Functional analysis of Histoplasma capsulatum-reactive T-cell hybridomas

Abstract

Activation of CD4+ T cells is a crucial step in the elimination of Histoplasma capsulatum yeast cells from tissues. However, only a limited amount of information exists concerning the immunobiology of H. capsulatum-reactive T cells that are CD4+. To facilitate the analysis of the functional activities of this T-cell subpopulation, we developed a panel of 10 murine T-cell hybridomas from splenocytes of immune C57BL/6 mice. All hybridomas reacted with monoclonal anti-CD4+ antibody and released interleukin-2 after stimulation with histoplasmin. Within 3 weeks, the reactivity of hybridomas to histoplasmin declined dramatically, yet the cells responded vigorously to yeast-phase preparations that were enriched for cytosol, cell wall, or cell membrane. Of 10 hybridomas studied, only one recognized heterologous fungal antigens. Responsiveness to yeast-phase antigens was restricted by I-Ab. We mapped determinants in cytosol and cell wall or cell membrane by the technique of one-dimensional T-cell immunoblotting. The patterns of responses of hybridomas to cytosol were nearly uniform. All hybridomas responded to two immunodominant regions in cytosol with masses ranging from less than or equal to 18 to 26 kilodaltons (kDa) and 35 to 39 kDa. All hybridomas tested responded to determinants in the cell wall or cell membrane preparation with masses of 35 to 39 kDa. These hybridomas provide a useful tool for defining yeast-phase antigens that trigger T-cell activation.