURE 3.
FIGURE 3.

Negative selection in mixed CD40L WT-CD40L KO bone marrow chimeras. CD40L WT-CD40L KO mixed chimeras were generated by reconstituting lethally irradiated B6.Ly5.1 × CBA/J (CD40L WT) host mice with equal numbers of bone marrow cells from B6.Ly5.1 × CBA/J (CD40L WT) and from B6.Ly5.2 × CBA/J (CD40L KO) males. CD40L-only chimeras were generated by reconstituting B6.Ly5.1 × CBA/J (CD40L KO) host mice with bone marrow from B6.Ly5.2 × CBA/J (CD40L KO) males. Thymocytes were obtained from chimeras 6–10 wk after reconstitution and stained with FITC-anti-Ly5.2, PE-anti-CD4, Cy5-anti-CD8, and biotin-conjugated anti-TCR Vβ mAbs. The percentages of Vβ-positive CD4 SP and CD8 SP thymocytes in the CD40L WT and CD40Lnull populations of the mixed chimera are shown separately (n = 7). CD40L KO-only bone marrow chimeras were analyzed by gating on Ly5.2+ thymocytes (n = 4). Thymocytes from 8- to 12-wk C57BL/6 mice (n = 4) are shown as a nondeleting control. CD40L KO-only chimera values are statistically different from both population values in the mixed CD40L WT/CD40L KO chimeras by t test (∗, p < 0.05; ∗∗, p < 0.005; ∗∗∗, p < 0.0005).

CD40L-dependent negative selection does not require B7-CD28 interactions

The results from the CD40L WT/CD40L KO chimeras suggest that the role of CD40L during negative selection may be to regulate expression of other costimulatory molecules and/or cytokines through engagement of CD40, the only known CD40LR. To test the role of CD40 directly, SAg-mediated negative selection was examined in BALB CD40 KO mice. BALB mice are I-E+; express Mtv Ags 6, 8, and 9; and delete thymocytes expressing a number of Vβs including Vβ5, Vβ11, and Vβ12. Comparison of Vβ5, Vβ11, and Vβ12 frequencies in thymocytes from B6 (nondeleting strain), BALB CD40 WT, and BALB CD40 KO controls revealed impaired negative selection in CD40 KO mice (Fig. 4⇓) similar to the defect observed in CD40L KO mice. These findings indicate that interaction of CD40 with CD40L is required for intrathymic negative selection.

FIGURE 4.
FIGURE 4.

Negative selection is defective in BALB CD40 KO mice. Thymocytes from 8- to 12-wk-old C57BL/6 (n = 5), BALB CD40 WT (n = 4) and BALB CD40 KO (n = 5) mice were stained with FITC-anti-CD4, PE-anti-CD8, and biotin-conjugated anti-TCR Vβ Abs. The percent Vβ-positive CD4 SP and CD8 SP thymocytes in each strain is shown. BALB CD40 KO values are statistically different from BALB CD40 WT values by t test (∗∗, p < 0.005; ∗∗∗, p < 0.0005).

It has been reported that engagement of CD40 by CD40L induces increased expression of B7-1 and B7-2 on CD40-expressing cells (13). To determine whether CD40-CD40L interactions might function in negative selection by up-regulating B7, thus leading to enhanced B7-CD28 interactions, we examined SAg-mediated negative selection in CB6F1 (B6 × BALB) CD28 KO and in BALB B7-1-B7-2 DKO (B7 DKO) mice (10). SAg-mediated negative selection was found to be intact in the absence of CD28; the extent of deletion of Vβ5-, Vβ11-, and Vβ12-expressing thymocytes was similar in CB6F1 (I-E+, Mtv-6+, Mtv-8+, Mtv-9+) CD28 KO and CB6F1 CD28+/− mice (Fig. 5⇓). SAg-mediated negative selection in BALB B7 DKO mice was assessed to determine whether the failure to observe effects on negative selection in CD28 KO animals might reflect the use of alternate B7 receptors such as CTLA-4. However, as shown in Fig. 6⇓, deletion of Vβ5-, Vβ11-, and Vβ12-bearing thymocytes proceeds in the absence of both B7-1 and B7-2. A small, but statistically insignificant, increase in the percentage of CD4+ SP thymocytes expressing Vβ5, Vβ11, and Vβ12 is observed in the B7 DKO mice, but the levels of these cells are significantly lower than those observed in the absence of CD40 or CD40L. Thus, the failure to observe a substantial effect on SAg-mediated thymic deletion in the absence of either CD28 or B7 indicates that CD40-CD40L interactions do not rely solely on signals derived from B7-CD28 interactions to affect negative selection.

