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The Journal of Immunology, 1999, 163: 5250-5256.
Copyright © 1999 by The American Association of Immunologists

CD4+ T Cell Responses to CD40-Deficient APCs: Defects in Proliferation and Negative Selection Apply Only with B Cells as APCs1

Minette E. Ozaki*, Barbara A. Coren*, Tracy N. Huynh*, Deborah J. Redondo*, Hitoshi Kikutani2,{dagger} and Susan R. Webb3,*

* Ozaki, Coven, Huynh, Redondo and Webb-Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037; and {dagger} Kikutani-Department of Molecular Immunology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
During T-APC interactions in vivo, interfering with CD40-CD154 interactions leads to reduced T cell priming, defects in effector function, and, in some cases, T cell tolerance. As shown here, however, presentation of conventional peptide Ags by CD40-deficient spleen APC in vitro leads to normal CD4+ T cell proliferative responses. By contrast, responses to the same peptides presented by purified B cells were markedly reduced in the absence of CD40. Thus, the requirement for CD40-CD154 interactions appears to be strongly influenced by the type of APC involved. Analysis of responses to endogenous superantigens, which are known to be strongly dependent on B cells for presentation, indicated that CD4+ responses to strong Ags are less dependent on CD40 than are responses to weak Ags. Similar findings applied to negative selection in the thymus. Thus, deletion of potentially autoreactive cells depended on CD40 expression when B APC were involved, and this requirement was most pronounced when negative selection was directed to weak Ags.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Aprominent member of the TNF receptor family, CD40, is widely expressed in vivo and is detectable on APC (i.e., dendritic cells (DC),4 monocytes/macrophages, and B cells), endothelial cells, epithelial cells, mast cells, fibroblasts, and even smooth muscle cells (reviewed in Refs. 1, 2, 3, 4, 5). The ligand for CD40, CD154, is also widely expressed, notably on T cells, NK cells, basophils, mast cells, eosinophils, platelets, and others. Given this broad pattern of distribution of both receptor and ligand, it is not surprising that CD40-CD154 interactions are thought to play key roles in a diverse array of in vivo activities during typical immune responses. Ligation of CD40 leads to cell type-specific responses, which frequently include proliferation, maturation, increased survival, increased expression of cell surface proteins involved in the regulation of immune responses (e.g., CD80, CD86, CD54, and others) and production of important soluble mediators such as cytokines (e.g., IL-6, TNF-{alpha}, IL-12, IL-10, and IL-1) and chemokines (e.g. macrophage inflammatory protein-1{alpha}) (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14).

The absence of CD40-CD154 interactions during the initiation of T-dependent immune responses in vivo has marked effects on these responses. Thus, Ab to CD154 inhibits T-dependent humoral responses (15), graft-vs-host disease (16), tissue rejection (17, 18, 19, 20, 21), as well as disease in a variety of murine models for autoimmunity (22, 23, 24, 25, 26). Similarly, CD154-deficient mice fail to mount normal proliferative responses to protein Ags administered in adjuvant (27, 28), are unable to generate effective CTL responses to some Ags (29, 30, 31, 32), and do not develop autoimmune disease in a murine experimental allergic encephalomyelitis model (33). Given the many potential activities of CD40-CD154 in vivo mentioned above, it is difficult to definitively associate particular activities with particular defects in T cell function. To simplify the analysis of T-APC interactions to gain additional insight into the role of CD40 in inducing CD4+ T cell proliferative responses, we examined the role of CD40 in in vitro responses of naive CD4+ cells to Ags presented predominantly by conventional APC (DC and macrophages) or B cells.


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

BALB/c, C57BL/6J, B10.BR, AKR/J, and D1.LP mice (6–12 wk) were obtained from The Jackson Laboratory (Bar Harbor, ME). BALB.D2, D011 TCR transgenic mice, and CD40-deficient mice were bred here at The Scripps Research Institute. CD40-deficient mice (34) were bred to AKR/J or D1.LP mice for five generations and intercrossed to obtain homozygous mtv-7-positive, CD40-deficient offspring. D011 TCR transgenic mice were similarly crossed to CD40-/- mice and after four to seven generations intercrossed to obtain transgenic, CD40-/- mice.

