CD154CD40-independent up-regulation of B7-2 on splenic antigen-presenting cells and efficient T cell priming by staphylococcal enterotoxin A
Koji Eshima1,3,
Yongwon Choi4 and
Richard A. Flavell1,2
1 Section of Immunobiology and 2 Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06520, USA 3 Department of Immunology, Kitasato University School of Medicine, 1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan 4 Abramson Family Cancer Research Institute, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
Correspondence to: R. A. Flavell; E-mail: richard.flavell{at}yale.edu
Transmitting editor: H. Karasuyama
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Abstract
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It has been demonstrated that in vivo T cell priming requires CD154CD40 interaction, which is suggested to be critical in the induction of co-stimulatory activities on antigen-presenting cells (APC). In the current study, we demonstrate that in vivo administration of a high dose of a superantigen, staphylococcal enterotoxin A (SEA), could up-regulate B7-2 on most splenic APC independently of the CD154CD40 interaction, followed by efficient expansion of SEA-reactive Vß3+ T cells in CD154- or CD40-deficient mice. However, the CD154CD40 interaction may be involved in SEA-mediated T cell activation, since a contribution of the CD154CD40 interaction was observed when a lower dose of SEA was injected. CD154-independent T cell priming by SEA appeared also independent of the TRANCERANK pathway, which was shown to be capable of mediating CD154-independent activation of naive T cells during the infection of some viruses. These results indicate that SEA, which provokes rapid and efficient T cell responses without adjuvant, could utilize multiple CD154/TRANCE-independent pathways, to prime T cells.
Keywords: cellular activation, co-stimulation, superantigen, T lymphocyte
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Introduction
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Among the molecules involved in contact-dependent interactions between T cells and antigen-presenting cells (APC), CD154 and its receptor CD40 have been shown to be one of the most critical molecular pairs in the initiation and development of cell-mediated immunity [reviewed (13)]. In the absence of CD154CD40 interaction, T cell-dependent in vivo immune responses were shown to be defective (112). Several studies have demonstrated that blockade of CD154CD40 interaction by antibody administration or by gene-targeting technology could successfully prevent the onset and/or progress of various T cell-mediated autoimmune diseases (1319). For example, the development of experimental allergic encephalomyelitis was completely blocked in myelin basic protein (MBP)-specific TCR transgenic mice, when examined on the CD154-deficient background (16). In these mice, however, CD4 T cell response (to the MBP peptide), as well as disease development, were restored by reconstitution with B7-1-expressing APC prepared from B7-1 transgenic mice, suggesting that CD40-mediated induction of co-stimulatory molecules on APC is crucial for the priming of CD4 T cells (16).
In contrast, it was shown that CD40- or CD154-deficient mice challenged with lymphocytic choriomeningitis virus (LCMV) or Pichinde virus could mount substantial protective CD4 T cell responses, suggesting the existence of an alternative pathway to prime T cells in the infections with these viruses (20,21). This CD154CD40-independent pathway of T cell priming has been found to be mediated by the interaction of another tumor necrosis factor (TNF) family member, TNF-related activation-induced cytokine (TRANCE) (22,23), with its receptor [TRANCE-R or receptor activator of NF-
B (RANK)]. Although blockade of this interaction by soluble RANKhuman Ig Fc fusion protein did not have a significant effect in anti-virus responses in wild-type mice, maximal inhibition of T cell response was observed in CD40-deficient mice, indicating that there indeed exists two independent routes for in vivo T cells priming (24).
In the current study, we demonstrate that a superantigen, staphylococcal enterotoxin A (SEA), is capable of efficiently priming T cells in CD154-knockout (KO) mice in vivo. Administration of a high dose of SEA up-regulated B7-2 expression on most splenic APC in the absence of CD154CD40 interactions. CD154-independent APC activation and T cell priming mediated by SEA also appears to be independent of TRANCERANK interaction since injection of RANKFc to block this interaction did not affect SEA-mediated T cell priming in CD154-KO mice. The possible mechanism for this SEA-mediated T cell priming and the more general requirements for optimal naive T cell activation are discussed in the context of the roles of CD154.
