CD4+ T cell interactions through CD28/B7 molecules affects their Th1/Th2 phenotype

The Th1/Th2 phenotype of the immune response generated against a pathogen or disease can have a profound impact upon the survival of the host. Thus, a complete and accurate model for the generation of Th1 versus Th2 cells is critical for the development of effective immunotherapeutic strategies aimed against diseases including cancer and tuberculosis. Numerous factors are considered to be important in the differential development of Th1 and Th2 cells. These include the strength of stimulation through the T cell receptor, the cytokine environment, the dose of antigen, and the types of co-stimulatory molecules expressed by the antigen-presenting cells presenting the T cell’s cognate antigen. A common thread found in the majority of studies on the generation of Th1 and Th2 cells is that naïve CD4+ T cells are thought to be passive players in this process. In contrast, our laboratory has demonstrated, in a number of in vivo systems, that the density of the responding naïve CD4+ T cells, that is to say the frequency of responding CD4+ T cells in an animal, has a significant impact on their Th1/Th2 phenotype. In particular, our laboratory has reported that in response to antigen, a low density of responding naïve CD4+ T cells give rise to Th1 cells while a high density of responding CD4+ T cells give rise to Th2 cells. Although we have made these observations, we had not, prior to the work described herein, elucidated the mechanism(s) surrounding this phenomenon. This was the impetus for the experiments detailed in this thesis. In order to investigate the mechanism(s) mediating the effect that the density of responding CD4+ T cells has on the Th1/Th2 phenotype, we have developed an in vitro system that employs a physiological number of naïve TCR transgenic CD4+ T cells (DO11.10) specific for the OVA323-339 peptide in the context of I-Ad. My observations in this in vitro system mirror previous in vivo observation in that a low frequency of naïve CD4+ DO11.10 T cells cultured with syngeneic APC and the OVA323-339 peptide, acquire a Th1 phenotype while naïve CD4+ DO11.10 T cells cultured at high frequencies, under identical conditions, acquire a Th2 phenotype. By controlling the type of APC in our culture system, we have ascertained that the effect of CD4+ T cell density on the differential generation of Th1 and Th2 cells is mediated by B cells, but not dendritic cells. We also found that the addition of an agonistic anti-CD28 antibody skews the phenotype of T cells cultured at low densities towards the Th2 pole while partially blocking of CD80 and CD86, through the addition of CTLA4-Ig to high density T cell cultures, resulted in the development of Th1 cells. In contrast, modulating the interactions between CD40/CD40L and OX40/OX40L, two co-stimulatory molecule/ligand pairs known to affect Th1/Th2 differentiation, through the addition of agonistic and antagonistic antibodies in high or low density CD4+ T cell cultures did not alter the Th1/Th2 phenotype of the newly activated DO11.10 T cells. Thus, it appears that the level of CD80 and CD86 in culture affects the Th1/Th2 differentiation of cultured naïve T cells. However, the level of CD80 and CD86 on the surface of B cells from high density CD4+ T cell cultures never exceeded that of B cells from low density CD4+ T cell cultures. We propose that the additional CD80 and CD86 in high density cultures are found on the surface of the responding CD4+ T cells. This hypothesis is based on the fact that CD4+ T cells are known to express CD80 and CD86 on their surface shortly after TCR ligation, which we have confirmed in our system using flow cytometry. Therefore, we suggest that the Th2 phenotype observed in cultures containing a high density of responding CD4+ T cells is due to the additional CD28 stimulation responding T cells receive through increased CD28-CD80/86 mediated CD4+ T cell-T cell interactions