Reagents

Hybridomas secreting Abs to Thy-1 (J1j), CD8 (3.168), CD4 (RL172), D011 TCR (KJ1.26), HSA (J11d), IAd (MKD6), and Vß6 (RR47) are maintained in our laboratory as a source of Ab for cell purification and flow cytometry. Directly conjugated, anti-CD4-FITC, anti-CD4-APC, and anti-CD8-PE were purchased from PharMingen (San Diego, CA) or Life Technologies (Gaithersburg, MD), mouse anti-rat Ig-FITC was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA), and streptavidin coupled to FITC, PE (Life Technologies), or APC was obtained from PharMingen.

Cell purification and culture

D011 CD4+ T cells were purified from pooled lymph nodes (LN) by passage over nylon wool columns followed by Ab- and C'-mediated cytotoxicity using anti-CD8, anti-HSA, anti-H2-Ad, and complement (Rockland, Gilbertsville, PA) as previously described (35). Cells (4–5 x 104) were cultured with 5 x 105 mitomycin C-treated spleen cells depleted of T cells by treatment with anti-Thy-1, anti-CD8, anti-CD4 mAb, and complement. The culture medium was RPMI 1640 supplemented with glutamine, 5% NCTC 109, 10% FCS, 5 x 10-5 M 2-ME, and antibiotics. Proliferation was assessed by measuring the uptake of [3H]thymidine during an 18-h pulse at the indicated times. All cultures were performed in triplicate.

For responses to mtv-7, CD4+ cells were enriched from C57BL/6 or B10.BR mice using Ab- and C'-mediated cytotoxicity only. CD4+ cells (1–2 x 105) were cultured with 5 x 105 T-depleted spleen stimulators as described above.

B cells were purified from CD40-/- or CD40+/+ mice by sequential passage over G10 columns, followed by Ab and complement-mediated cytotoxicity using anti-Thy-1, anti-CD4, and anti-CD8 mAbs as described above, and finally separation on discontinuous Percoll gradients as previously described (36).

Flow cytometry

Cells were incubated sequentially for 20 min with the appropriate concentration of the indicated Abs. Cells were washed with PBS containing 2.5% {gamma}-globulin-free horse serum and 0.1% NaN3 between incubations. Normal rat serum was used to block potential cross-reactivity of secondary and tertiary reagents as necessary, and propidium iodide was added to permit exclusion of dead cells. FACSort, FACSCalibur, or FACScan was used for collecting data on stained cells and WINMDI software (Joe Trotter, The Scripps Research Institute) used for data analysis.

Fetal thymic organ culture (FTOC)

Thymus lobes were harvested from embryos on days 14–15 of gestation, before the emergence of mature single-positive (SP) thymocytes. The lobes (about four per culture) were cultured as previously described (37) on floating rafts of Gelfoam sponges (Upjohn, Kalamazoo, MI) in RPMI 1640 culture medium (see above). After 5–6 days, cell suspensions were assessed for cell number and analyzed by flow cytometry.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD40-deficient splenic APC stimulate strong primary CD4+ T cell proliferative responses