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Methods
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Mice
CD154-KO mice were generated as described previously (4) and the mice crossed with C57BL/6 mice for seven generations were used in this study. CD40-KO mice on the C57BL/6 background were purchased from Jackson Laboratories (Bar Harbor, ME). CD154-deficient or wild-type mice bearing transgenic TCR specific for an N-terminal peptide of MBP (ASQKRPSQRSK) were prepared as described previously (16,25).
Reagents
FITC-labeled anti-TCRVß6 antibody, phycoerythrin (PE)- labeled anti-TCRVß3 antibody, FITC-labeled anti-IFN-
antibody, PE-labeled anti-IL-4 antibody, PE-labeled anti-B7-2 antibody and FITC-labeled anti-CD3 antibody were purchased from PharMingen (San Diego, CA). SEA was purchased from Toxin Technology (Sarasota, FL) and re-suspended in PBS to prepare 1 mg/ml solution. RANKFc, a fusion protein of RANK and human IgG1 Fc, was prepared as described previously (24). FITC-conjugated goat IgG to human IgG Fc was purchased from ICN Biomedicals (Aurora, OH) and purified anti-mouse B7-2 antibody (GL1, rat IgG2a) was obtained from R & D Systems (Minneapolis, MN). Anti-Thy1.2 antibody (21-12, mouse IgM; ATCC, Manassas, VA), anti-CD4 antibody (GK1.5, rat IgG2b; ATCC), anti-CD8 antibody (83-12-5, mouse IgM; ATCC), anti-CD11b (M1/70, rat IgG2b; ATCC), anti-B220 (RA3-3A1/6.1, rat IgM; ATCC), anti-I-Ab (AF6-120.1.2; mouse IgG) and murine dendritic cell-specific antibody (33D1, rat IgG2b; ATCC) were used to remove subpopulations of splenocytes. mAb to IL-4 (11B11) and to IFN-
(XMG1.2) were obtained from ATCC. Anti-TNF-
polyclonal antibody, an IgG fraction purified from rabbit antiserum (Genzyme, Piscataway, NY), was generously provided by Dr H. Takayama (Mitsubishi Kasei Institute of Life Science, Tokyo).
T cell proliferation assay
Spleen cells or purified CD4 T cells with different numbers of APC were cultured in the presence of SEA or MBP peptide respectively for the indicated time periods in flat-bottomed 96-well plates, and incorporation of [3H]thymidine for the last 18 h measured by liquid scintillation counter. Dendritic cells were induced from bone marrow cells by culturing them in the presence of granulocyte macrophage colony stimulating factor for 8 days as described elsewhere (26). Mature dendritic cells, used as APC, were sorted by FACS for high B7-2 expression.
Measurement of IL-2 and IFN-
After depleting CD8 T cells by antibody plus complement, spleen cells (5 x 105/well) from CD154-deficient or -sufficient mice injected with H-2d tumor cells were stimulated with the same number of irradiated BALB/c spleen cells. The supernatants from 18- or 48-h cultures were harvested to measure IL-2 or IFN-
respectively by ELISA. The amount of IFN-
secreted by spleen cells from the mice injected with PBS or SEA was also measured by ELISA.
Flow cytometric analyses
Cells were incubated with various antibodies diluted in 100 µl of staining buffer (PBS with 1% FCS) on ice for 30 min. After washing cells 3 times with 2 ml staining buffer, cells were re-suspended in 400 µl of staining buffer for analysis on a FACSCalibur (Becton Dickinson, Mountain View, CA). For intracellular staining of cytokines, spleen cells prepared from PBS- or SEA-injected mice were re-stimulated in vitro with 1 µM ionomycin (Calbiochem, La Jolla, CA) and 10 nM phorbol myristate acetate. After 2 h, monensin (2 µM, Golgi-Stop; PharMingen) was added and cells were incubated for a further 4 h before intracellular staining performed using the Cytofix/Cytoperm kit (PharMingen).
Fixation of T cells
After culturing whole splenocytes with or without SEA (5 µg/ml) for 15 h, cells were harvested and depleted APC using antibodies against class II MHC, B220, 33D1 and CD11b with rabbit complement. The cells obtained, most of which were CD3+ (8090%), were washed and re-suspended with PBS. Then 1 x 107 cells were fixed in 1 ml 0.5% paraformaldehyde (Nacalai Tesque, Kyoto, Japan) at room temperature for 1 min.
