IN-VIVO CD40-GP39 INTERACTIONS ARE ESSENTIAL FOR THYMUS-DEPENDENT HUMORAL IMMUNITY .1. IN-VIVO EXPRESSION OF CD40 LIGAND, CYTOKINES, AND ANTIBODY-PRODUCTION DELINEATES SITES OF COGNATE T-B-CELL INTERACTIONS

T-B cell interactions have a central role in the development of antibo dy responses. Upon activation, T helper (Th) cells express the ligand for CD40, gp39, which is essential for Th cell-dependent B cell activa tion. The cytokines produced by activated Th cells have a regulatory r ole in B cell differentiation. In this study, we investigated, using i mmunohistochemical techniques, the in vivo time course and localizatio n of gp39 expression and cytokine production in relation to the specif ic antibody production. Both the immunization with keyhole limpet hemo cyanin (KLH), a thymus-dependent (TD) antigen, and trinitrophenyl (TNP )-Ficoll, a thymus-independent type 2 (TI-2) antigen, induced Th cells to express gp39. The expression of gp39 was restricted to Th cells in the outer periarteriolar lymphocyte sheaths (outer-PALS) and around t he terminal arterioles (TA). Incidentally, gp39+ Th cells were found i n the corona of follicles, whereas gp39+ cells were never found in the germinal centers or marginal zones of the spleen. Maximum frequencies of gp39+ cells were observed 3 and 4 d after primary and secondary im munization with KLH. After injection of TNP-Ficoll, a marked increase in gp39+ cells was observed, confirming previous observations that act ivated T cells are involved in TI-2 antibody responses. Analysis of th e in vivo cytokine production revealed that interleukin 2 (IL-2)-, IL- 4- and interferon gamma (IFN-gamma)-producing cells (IFN-gamma-PC) dev eloped according to similar kinetics as observed for gp39+ cells. IL-2 -PC and IL-4-PC were present in higher frequencies as were IFN-gamma-P C in the immune response against TNP-KLH. Double staining experiments revealed gp39+ Th cells producing IL-2, IL-4, or IFN-gamma suggesting that these cells were involved in both the initial activation as well as the differentiation process of B cells into antibody-forming cells. Dual immunohistochemical analysis revealed gp39+ T cells and cytokine -PC in close proximity to antigen-specific, antibody-forming B cells. In conclusion, this study shows that in vivo gp39 is expressed on acti vated Th cells after immunization with TD and TI-2 antigens. Furthermo re, the time course and compartmentalization of gp39+ expression, cyto kine production and antibody formation after immunization suggest that cognate T-B cell interactions and T cell-regulated B cell differentia tion occur in the outer-PALS and around the TA of the spleen.