To examine the CD40 dependence of CD4+ T cell proliferative responses in vitro, we used splenic APC from normal CD40+/+ mice vs CD40-deficient, CD40-/- mice. CD4+ T cells were purified from OVA peptide-specific D011 TCR transgenic mice and cultured with OVA and T-depleted splenic APC from CD40+/+ vs CD40-/- H2-Ad-positive mice. As illustrated in Fig. 1Go, A and B, CD40-/- spleen APC induced strong primary proliferative responses that were equivalent to or slightly higher than responses to CD40+/+ spleen cells; these findings are representative of eight experiments and applied regardless of the number of APC added or the concentration of OVA peptide used. Responses induced using CD40-/- splenic APC were higher than those induced by normal APC in approximately half the experiments. Although we have no explanation for this variability, the key point is that proliferation was never reduced in the absence of CD40 expression on APC. The data confirm a previous report in which high in vitro peptide-specific proliferative responses by TCR transgenic CD4+ cells occurred in the absence of CD40-CD154 interactions (27). CD40-/- spleen APC are thus not intrinsically defective in stimulating naive CD4+ T cells.



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  		FIGURE 1. Spleen APC from CD40-/- mice stimulate strong primary CD4+ T cell proliferative responses. D011 CD4+ T cells (5 x 104/well), purified as described in Materials and Methods, were cultured with a titration of OVA peptide (from 0.01 to 10 µM) and T-depleted, mitomycin C-treated spleen cells (5 x 105) from either normal CD40+/+ or CD40-/- mice. Proliferation was measured on days 3–5, and representative responses in two separate experiments are illustrated (A, day 4; B, day 3). These assays included titration of splenic APC number; at no concentration were significant differences in the APC function of CD40-/- and CD40+/+ APC noted.

 
Resting B cells from CD40-deficient mice fail to induce primary peptide-dependent proliferative responses

The above data refer to responses directed to T-depleted spleen APC, a mixture of DC, macrophages, and B cells. With purified B cells as APC, the results were quite different (Fig. 2Go). Thus, proliferative responses of D011 CD4+ cells were high with CD40+/+ B cells as APC, but low with CD40-/- B cells. With OVA peptide at 1 µM, responses elicited by CD40-/- B cells were minimal on days 2–5 of culture (Fig. 2GoB); by contrast, with CD40-/- T-depleted spleen as APC, responses on days 2–5 were as high as those with CD40+/+ spleen APC (Fig. 2GoA). With high concentrations of OVA peptide (10 µM), detectable responses with CD40-/- B cells were seen on day 5. In the experiment shown, these responses were far lower than those with CD40+/+ B cells (Fig. 2GoC), although still highly significant (52,800 vs <100 cpm in the absence of OVA peptide). In another experiment, the responses induced by CD40-/- B cells were somewhat higher, although, again, the peptide concentration required for these responses was 100-fold higher than that for CD40+ B cells.



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  		FIGURE 2. CD40-deficient B cells are poor APC for D011 CD4+ proliferative responses. D011 CD4+ cells (4 x 104) were cultured with a titration of OVA peptide (from 0.01–10 µM) and either T-depleted splenic APC (A) or purified B cells (B and C). Proliferation was measured on days 2–5. The kinetics of the response to 1 µM OVA is illustrated in A and B, and the dose response on day 5 is illustrated in C.

 
The capacity of CD40-/- B cells to induce proliferative responses depends on the strength of the Ag stimulus

To obtain further information on the role of CD40 in the APC function of B cells, we studied primary proliferative responses of normal, nontransgenic CD4+ T cells to mtv-7-encoded superantigens (SAG) (4). Here it is well established that mtv-7 SAGs are presented primarily if not exclusively by B cells (36); conversely, presentation of MHC alloantigens is controlled largely by DC. Presentation of mtv-7 SAGs to CD4+ cells is MHC class II restricted and is strong with H2-E+ APC and weak, although significant, with H2-E- (H2-A+) APC (38).

The strong response of B10.BR (H2-Ak) CD4+ T cells to mtv-7 SAG presented by H2-A-compatible, H2-E+ AKR/J T-depleted spleen APC is shown in Fig. 3GoA. Proliferative responses to AKR/J APC were very high with CD40+/+ APC and lower, but significant, with CD40-/- APC. By contrast, CD40-/- AKR/J APC were fully capable of eliciting responses directed to MHC alloantigens (Fig. 3GoB); thus, for stimulating H2-different, mtv-7-compatible BALB.D2 (H2d, mtv-7+) CD4+ cells, CD40-/- AKR/J APCwere as effective as CD40+/+ APC. These findings are representative of five individual experiments. Confirming that the residual mtv-7-specific proliferation to AKR/J CD40-/- APC depended on B cells, depletion of B cells using anti-Ig-coated magnetic beads removed the capacity to stimulate proliferative responses (Fig. 3GoC).