In vivo depletion of CD4 T cells
Anti-CD4 mAb GK1.5 from ascites was injected in C57BL/6 mice i.p. (150 µl per mouse for each injection) every other day for 6 days (3 times in total). One week later, these mice were bled to confirm by flow cytometry that CD4 T cells were depleted.
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Results
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Impairment of CD4+ T cell priming in the absence of CD154
As we previously demonstrated, in vivo T cell priming by immunization with a foreign protein antigen (8) or with allogeneic cells is defective in CD154-KO mice (Fig. 1A). Although in vitro responses of CD154-deficient T cells were shown to be relatively normal when spleen cells from CD154-KO mice were stimulated with polyclonal activators such as anti-CD3 antibody or concanavalin A (8), CD154-KO CD4 T cells exhibited significantly lower responses as compared with wild-type T cells when they were stimulated with smaller numbers of splenic APC (data not shown) or mature dendritic cells (Fig. 1B) in the presence of concanavalin A or the antigenic peptide respectively. These results indicate that the response of CD154-deficient CD4 T cells is largely dependent on the number of APC and that the in vitro response of CD4 T cells is impaired in the absence of CD154 when the number of APC is limited.

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Fig. 1. Impaired T cell response in CD154/ mice to an allogeneic B lymphoma (A) and to an antigenic peptide presented by mature dendritic cells (B). BALB/c-derived B lymphoma A20.2J was injected (2 x 107/mouse) i.v. into CD154/ mice (B6 background) and normal B6 mice. Seven days later, spleen cells depleted of CD8 T cells were re-stimulated in vitro with irradiated BALB/c splenocytes to measure [3H]thymidine incorporation and cytokine production (A). FACS-sorted naive CD4 T cells (1 x 105/well) from MBP-reactive TCR transgenic, CD154-deficient or sufficient mice were stimulated with the antigenic peptide presented by the indicated number of mature dendritic cells prepared from bone marrow cells. Cells were cultured for 23 days and incorporation of [3H]thymidine during the last 16 h was determined (B).
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CD154-independent, T cell-mediated up-regulation of B7-2 on splenic APC by SEA
Since it was shown that cross-linking of class II MHC molecules induces B cells to express B7 molecules (27), we explored the possibility that a superantigen, SEA, a possible cross-linker of class II MHC, could up-regulate B7 on APC without the requirement for the CD154CD40 interaction. One hundred micrograms of SEA was injected i.p. in CD154-KO mice or in wild-type mice and the expression of B7 molecules on CD3 splenocytes (splenic APC, most of which are B cells) was examined on the next day. As shown in Fig. 2, B7-2 but not B7-1 was up-regulated on most splenic APC in CD154-KO mice as well as in wild-type mice. Up-regulation of B7-2 was also observed when whole spleen cells were cultured in vitro with SEA (Fig. 3). Depletion of T cells abrogated the up-regulation of B7-2 (Fig. 3A), indicating that binding of SEA to class II MHC on APC alone is not sufficient to activate APC to enhance B7-2 expression. Another experiment using CD40-KO mice confirmed that up-regulation of B7-2 on splenic APC is entirely independent of CD40 signaling (Fig. 3B).

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Fig. 2. B7-2, but not B7-1, is up-regulated on splenic APC by SEA injection in CD154/ mice. Spleen cells from CD154/ or wild-type mice were analyzed for B7 expression on the next day of SEA (100 µg/mouse) or PBS injection. CD3+ cells were gated out at FACS analyses.
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Fig. 3. SEA-mediated B7-2 up-regulation on splenic APC is T cell-dependent, but is independent of CD40 signaling. Whole spleen cells or T cell-depleted splenocytes (8 x106/well) from normal B6 mice were cultured in a 24-well plate in the absence or presence of SEA (10 µg/ml). B7-2 expression on CD3 cells after 18 h culture is shown (A). Whole spleen cells from normal B6 mice or CD40/ mice were cultured with or without 10 µg/ml SEA for 18 h and B7-2 expression on CD3 cells are compared (B).