FIGURE 5.
FIGURE 5.

Negative selection is intact in CB6F1 CD28 KO mice. Thymocytes from 8- to 12-wk-old C57BL/6 (n = 5), CB6F1 CD28+/− mice (n = 4), and CB6F1 CD28 KO (n = 4) mice were stained with FITC-anti-CD4, PE-anti-CD8, and biotin-conjugated anti-TCR Vβ Abs. The percent Vβ-positive CD4 SP and CD8 SP thymocytes in each strain is shown. Values for CD4 and CD8 SP thymocytes in CB6F1 CD28+/− and CB6F1 CD28−/− mice were not statistically different as determined by t test.

FIGURE 6.
FIGURE 6.

Absence of both B7-1 and B7-2 has minimal effect on negative selection. Thymocytes from 8- to 12-wk-old C57BL/6 (n = 5), BALB B7 WT (n = 4), and BALB B7 DKO (n = 6) mice were stained with FITC-anti-CD4, PE-anti-CD8, and biotin-conjugated anti-TCR Vβ Abs. The percent Vβ-positive CD4 SP and CD8 SP thymocytes in each strain is shown. BALB B7 DKO values are not statistically different from BALB B7 WT values by t test.

CD40L-independent deletion of mature T cells

The fate of potentially self-reactive thymocytes that escape SAg -mediated deletion in the thymus of CD40L KO mice was explored by determining frequencies of Vβ5-, Vβ11-, and Vβ12-expressing T cells in the spleens of CB6F1 CD40L KO mice. Interestingly, despite the failure to observe negative selection in CD4+ SP thymocytes, peripheral CD4+ T cells expressing self-reactive Vβs were deleted in CD40L KO mice (Fig. 7⇓). This was not the case for peripheral CD8+ T cells where deletion did not occur and where percentages of Vβ5, Vβ11, and Vβ12 remain elevated in CD40L KO mice (Fig. 7⇓). Thus, CD8+ T cells that normally would be deleted in mice expressing endogenous SAgs were present in the periphery of CD40L mice, whereas CD4+ T cells expressing these same Vβs were deleted. Analysis of CD40 KO mice yielded similar results (data not shown). These results suggest that the deletion of SAg-reactive cells is occurring in CD4+ cells subsequent to down-modulation of CD8, either late in thymic SP development or in the periphery. To distinguish between these two possibilities, CD4+ SP thymocytes from CB6F1 CD40L KO mice were stained with HSA to differentiate mature (HSAlow) from immature (HSAhigh) SP cells (14). Fig. 8⇓ shows that the frequencies of CD4+ SP thymocytes expressing Vβ5, Vβ11, and Vβ12 are significantly decreased as cells progress from the HSAhigh to HSAlow stage in the thymus and are further decreased in the periphery. HSAlow CD4+ SP thymocytes comprise a minor proportion of total CD4+ SP thymocytes; thus, deletion in this population is not apparent without gating on HSAhigh and HSAlow subsets. The frequency of Vβ10-expressing cells, which are not subject to SAg-mediated deletion, is similar at all maturational stages. These data indicate that a pathway exists for CD40-CD40L-independent negative selection of mature SAg-reactive CD4+ T cells.

FIGURE 7.
FIGURE 7.

Late negative selection of SAg-reactive CD4 T cells occurs in CD40L KO mice. Thymocytes and splenocytes from 8- to 12-wk-old CB6F1 CD40L KO mice (n = 4) were stained with FITC-anti-CD4, PE-anti-CD8, and biotin-conjugated anti-TCR Vβ Abs. The percent Vβ-positive CD4 SP and CD8 SP thymocytes and splenocytes is shown. CB6F1 CD40L KO CD4 SP splenocyte values are statistically different from thymocyte values as determined by t test (∗, p < 0.05; ∗∗∗, p < 0.0005). CD8 SP splenocyte and thymocyte values were not statistically different.

