Many studies indicate that B cell and T cell responses are coordinated to orchestrate cross regulation of expansion, survival, and differentiation via cooperative interactions. Although the essential role of CD4 cell–derived cytokines in B cell differentiation and responses has been widely studied, mechanisms that underlie the regulation of CD4 cell responses by B cells have not yet been fully explored. In this study, we determined that B cells can directly affect CD4 effector responses through costimulation by OX40L. We show that in the absence of B cells that express OX40L, Ag-induced expansion of CD4 cells and development of effectors that secrete Th2 cytokines are impaired.
Several studies have identified a link between B cell Ag-presentation and the induction of IL-4 secretion by CD4 cells (31
) but did not reveal whether additional signaling pathways were required. Although a recent report suggests that B cells can provide a source of IL-4 for differentiation of Th2 cells (13
), this does not appear to be the sole mechanism to support Th2 responses since our results show that IL-4–deficient B cells were fully capable of eliciting Th2 cytokines. However, it is striking that several costimulation molecules have profound effects on the development of CD4 cells. It has been proposed that three major costimulatory pathways, CD28/B7, CD40L/CD40, and OX40/OX40L, act sequentially and synergistically to regulate CD4 cell responses from initial activation to optimal expansion, survival, and migration into B cell follicles (6
). Although CD4 cell signaling through CD28, CD40L, or OX40 are associated with IL-4 induction (6
), in vivo studies suggest that IL-4 production and responses that require IL-4 may be particularly dependent upon the OX40/OX40L pathway (27
). Our data reveal that B cells can be an essential population to provide signals via OX40L to CD4 cells.
We envision that following CD4 cell activation by TCR-interactions with MHC/peptide complexes and CD28 engagement of B7 molecules, receptors such as CD40L and OX40 are induced and serve to amplify TCR signaling, as well as enable responding T cells to interact with B cells which constitutively express CD40 and up-regulate OX40L in response to activation. Temporally, OX40 expression on T cells coincides with induction of OX40L on B cells (27
). Although interactions with DCs can initially support the response, they become limiting as T cells proliferate, and without further recruitment of DC, usage of B cells can become necessary to sustain the response. Our data suggest that CD4 cells can be maintained in this physiologic context by OX40 interactions with OX40L on B cells. Although this pathway can also contribute to the early activation phase of CD4 responses via DCs (15
), our study of the knockout animals as recipients of Ag-specific CD4 cells shows that although priming occurs, Th2 responses are limited. Importantly, normal B cells are sufficient to restore Th2 effector development.
Our previous studies suggest that other APC can fulfill the role of B cells in CD4 cell expansion and Th2 development if made available in sufficient numbers during effector priming (4
). In addition, provision of costimulation to T cells via signaling with an agonist antibody to OX40 (29
) is associated with increased frequencies of Ag-specific CD4 cells. Recent studies also implicate OX40 in survival of activated CD4 cells (35
), suggesting that expansion together with increased lifespan may allow effectors to participate in a response for an extended period. Our results where a high number of Ag-specific naive CD4 cells are present at the time of immunization show that the capacity to secrete several effector cytokines, with the notable exception of Th2 cytokines, can develop independently of expansion that is sustained via OX40/OX40L interactions.
In view of our finding that IL-4 producing effectors, though reduced in number appear to be primed normally, and previous reports that OX40/OX40L interactions maintain later rather than initial CD4 cell expansion, we envision an indirect role of costimulation in the development of Th2 responses through effects on survival (35
) and division (29
). The data support the concept that sustained responses are necessary for optimal Th2 development (19
). As the OX40 pathway has been associated with CD4 cell accumulation in germinal centers (36
), it is attractive to speculate that B cell regulation of CD4 cell survival and response by OX40 in turn facilitates a sustained B cell response (37
). Although cytokines produced by APC including IL-13, IL-1, and IL-6 can support Th2 differentiation, IL-4 is the key mediator to function in this capacity (38
). The lag in optimal priming for IL-4 synthesis in our model may in part be due to dependence of responding CD4 cells upon autocrine secretion and usage of IL-4 to direct Th2 development (39
) through induction of transcription factors that include Stat6, Gata3, and Gfl-1 (38
). Conditions of strong TCR stimulation and costimulation may be necessary to achieve sufficient endogenous IL-4 levels for commitment to a Th2 phenotype and achieve stable secretion after extended chromatin remodeling that accompanies cell division (41
Our study shows that in vivo, OX40L expressed on B cells can play a crucial role in determining the outcome of a response in terms of the frequency and cytokine commitment of effectors and, from our previous work and that of others, in turn on persisting memory. In the absence of B cell involvement, CD4 cell responses, at least to many protein Ag would most typically be biased toward Th1 cytokines, as is often observed in responses where Ag is limiting or of low affinity. The data indicate that modulation of costimulation through OX40 can provide a key checkpoint in the regulation of CD4 cell responses.