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CD154-independent T cell priming by SEA
In order to examine whether in vivo injection of SEA could induce not only B7-2 up-regulation on APC, but also efficient T cell priming in CD154-deficient mice, spleen cells from SEA-injected CD154-KO mice and wild-type mice were analyzed for the expansion of Vß3+ T cells which are reactive to SEA. As shown in Fig. 4(A), Vß3+ T cells were expanded in CD154-KO mice to an equivalent level as in wild-type mice upon SEA administration (5.70 ± 1.00% in wild-type mice versus 5.43 ± 0.95% in CD154-KO mice, n = 5). The proportion of Vß3+ cells among both CD4 and CD8 T cell subsets was equally increased (data not shown). On the other hand, the proportion of Vß6+ cells, which are not responsive to SEA, was not affected (or sometimes decreased, probably due to the overall increase of total spleen cell number), indicating the specificity of the SEA-induced T cell response. When spleen cells from wild-type and CD154-KO mice which had been injected with SEA 3 days previously were re-stimulated with SEA in vitro, they both showed a similar level of proliferative responses (Fig. 4B). These results suggest that the lack of CD154CD40 interaction did not result in the induction of anergy, but rather in the proper activation of T cells when they were primed by a high dose of SEA.

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Fig. 4. CD154 is dispensable in in vivo priming of Vß3+ T cells by SEA injection. CD154/ mice (B6 background) and normal B6 mice were i.p. injected with PBS or 100 µg SEA. Three days after injection, the proportion of Vß3+ cells (reactive to SEA) and Vß6+ cells (for control) in the spleens was analyzed (A). Three days after SEA or PBS injection, whole spleen cells (5 x 105/well) from CD154/ or wild-type mice were stimulated in a flat-bottomed microtiter plate with the indicated concentrations of SEA for 24 h. Incorporation of [3H]thymidine during the last 6 h was determined (B).
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CD40 stimulation on splenic APC is important in B7-2 up-regulation by low-dose SEA
The above results raised the possibility that CD154 (or CD40) was not involved in the activation of APC by SEA. We therefore next investigated whether the involvement of the CD154CD40 interaction could be observed in B7-2 up-regulation by the injection of a lower dose of SEA. We administrated different amounts of SEA to analyze B7-2 expression on CD3 splenocytes on the next day. As shown in Fig. 5, the level of B7-2 expression was lower in CD40-KO mice as compared to wild-type B6 mice, when 10 (but not one 100) µg SEA was injected, resulting in the impaired expansion of Vß3+ cells in CD40-KO mice (Fig. 6B). Impaired activation of CD40-KO APC was also observed in vitro when splenocytes were stimulated with lower concentrations of SEA (data not shown). These results suggest that CD154CD40 could contribute to APC activation and to T cell proliferation induced by a lower dose of SEA, but is dispensable when a high dose of SEA was injected.

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Fig. 5. Contribution of CD40 stimulation in APC activation by a lower dose of SEA. B7-2 expression was analyzed on CD3 splenocytes from CD40-KO or wild-type B6 mice which had been injected with PBS (top), 10 µg SEA (middle) or 100 µg SEA (bottom) 1 day before analyses.
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Fig. 6. No significant effect of RANKFc on CD154/CD40-independent T cell priming by SEA. (A) CD40-KO splenocytes (1 x 107/well in 24-well plate) were cultured in the presence (right) or absence (left) of 5 µg/ml SEA for 15 h, following which cells were stained with PEanti-CD8 (top) or PEanti-CD4 antibody (bottom), together with RANKFc (5 µg/ml) plus FITCanti-human IgG antibody. Percentages of TRANCE+ cells among CD8 or CD4 T cells are indicated. (B) Proportion of Vß3+ cells in spleens from CD40-KO or wild-type C57BL/6 mice 3 days after injection of PBS or of 10 µg SEA. Four CD40-KO mice were injected with 100 µg RANKFc protein on day 0, 1 and 2 before sacrificing on day 3. Bars show the mean of each experimental group.
