Characterization of the Immune Response to Alloantigens: An in vitro Model System for Cognate Regulation of CD4 T cell Differentiation by CD8 T cells
ABSTRACT It is accepted that CD4 T cells are required for the activation of naïve CD8 T cells. In this study, we have investigated the “reverse reaction” that of the effect of CD8 T cells have on the differentiation of CD4 T cells while present at the initiation of the immune response. Our experimental system consists of One Way Mixed Lymphocyte Reaction, MLR, where parental spleen cells homozygous at the Major Histocompatibility Complex, MHC, are activated by irradiated allogeneic spleen cells differing at the MHC loci. Differentiation of CD4 and CD8 T cells was assessed by their expression of distinct signature cytokines, IFN-γ and IL-4 by employing the single T cell ELISPOT assay. CD4 and CD8 T cell sorting and identification was achieved by Magnetically Activating Cell Sorting technology, MACs, and Flow-Cytometry, respectively. This methodology, allowed us to explore the mechanism by which CD8 T cells regulate the differentiation of naïve CD4 T cells towards either a Th1(IFN-γ) or a Th2(IL-4) phenotype. We have found that distinct CD4:CD8 T cell ratios of the spleens of different strains of mice correlate with a Th1(IFN-γ), a Th2(IL-4) or a mixed Th1(IFN-γ)/Th2(IL-4) type immunity when these cells are stimulated with spleen cells from mice differing at the MHC loci. In contrast, similar cultures depleted of CD8 T cells, invariably generate Th2(IL-4), CD4 T cells in all mouse strains tested. Both, unprimed and effector CD8 T cells suppress the Th2(IL-4) development in an antigen specific manner. They do so by means of an allo-antigen dependent secreted soluble factor. This factor is IFN-γ. Finally, the employment of the ELISPOT assay for enumerating and characterizing effector CD4 and CD8 T cells allowed us to verify, with contemporary methodology, the extraordinary high frequency of alloreactive T cells, a phenomenon described by Simonsen in 1957. Thus, in these studies we have revisited the mechanism by which CD8 T cells were known to “suppress” the antibody response. Our findings indicate that CD8 T cells inhibit in a cognate manner the CD4 Th2 (IL-4) differentiation, the subset required for antibody development. Further on, by extrapolation our data may explain the well-known in vivo phenomena in which distinct naturally occurring CD4:CD8 T cell ratios from two different parental mouse strains, promote either Th1 immunity and graft rejection, or a Th2-type response ineffective at clearing the graft, upon allo-transplantation of parental spleen cells into F1 mice. Consequently, a persistent Th2 response results in time in antibody-mediated generalised autoimmunity resembling Systemic Lupus Erythematosus.
CD8 T cells, CD4 T cells, cytokines, Th1, Th2
Doctor of Philosophy (Ph.D.)
Microbiology and Immunology
Microbiology and Immunology