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  		FIGURE 3. CD4+ T cell proliferative responses to mtv-7 SAGs presented by CD40+/+ vs CD40-/- APC. CD4+ T cells (1.5 x 105) were cultured with T-depleted, mitomycin C-treated splenic APC, and proliferation was measured on days 3–5. A, B10.BR (H2-Ak, mtv-7-negative) CD4+ cells were cultured with the indicated number of T-depleted spleen cells from mtv-7-positive, H2-Ak-matched AKR/J mice; the proliferative response to mtv-7 SAG measured on day 4 is illustrated. B, BALB.D2, H2-Ad, mtv-7-positive CD4+ cells were cultured with H2-A-mismatched, mtv-7-matched AKR/J spleen cells; the day 4 response to allogeneic class II Ags is shown. C, B10.BR CD4+ T cells were cultured with B10.BR or AKR/J T-depleted spleen cells or with AKR/J T-S cells after removal of Ig+ cells using sheep anti-mouse Ig-coated magnetic beads (Dynabeads, Dynal, Chantilly, VA). [3H]Thymidine was measured on days 2–5; the results from day 3 are shown. D, CD4+ cells from H2-Ab, mtv-7-negative C57BL/6 mice were cultured with T-depleted spleen APC from H2-Ab-matched, mtv-7-positive D1.LP mice; the response to mtv-7 SAG using 5 x 105 APC is illustrated. E, C57BL/6 CD4+ T cells were stimulated with LPS blasts (24-h culture) from D1.LP mice; the peak response to mtv-7 SAG (measured on day 4) is shown.

 
The above data indicate that the capacity of CD40-/- APC to present mtv-7 SAGs in association with strongly stimulatory H2-E molecules is reduced (relative to that of CD40+/+ APC), but is clearly significant. With responses directed to mtv-7 presented by weakly stimulatory H2-E- APC, the results were different. Thus, for proliferative responses of C57BL/6 (H2-Ab, mtv-7-) CD4+ cells to D1.LP (H2-Ab, mtv-7+) APC, responses were high for CD40+/+ APC, but undetectable with CD40-/- APC (Fig. 3GoD). The poor stimulatory capacity of D1.LP CD40-/- B cells was observed in five experiments. These data corroborate the above findings on peptide presentation by B cells and indicate that the requirement for CD40 expression on B cells as APC is most pronounced for weak Ags.

With B cells as APC, the requirement for CD40-CD154 interactions could reflect the need to induce expression of important costimulatory molecules such as B7.1, B7.2, CD44, and ICAM-1 on B cells, molecules well known to be up-regulated by CD40 signals (39, 40, 41, 42, 43). If so, inducing prior up-regulation of these molecules by pretreating B cells with LPS would be expected to bypass the need for CD40 expression. As shown in Fig. 3GoE, however, proliferative responses of B6 CD4+ cells to D1.LP LPS-blasts were very low, suggesting that factors other than costimulatory molecule expression contributed to the poor immunogenicity of CD40-/- D1.LP B cells.