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No significant effect of blocking TRANCERANK interaction on SEA-mediated T cell priming in the absence of CD154
Next, we investigated the possible involvement in the SEA-mediated T cell activation of the TRANCERANK pathway, which is, so far reported, the only pathway capable of compensating for the lack of CD154CD40 interaction in T cell priming (24). In the latter study, employing LCMV infection, three injections of 100 µg RANKFc protein during the 8 days after virus infection was effective in blocking the CD4 T cell responses in CD40-KO mice. However, despite the observation that TRANCE induction was observed especially on CD4 T cells by SEA stimulation in vitro (Fig. 6A), when CD154-KO mice were administrated with 200 µg RANKFc on the day of SEA injection (100 µg/mouse), no significant impact on the expansion of Vß3+ T cells either in CD154-KO or in wild-type mice was observed (data not shown). This was also true when a lower dose of SEA (10 µg) was used to prime T cells in CD40-KO mice (Fig. 6B), even though, in this experiment 100 µg RANKFc protein was injected every day (on day 0, 1 and 2). In addition, significant effects were not seen either in SEA-mediated B7-2 up-regulation or in T cell proliferation by adding RANKFc (at 10 µg/ml) in in vitro culture of CD40-KO splenocytes (data not shown). These results indicate that both CD154CD40 and TRANCERANK interactions may be dispensable simultaneously in SEA-mediated T cell priming.
Involvement of both contact-dependent and -independent pathways in the up-regulation of B7-2 on splenic APC by SEA
In order to elucidate whether SEA-mediated APC activation is induced via signals triggered by cross-linking class II MHC or is still dependent on some molecules that are newly induced on T cells upon activation, we examined the ability of fixed T cells to activate splenic APC to up-regulate B7-2. As shown in Fig. 7, resting T cells fixed with paraformaldehyde could not up-regulate B7-2 on APC even in the presence of SEA. On the other hand, fixed T cells that had been pre-activated with SEA had the ability to up-regulate B7-2. Although this activity was lower on the APC from CD40-KO mice than on wild-type APC, some B7-2 up-regulation was always observed on CD40-deficient APC, suggesting that not only CD154, but also other molecules inducible on activated T cells are involved in B7-2 up-regulation. To investigate if the up-regulation of B7-2 would also be mediated via soluble factors secreted during the responses to SEA, we examined the activity of culture supernatants of whole splenocytes cultured with SEA for 15 h to activate APC. The results in Fig. 7 show that soluble factors are also involved in B7-2 up-regulation on APC. Taken collectively, SEA-mediated B7-2 up-regulation may be induced via multiple pathways, including both cognate interaction-dependent and -independent pathways.

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Fig. 7. Involvement of both contact-dependent and -independent pathways in SEA-mediated B7-2 up-regulation on splenic APC. Splenic APC were prepared from whole spleen cells of wild-type C57BL/6 (ae) or CD40-KO mice (fj) by depleting T cells with anti-Thy1.2, anti-CD4 and anti-CD8 antibody plus complement. These APC (5 x 105/well) were cultured in flat-bottomed 96-well plates with fixed T cells (3 x 105/well) in the absence (a, f, c and h) or presence (b and g) of 5 µg/ml SEA for 20 h. Fixed resting T cells and SEA-activated T cells were prepared as described in Methods. In (e) and (j) the culture supernatants from SEA-stimulated whole splenocytes (15 h culture of wild-type splenocytes in the presence of 5 µg/ml SEA) were added and APC were cultured for a further 20 h. Since this supernatant contained 5 µg/ml SEA, APC cultures supplied with 5 µg/ml SEA were prepared as control (d and i). B7-2 expression on CD3 cells is indicated.
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CD154-independent differentiation of CD8 T cells into IFN-
-producing Tc1-type cells
To analyze the effect of CD154CD40 interaction on differentiation of CD8 or CD4 T cells into effector cells, we next investigated the cytokine profiles of T cells that were primed in CD40-KO mice injected with SEA. Splenocytes from CD40-KO mice or wild-type B6 mice were prepared 3 days after SEA administration (100 µg/mouse) and were re-stimulated in vitro with SEA to measure cytokine production by T cells. Although no IL-4 production was detected either in CD40-KO mice or wild-type mice (data not shown), a similar amount of IFN-
production was observed in CD40-KO and wild-type mice (Fig. 8A). However, the production of IFN-
was abrogated when CD8 T cells, but not CD4 T cells, were depleted before in vitro re-stimulation with SEA, indicating that it is CD8 T cells that secreted IFN-
. Intracellular staining confirmed this result, showing that a significant increase in the frequency of IFN-
-producing cells was observed in CD8 T cells, but not in CD4 T cells, and that there was no difference in this increase between wild-type and CD154-KO mice (data not shown). These results demonstrate that the co-stimulatory requirement for differentiation into IFN-
-producing cells is different between CD4 T cells and CD8 T cells, and that the generation of Tc1-type, IFN-
-producing CD8 T cells in vivo does not require any CD40-mediated events.
