The fate of self-reactive T lymphocytes bearing alpha-beta T cell receptors (TcR) in the thymus and periphery, as well as their autoimmune potential, have been the subject of many investigations. In contrast, very little is known about the ontogeny or autoimmune potential of self-mutative gamma- delta TcR-bearing lymphocytes. The recent literature has suggested that there are significant numbers of self-mutative gamma-delta lymphocytes in normal mice. The objective of this proposal is to identify and characterize self-self interactions mediated by gamma-delta TcR-bearing subsets. We will focus on those interactions which alter lymphoid tissue architecture, either to its detriment or to promote T cell maturation. T cell clones and hybridomas will be derived from gamma-delta T cell- enriched cultures isolated from fetal and neonatal thymus, spleen and gut of normal and Vgamma1.1Jgamma4Cgamma4-C-gamma-4 transgenic mice. These gamma-delta lines will be selected for reactivity against autologous primary stromal (epithelium, dendritic, etc) populations and cell lines isolated from the same tissue at the same time during development. The fine specificity of these clones will be determined by stimulation with antibody-enriched lymphoid stromal cell subsets and characterized lymphoid stromal cell lines. T cell receptors from these stromal-specific clones and hybridomas will be cloned by the polymerase chain reaction (PCR) and sequenced to identify any biased expression of TcR chain segments and for uses as trans genes. T cell clones expressing gamma-delta TcRs and recognizing autologous lymphoid stromal subsets, will be further characterized in vitro for cytotoxicity and elaboration of hematopoietic growth factors. The requirement for presentation by MHC class Ia, Ib, and II antigens will be assessed. Lastly, the stromal specific gamma-delta clones and hybridomas will be tested for their ability to specifically respond to heat-shock proteins (HSP), a class of highly conserved molecules that are hypothesized to be a major endogenous and exogenous antigen for gamma-delta TcRs. Selected T cell clones capable of secreting hematopoietic growth factors (i.e., IL-2, IL-3, IL-4, interferon-gamma) will be introduced into immune deficient (irradiated and SCID) by intravenous or direct intra-organ injection. To reproduce the fetal thymic microenvironment, these stromal cell-specific clones will also be tested in fetal thymic organ cultures. Alternatively, gamma-delta TcR from stroma-specific hybridomas will be used to generate transgenic mice that will be bred onto the SCID background. These systems will assess directly the ability of stromal-specific gamma- delta lymphocytes to support development of lymphoid microenvironments in the absence of other TcR-bearing subsets. Previously, expression of a Vgamma1.1Jgamma4Cgamma4 TcR transgene in mice was shown to support expansion of stromal elements in the lymphoid organs which is associated with the early appearance of T cell reactivity. Mice carrying a disrupted Vgamma1.1Jgamma4Cgamma4 gene in their germline are currently being generated for this study. Absence of the Vgamma1.1Jgamma4Cgamma4 TcR gene product will directly address its putative role in cell-cell signalling within lymphoid stroma. These mice will also be excellent candidates for the introduction of autologous, stromal- specific clones or gamma-delta TcR transgenes that express a Vgamma1.1Jgamma4Cgamma4 TcR chain. In summary, this study will characterize self-self interactions between lymphoid stroma and gamma-delta T cell subsets at the functional and molecular level. The possibility that there is a component of T lymphocyte self recognition which functions to support normal development and/or reactivity of the immune system is a unique feature of this proposed study. The supportive or cytotoxic potential of the autologous, stromal-specific lymphocytes will serve to extend our knowledge of the biological role of the enigmatic gamma-delta TcR.