Requirement for CD40 in negative selection

The above data suggest that several factors may influence the requirement for CD40-CD154 interactions in T cell responses; these factors include the type of APC involved, the density of ligand on the APC, and the immunogenicity of the ligand concerned. To determine whether these factors influenced the requirement for CD40 in negative selection of thymocytes, we examined intrathymic deletion of mtv-7-reactive Vß6+ T cells in mtv-7+ CD40+/+ vs CD40-/- AKR/J (H2-E+) mice. Deletion of Vß6+ cells in normal mtv-7 mice is a highly reproducible and well-documented finding. Thus, in CD40+/+ AKR/J mice, deletion of Vß6+ cells was limited for CD4+8+ cells (data not shown), but was near complete at the level of SP CD4+8- cells (Fig. 4GoA). Deletion of Vß6+ cells was also prominent in CD40-/- AKR/J mice; whereas; CD4+ SP thymocytes from MHC-matched, mtv-7-negative B10.BR mice reproducibly contain around 7% Vß6+ cells, CD40-/- AKR/J CD4+ thymocytes contain only 2% Vß6+ cells; in LN, by contrast, Vß6+ cells were undetectable. There is some indication of defective deletion, however, since CD40+ AKR/J mice have virtually no Vß6+ CD4+ T cells (~0.1%) in either thymus or LN. Thus, for presentation of mtv-7 by strongly immunogenic H2-E molecules, CD40 plays only a minor role in negative selection. In H2-E- D1.LP mice, the results were quite different. Here, CD40 appears to play a decisive role in negative selection. Intrathymic deletion of Vß6+ cells is clearly apparent (though incomplete) in CD40+/+ D1.LP mice (1.6% Vß6+ CD4+ in D1.LP vs 6.5% for the MHC-matched, mtv-7-negative control strain, C57BL/6). However, we found little evidence for deletion of Vß6 cells in CD40-/- D1.LP mice (6.1% Vß6+, CD4+ cells vs 6.5% for the MHC-matched, mtv-7-negative control strain C57BL/6). The results shown in Fig. 4Go are the pooled data from three experiments. Thus, as for stimulation of mature T cells, the strength of the stimulus appeared to play a key role in determining the requirement for CD40 in negative selection. Interestingly, unlike the thymus, spleen and LN of CD40-/- D1.LP mice showed considerable depletion of Vß6+ cells, implying that these cells underwent deletion in the post-thymic environment.



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  		FIGURE 4. Negative selection of Vß6+ CD4+ cells in mtv-7-positive CD40+/+ vs CD40-/- mice. Thymus and LN cell suspensions from the indicated mice were stained with Abs to Vß6+ (biotin conjugated and detected with streptavidin-FITC), CD4 (directly conjugated to APC), and CD8 (conjugated to PE). The data indicate the proportion of CD4+, CD8- cells expressing Vß6 TCRs in the LN or thymus. No significant differences in the expression of control Vß8.2+ cells were noted in these experiments. B10.BR and C57BL/6 mice are mtv-7 negative and are used to illustrate the normal percentage of Vß6+ CD4+ cells in H2-Ak and H2-Ab mice, respectively. AKR/J (H2-Ak) and D1.LP (H2-Ab) are mtv-7 positive. The data are pooled from three experiments each, and SDs are indicated.

 
To examine the requirement for CD40 expression in negative selection of thymocytes to conventional Ags, we used a fetal thymus organ culture (FTOC) system. Day 14–15 fetal thymi from D011 TCR transgenic mice were cultured with a titration of OVA peptide, and the proportion of TCR Id+ cells was enumerated after 5–6 days. As illustrated in Fig. 5GoA, significant numbers of Id+ cells were generated during the culture period regardless of whether the fetal thymi were taken from CD40+/+ or CD40-/- mice. With CD40+/+ thymi, addition of 0.1 µM OVA peptide caused marked deletion of both CD4+8+ and CD4+8- Id+ thymocytes (Fig. 5Go, A and B). Significantly, essentially identical deletion of Id+ cells occurred in CD40-/- thymi. Thus, even at limiting concentrations of OVA peptide, CD40 deficiency was not associated with defects in negative selection (Fig. 5GoC); these findings are representative of the results of seven distinct experiments.