These results raised the possibility that CD8 T cells could be differentiated without help from CD4 T cells, since differentiation of naive CD8 T cells into effector cells was suggested to be mediated by CD154 on CD4 T cells (2830). In order to investigate this possibility, we examined if CD8 T cells could be primed and differentiated into IFN-
producing cells in mice depleted of CD4 T cells. We prepared CD4-depleted mice by in vivo injection of anti-CD4 antibody and administrated 50 µg SEA in those mice as well as in control mice (with no treatment). As shown in Fig. 8(B), the proportion of Vß3+ cells among CD8 T cells was increased efficiently even in mice depleted of CD4 T cells. Furthermore, when splenocytes were re-stimulated with SEA in vitro, they produced significant amounts of IFN-
(Fig. 8C), indicating that CD8 T cells could be primed and differentiated in vivo by SEA, into Tc1 cells producing IFN-
independently of the help from CD4 T cells.
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Discussion
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Although CD154 was originally characterized as the T cell molecule that is important in cognate T and B cell interaction, and in T cell-dependent humoral immunity, it is now evident that the CD154CD40 interaction also plays multiple important roles in cell-mediated immunity (13). The possible requirement of CD154 in T cell responses was first suggested by the fact that the lack of functional CD154 in the patients of hyper-IgM syndrome is associated with increased susceptibility to infections of some opportunistic pathogens such as Pneumocystis or Cryptosporidium (31,32). Analyses of T cell responses in CD154-KO mice revealed that in vivo activation of antigen-specific naive T cells is indeed compromised in those mice (812,16). Further analyses suggested that T cell priming may be defective in CD154-deficient mice because CD154-mediated induction of co-stimulatory molecules on APC is crucial in the priming of CD4 T cells (12,16).
An apparent contradiction that we found, despite the obvious impairment of in vivo T cell priming in CD154-KO mice, was that in vitro responses of CD154-deficient T cells were relatively normal when total spleen cells were stimulated in vitro with antigenic peptides or mitogenic stimuli (8). This could be explained by the findings by Inaba et al. (33), who showed that splenic dendritic cells, which may be the primary APC in T cell priming, are spontaneously activated to express high levels of B7 molecules when cultured on plastic plates (without CD40 stimulation or other exogenous stimuli). This observation suggested the possibility that in in vitro culture, induction of co-stimulatory activities on dendritic cells does not require CD40 stimulation. However, as shown in the current study, the in vitro response of CD154-KO T cells was significantly lower as compared with wild-type T cells when the number of APC available to those cells was limiting. This result might indicate that the number of APC presenting antigens to cognate T cells is limited in physiological conditions in vivo. In addition, this result also suggests that provision of co-stimulatory activities to APC may not always be sufficient for the optimal priming of CD4 T cells. In the experiment shown in Fig. 1(B), we used mature dendritic cells, which express high levels of B7 molecules, as APC to stimulate naive CD4 T cells. However, the response was still impaired in CD154-deficient CD4 T cells when the number of mature dendritic cells was small. In addition, this impaired response was substantially restored by the addition of soluble CD154 to stimulate CD40 on mature dendritic cells, while the expression of B7 on dendritic cells was not significantly enhanced (our unpublished observation). It is therefore possible that CD154-mediated induction of cytokines or co-stimulatory molecules other than B7 may be important for optimal T cell priming. However, the effect of these molecules may not be so significant, considering the result showing that the reduced response of CD154-deficient T cells could be restored just by increasing the number of mature dendritic cells.