FIGURE 8.
FIGURE 8.

Late deletion of SAg-reactive CD4 T cells in CD40L KO mice occurs as thymocytes transition from HSAhigh to HSAlow SP phenotype. Thymocytes and splenocytes from 8- to 12-wk-old CB6F1 CD40L KO mice (n = 4) were stained with FITC-anti-HSA, PE-anti-CD4, Cy5-anti-CD8, and biotin-conjugated anti-TCR Vβ Abs. The percent Vβ-positive CD4 SP cells was determined after gating on HSAhigh and HSAlow populations. CB6F1 CD40L HSAhigh values are statistically different from HSAlow values by t test (∗, p < 0.05; ∗∗, p < 0.005).

Discussion

Recent studies have demonstrated that expression of CD40L is required for efficient thymic negative selection (3, 4, 5). However, it has not been previously determined whether CD40L expression is required on the cell undergoing negative selection or whether its role in this process is indirect, resulting in modulation of downstream effector events. In our studies, we have demonstrated that CD40-CD40L-dependent negative selection does not require that CD40L be expressed on those thymocytes that are themselves undergoing negative selection. This was established using bone marrow chimeras that had been reconstituted with a mixture of CD40L WT and CD40L KO bone marrow cells. We found that deletion of thymocytes bearing TCRs reactive to endogenous Mtv Ags proceeded efficiently in both the CD40L WT and CD40L KO populations. This finding suggests that CD40-CD40L interactions can function indirectly during negative selection to regulate downstream events such as increased expression of costimulatory molecules and/or cytokines that promote negative selection. Thus, while it remains possible that direct signaling through CD40L may play a role during negative selection, our results indicate that the effects of any such signaling can be masked by non-cell-autonomous effects of CD40-CD40L interactions.

The identity of the CD40- and CD40L-expressing cells which participate in the negative selection process is not yet well defined. Expression of CD40 has been demonstrated on thymic cortical epithelial cells as well as medullary epithelial and dendritic cells (15, 16, 17). However, it has been reported that CD40L expression appears to be restricted to relatively mature thymocytes localized to the medulla (15), suggesting that CD40-CD40L interactions participating in negative selection would have their effect at this stage of development. As discussed below, this is consistent with the observation that SAg-mediated deletion in CD40-CD40L-intact mice occurs in relatively mature thymocytes transiting from the double-positive to the SP stage (18, 19).

Among the known effects of CD40 signaling on APCs is the induction of increased expression of B7-1 and B7-2 molecules, the two known ligands for CD28. Foy et al. (3), in their original study of deficient negative selection in CD40L KO mice, found that thymic expression of B7-2 was substantially reduced in CD40L KO compared with WT mice. These authors raised the possibility that the role of CD40 signaling during negative selection might be to increase expression of B7 molecules, leading to increased interaction of B7 with CD28 and/or CTLA-4 on developing thymocytes. However, consistent with previous reports (20, 21), we have found that SAg-mediated negative selection proceeds normally in CD28 KO mice. Recently, CTLA-4 has been implicated in negative selection (22) and evidence to suggest the existence of other, yet undefined, B7 receptors has been obtained in studies of CD28-CTLA-4 DKO (23, 24). These findings suggested that, although negative selection is intact in CD28 KO mice, defects in negative selection might be observed in the absence of both B7-1 and B7-2. We have found, however, that SAg-mediated negative selection is not significantly affected in B7 DKO mice, demonstrating that B7-1 and B7-2 are not essential for negative selection and that the role of CD40-CD40L interactions during negative selection cannot be explained solely by increased interactions between B7 molecules and their ligands.