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  		FIGURE 5. Negative selection of Id+ D011 cells in FTOC. Day 15 fetal thymi were taken from CD40+/+ or CD40-/- D011 mice and cultured with or without OVA peptide at the indicated concentrations. After 6 days, the cultures were harvested, and the proportion of Id+ (KJ1.26+) cells among CD4+ CD8+ double-positive (DP) or CD4+ SP cells was determined by flow cytometry as described in Fig. 4Go. The data indicate the number of Id+ cells of the indicated subpopulation recovered per thymus lobe. Two experiments are illustrated: A and B show deletion of SP and DP cells from one experiment, and C illustrates deletion of DP cells at lower peptide concentrations in a second experiment.

 
To address whether the dependence of negative selection on CD40 was influenced by the affinity of TCR-peptide/MHC interactions, thymic lobes were cultured with a partial agonist OVA peptide (OVA337A->L), which shows a 10- to 100-fold decrease in its ability to stimulate mature D011 CD4+ cells. Even with this poorly stimulatory peptide, negative selection of Id+ CD4+8+ and CD4+8- thymocytes was equivalent in CD40-/- vs CD40+/+ FTOC (Fig. 6Go, A and B).



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  		FIGURE 6. Partial agonist OVA peptide fails to reveal a role for CD40 in negative selection of CD4+ cells even at reduced TCR-peptide/MHC affinity. Day 15 fetal thymus lobes were cultured with native OVA peptide or OVA337A->L for 6 days as described in Fig. 5Go. The number of Id+ double-positive (DP) cells (A) or SP (B) cells recovered from these cultures is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the experiments described here, CD40-deficient APC are not inherently defective in their ability to induce strong CD4+ T cell proliferative responses. Indeed, responses to conventional alloantigens or to defined peptide Ags, e.g., OVA, were indistinguishable when presented by CD40+/+ vs CD40-/- spleen APC. By contrast, prominent differences were found when resting B cells were used as a source of APC. Confirming an important role for CD40 in B APC function (43, 44, 45), purified CD40-/- B cells induce poor peptide-specific proliferation of D011 CD4+ cells and require higher concentrations of peptide, whereas CD40+/+ B cells induce strong responses at low concentrations of peptide. Likewise, the B cell-dependent proliferative response of T cells to mtv-7 superantigens was markedly reduced with CD40-deficient APC. These studies suggest that several different factors influence the APC function of CD40-/- B cells. The lack of response to OVA peptide + CD40-/- B cells could be partially overcome by increasing the concentration of peptide (Fig. 2GoC), indicating that the requirement for CD40 in B cell Ag presentation is influenced by Ag concentration. Further, for responses to mtv-7 SAG, the requirement for CD40 was much more stringent for weakly stimulating H2-Ab (H2-E-) APC than for strongly stimulating H2-Ak (H2-E+) APC. Collectively, these findings suggest that the requirement for CD40 interactions for proliferative responses of CD4+ cells depends on 1) the type of APC used, 2) the ligand density, and 3) the affinity of interactions among TCR, Ag, and MHC molecules.

Some studies have shown only minimal effects of CD40 deficiency on in vivo responses (46, 47, 48, 49, 50). Nonetheless, the vast majority of studies suggest that in vivo responses are strongly dependent on CD40-CD154 interaction (see Refs. 51 and 52 for review). In view of the important role of CD40 signaling in promoting DC activity (2, 13), defective DC function has been implicated as a contributing factor in the poor responses seen in the absence of CD40-CD154 interactions. At face value, this line of reasoning is difficult to reconcile with the present finding that CD40-deficient professional APC can stimulate strong T proliferative responses in vitro. However, this was clearly not the case with B cells as APC. Hence, it is possible that the stringent requirement for CD40-CD154 interaction for in vivo responses reflects the fact that in vivo responses are B cell dependent.