The study by Lanzavecchia et al. demonstrated that naive CD4 T cells require a much longer interaction with APC to be committed to proliferate, as compared with primed T cells (34). Thus, it is quite possible that one of the important roles of CD154 in T cell priming would be the maintenance of competent APC which present antigenic peptides and have high co-stimulatory activity, by increasing the half-life of those competent APC such as mature dendritic cells, possibly via induction of Bcl-xL in them (22). This function of CD154 was shown to be shared with TRANCE (22,35).
Since CD154CD40 interaction is normally required for the activation of APC to confer co-stimulatory activities, this deficiency would lead to a failure of T cell responses. In the present study, however, we have shown that SEA could up-regulate B7-2, when injected with a high dose (100 µg), on most CD3 splenocytes (splenic APC), which are mostly B cells, either in CD154- or CD40-deficient mice. The up-regulation of B7-2 is likely to be essential for efficient T cell priming by SEA, since it was shown that interaction of CD28 with B7 molecules is necessary for T cell expansion induced by staphylococcal enterotoxins (3639). In fact, in vitro studies indicated that the proliferative response of splenocytes induced by SEA was inhibited by anti-B7-2 antibody (data not shown), which indicates that B7-2 up-regulated on splenic B cells is important for the optimal T cell activation by SEA. However, the inhibition by anti-B7-2 antibody was always partial, leaving the possibility that T cells could be activated by SEA to some extent independently of B7-2. Additionally, the contribution of B cells in SEA-mediated T cell priming has remained to be formally evaluated, using, for example, mutant mice which lack B cells, in future studies.
Co-stimulation through CD28 is well known to be able to lower the threshold for T cell activation. However, some publications, especially using human T cells, indicate that the CD154CD140 interaction is bidirectional and that CD154 also could deliver functional signals similar to CD28 (4042). In our hands, however, such signaling from CD154 into murine CD4 T cells could not be detected as assessed by proliferation, cytokine production or differentiation (our unpublished observation). Thus, in the priming of murine CD4 T cells, the role of CD154CD40 co-stimulation would be primarily to activate APC to make them more efficient mediators of T cell activation, rather than to send signals from CD154. In the present study, we have shown that the lack of CD154CD40 interaction impaired the response to a lower dose of SEA and that this was correlated with inefficient enhancement of B7-2 up-regulation on APC. Thus, it is possible that the CD154CD40 interaction could lower the threshold for T cell activation (or the antigen dose necessary for efficient T cell responses) by augmenting the expression of CD28 ligands on APC.
Although we could not determine the exact mechanism for the up-regulation of B7-2 in CD154- or CD40-KO mice, it is apparently TRANCE independent, since injection of soluble RANKFc to block TRANCERANK interaction had no inhibitory effect on the expansion of Vß3+ cells even in the absence of CD154 or CD40. Thus, in SEA-mediated T cell priming, other TNF family molecules (such as 4-1BBL or OX40L) might play important roles. Although the rationale at the start of this study, signals transduced from class II MHC may not be relevant for the up-regulation of B7-2 on splenic APC, since paraformaldehyde-fixed resting T cells failed to induce B7-2 up-regulation on APC when co-cultured with SEA. In keeping with this, we did not observe significant B7-2 up-regulation when class II MHC molecules on APC were cross-linked by mAb (data not shown). B7-2 up-regulation was apparently mediated by multiple mechanisms involving contact- dependent pathways (which include both CD154CD40-dependent and -independent pathways), as well as contact-independent pathways. We are currently seeking the molecules responsible for B7-2 up-regulation on APC, but the soluble components appeared unlikely to be either TNF-
, IFN-
or IL-4, since antibodies to these molecules, even in combinations of two, did not significantly block the activity of culture supernatant of SEA-stimulated splenocytes to up-regulate B7-2 in vitro (data not shown). The transmembrane form of TNF-
(mTNF-
) was a candidate for the responsible molecules induced on SEA-activated T cells, since it was shown that some cloned CD4 T cells could provide help for B cells via mTNF-
(4345). However, anti-TNF-
antibody did not block the B7-2 up-regulation by fixed, pre-activated T cells, while the same concentration (10 µg/ml) of this antibody could neutralize at least 20 ng/ml TNF-
, as assessed by cytotoxicity assay using fibroblasts (data not shown).