Previous studies in WT mice have indicated that negative selection of thymocytes specific for SAgs occurs during the double-positive to SP transition at a stage where the cells undergoing selection express both CD4 and CD8 (18, 19). This observation has been invoked to provide an explanation for the deletions observed in both CD4+ SP and CD8+ SP lineages in response to SAg that appear to be recognized by CD4-dependent MHC II-restricted T cells. Consistent with previous reports, we have demonstrated that the defect in negative selection observed in the absence of CD40 or CD40L results in dramatic increases in the numbers of self-reactive thymocytes maturing to both the CD4+ and CD8+ SP lineages (3, 4, 5). Interestingly, a late-acting deletional mechanism appears to prevent substantial accumulation of CD4+ cells in the periphery. The late deletion which occurs in CD40 and CD40L KO mice is not apparent until the transition from HSAhigh to a more mature HSAlow phenotype. It affects CD4+ but not CD8+ SP cells, plausibly because CD8+ T cells, lacking expression of CD4, are not susceptible to deletion in response to SAgs presented in the context of MHC class II molecules. In preliminary experiments, we find that this late CD40L-independent deletion can occur in the absence of CD28 or of both B7-1 and B7-2 molecules, as assessed in mice in which both the CD40-CD40L and CD28-B7 pathways have been genetically disrupted (data not shown).

It has been postulated that differences in the avidity of interactions between TCR and MHC plus Ag complexes could alter the stage of thymic differentiation during which negative selection occurs, with stronger interactions promoting earlier deletion (25). CD40-CD40L interactions could impact signals received by the TCR by affecting expression of MHC plus Ag complexes. Stimulation of CD40 on APCs results in enhanced presentation of MHC-II-Ag complexes due to increases in both MHC II levels and enhanced peptide loading of MHC II molecules (13, 26, 27). Thus, the absence of CD40 signaling could result in decreased presentation of superantigen as well as peptide-derived Ag and a subsequent delay in deletion of self-reactive T cells. CD40-CD40L interactions have previously been shown to be critical to deletion of TCR-transgenic thymocytes developing in the presence of cognate Ag (3). This indicates that the effects, if any, of CD40-CD40L interactions on Ag presentation within the thymus are not restricted to the SAg system.

Several recent reports have provided compelling evidence for the existence of multiple, redundant costimulatory pathways which may operate to promote deletion of SP thymocytes. Kishimoto and Sprent (2) reported that in vitro stimulation of immature (HSAhigh) CD4+ SP thymocytes with anti-TCR Ab plus mAbs specific for several potential costimulatory molecules (CD5, CD43, or CD28) resulted in increased cell death compared with treatment with anti-TCR alone. Thus, it would be expected that modulation of in vivo negative selection resulting in increased numbers of autoreactive, HSAlow mature thymocytes would require inhibition of several costimulatory pathways. This expectation is supported in recent work by Page (28), who found that accumulation of autoreactive, HSAlow, mature thymocytes was indeed apparent only when multiple costimulatory pathways were inhibited concurrently using blocking Abs directed to CD5, B7 and TNFR. The results of our studies presented here extend these previous findings by showing that deletion of SP thymocytes can proceed in the absence of CD40-CD40L interactions, indicating that the role of these costimulatory molecules, if any, is also redundant at this late deletional stage.

Thus, we conclude that negative selection during thymic development can proceed by at least two apparently distinct pathways that differ both in the stage in which deletion is manifest and in the requirement for CD40-CD40L interactions. In the first, deletion occurs before the SP stage and is dependent on CD40-CD40L interactions but does not require cell autonomous expression of CD40L. The second occurs at a later stage of differentiation as thymocytes transition from the HSAhigh to HSAlow maturational stage and does not require CD40-CD40L interactions.

Acknowledgments

We thank Alfred Singer, Avinash Bhandoola, Joanne Lumsden, and Karen Hathcock for critical reading of the manuscript. We thank Genevieve Sanchez and staff at Bioqual for excellent animal care and husbandry and thank Arlene Sharpe for generously providing BALB B7 DKO mice.

Footnotes

  • ↵1 Address correspondence and reprint requests to Dr. Joy A. Williams, Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, 10 Center Drive, Room 4B-10, Bethesda, MD 20892. E-mail address: williajo{at}mail.nih.gov

  • ↵2 Abbreviations used in this paper: CD40L, CD40 ligand; SP, single-positive; KO, knockout; DKO, double-KO; WT, wild type; HSA, heat-stable Ag; SAg, superantigen; Mtv, mammary tumor virus.

  • Received November 8, 2001.
  • Accepted January 14, 2002.

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