The role of B cells in T cell priming has been hotly debated for a number of years. The early studies of Ron and Sprent and others clearly demonstrated that in B cell-deficient (anti-µ-treated) mice, B cells were required for optimal T cell responses to several protein Ags, including F{gamma}G and keyhole limpet hemocyanin (53, 54, 55, 56). The main conclusion from these studies was that B cells may not initiate T cell priming but do play an important role in augmenting the clonal expansion of T cells when professional APC become limiting. This scenario fits well with our data here on CD40-/- APC. Although a number of studies using µMT B cell-deficient mice confirmed the importance of B cell APC (57, 58, 59, 60, 61, 62), other studies contradicted these results (63, 64, 65, 66, 67, 68). Recent studies (Y. Ron, personal communication) suggest that these contradictory results may reflect an intrinsic APC abnormality in µMT mice. This issue will be further examined in future studies.

The above possibility does not exclude an important role for CD40-mediated signaling of DC and/or macrophages for production of IL-12 and other important inflammatory mediators (14, 69, 70, 71, 72, 73, 74). Indeed, the available data indicate that, at least in some systems, the absence of CD40 signaling leads to poor induction of Th1-type responses, which could be an important means by which the inhibition of CD40 signaling prevents autoimmune disease. In our studies we have seen some evidence, even at the level of these primary responses, for reduced production of IFN-{gamma} in cultures stimulated with CD40-deficient APC.

In addition, our studies do not address the issue of how exposure to CD40-deficient APC induces T cell tolerance in vivo as previously reported (17, 44, 45). In our hands, resting B cells, rigorously depleted of DC, macrophages, and B cell blasts, can induce peptide-dependent CD4+ primary T cell responses; however, these responses are strongly dependent on CD40 expression. Although the explanation for these differences is not yet clear, the findings raise the possibility that induction of B cell-induced T cell tolerance involves additional, not yet well-defined, contributing factors.

CD40-CD154 interactions have also been implicated in controlling negative selection of developing autoreactive thymocytes (75). This idea is based in part on studies using CD154-deficient mice crossed to mtv-7+ CBA/J (H2-AK) mice. These F1 mice presumably expressed both weakly stimulating H2-Ab molecules as well as strongly stimulating H2-Ak molecules, each at reduced levels. Thus, the difference in the apparent requirement for CD40-CD154 in the negative selection of Vß6+ cells in our H2-Ak strain studies and the reported studies could involve a gene dose effect of the strongly presenting H2-Ak molecules. The previous study also examined deletion of peptide-specific TCR transgenic cells by injecting soluble peptide into adult mice. Although no CD40-specific effects were reported in these experiments, the relevance of these experiments to negative selection are questionable, as it is now known that this approach leads to a steroid/cytokine-dependent collapse of the thymus resulting from activation of mature T cells (76, 77) (C. Surh and J. Sprent, personal communication). To avoid this problem, we studied the influence of CD40 expression on deletion of peptide-specific TCR transgenic cells in FTOC. Extensive titrations of peptide and analysis of peptides with varying affinities suggested that deletion of these T cells did not require CD40 in the thymus. Although these findings do not exclude a possible role for CD40 in lower affinity interactions, our data are consistent with the idea that negative selection, as for stimulation of mature T cells, is dependent on CD40 when B cells are the dominant APC.


   Acknowledgments
 
We thank Ms. Barbara Marchand for typing the manuscript.


   Footnotes
 
1 This work was supported by Grants CA41993, CA25803, and AI39664 from the U.S. Public Health Service and a grant from the Juvenile Diabetes Foundation. Publication no. 12420-IMM from The Scripps Research Institute. Back

2 Current address: Department of Molecular Immunology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan. E-mail address: Back

3 Address correspondence and reprint requests to Dr. Susan R. Webb, Department of Immunology, IMM4, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: Back

4 Abbreviations used in this paper: DC, dendritic cells; SAG, superantigens; FTOC, fetal thymic organ culture; SP, single positive; LN, lymph node. Back

Received for publication May 21, 1999. Accepted for publication August 30, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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