The CD154CD40 interaction has been implicated in the differentiation of CD4 T cells into Th1/Th2 cells (3,15,41). The use of SEA was expected to provide us an opportunity to evaluate the role of the CD154CD40 interaction in CD4 T cell differentiation, since SEA could prime T cells successfully without the CD154CD40 interaction. However, splenic CD4 T cells expanded by the stimulation with SEA in vivo did not differentiate into either Th1 or Th2 cells even in CD154-sufficient mice, as assessed by their cytokine production. We examined CD4 T cell differentiation 3 days after SEA injection, after which SEA-primed T cells start to be deleted (38) and therefore we did not study later time points. Thus, it is possible that 3 days might be too short for CD4 T cells to be differentiated into Th1 or Th2 cells. It is also possible that the failure of CD4 T cell differentiation in vivo by SEA may be because the cytokines that are required for efficient CD4 T cell differentiation, such as IL-12 or IL-6 (46), may not be sufficiently produced during in vivo response to SEA.
In contrast to CD4 T cells, CD8 T cells could be efficiently activated and differentiated to IFN-
-producing Tc1 cells without the CD154CD40 interaction (Fig. 8A). Activation and differentiation of CD8 T cells, in most situations, require CD4 T cell help, which has been suggested to be mediated via CD154 to activate APC (3739). The lack of any requirement of CD154 in the differentiation of CD8 T cells suggested that CD4 T cell help could be dispensable in SEA-mediated priming and differentiation of CD8 T cells. The results in Fig. 8(B) showed that this was indeed the case. CD8 T cells could be primed efficiently in vivo by SEA to be differentiated into effector cells without help from CD4 T cells.
CD154-independent priming of CD8 T cells by SEA appears similar to the model of LCMV infection (21). A primary CD8 T cell response against LCMV was demonstrated to be unaffected by the absence of CD154 or CD40, while the CD8 response is still dependent on CD28 signals (47). In this regard, it is interesting to note that our preliminary data suggested that CD8 T cells alone (without CD4 T cells) could up-regulate B7-2 on splenic APC when cultured in vitro. The identification of its precise mechanism might hopefully lead to the determination whether or not the same pathways could be operative between SEA- and LCMV-mediated activation of CD8 T cells.
In conclusion, the results in the current study showed that SEA could activate most splenic APC via unknown pathways (probably distinct from CD154CD40 or TRANCERANK pathway), generating a large number of competent APC. It is tempting to speculate that the successful in vivo priming of T cells by a high dose of SEA in the absence of CD154CD40 interaction could result from the generation of competent APC expressing a high level of B7-2, in sufficient numbers, since the results in Fig. 1 indicate that the CD154-deficient T cell response could be affected by the number of competent APC. Further studies will be required to examine whether the important functions of CD154CD40 in T cell priming during the responses to conventional antigens involve the enhancement of survival of competent APC, mature dendritic cells in particular, to maintain a high level of class II MHC and co-stimulatory molecules at the site of T cell response.
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Acknowledgements
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We thank Dr C. A. Janeway, Jr for providing MBP-TCR transgenic mice, Dr D. Gray for the granulocyte macrophage colony stimulating factor-producing cell line, Dr H. Takayama for TNF-
and antibodies for it, and Dr N. Kim for RANKFc protein. We are also grateful to Dr E. Eynon for valuable discussions and suggestions, and to Dr N. Shinohara for supporting this study. We also thank F. Manzo for assistance with manuscript preparation. This work was supported by grants from the NIH 5 P01 AI36529 and the American Diabetes Association (R. A. F.), and in part by a Grant-in-Aid for Encouragement of Young Scientists from Japan Society for the Promotion of Science (K. E.). R. A. F. is an Investigator of the Howard Hughes Medical Institute.
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Abbreviations
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APCantigen-presenting cell
KOknockout
LCMVlymphocytic choriomeningitis virus
MBPmyelin basic protein
PEphycoerythrin
RANKreceptor activator of NF-
B
SEAstaphylococcal enterotoxin A
TNFtumor necrosis factor
TRANCETNF-related activation-induced cytokine
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