We propose the use two recently developed limiting dilution analysis (LDA) techniques to study the size, composition and stability of T cell subpopulations in the peripheral blood of healthy humans and selected surgery patients. These T cell subpopulations will be defined on the basis of their antigen- induced function: antigen-specific cytolytic activity (cytolytic T lymphocytes; CTL) or Interleukin-2 secretion (helper T lymphocytes; HTL). Cells bearing major histocompatibility complex (MHC) disparities will constitute the antigenic stimuli for the T cells in this study. We are unaware of any previous studies of this kind, so we will first study the T cell populations in healthy humans to establish baseline values. Hence, we propose to 1) determine how various major histocompatibility complex (MHC) disparities influence the frequency of alloantigen-reactive CTL or HTL when peripheral blood mononuclear cells (PBMC) from healthy humans are stimulated in limiting dilution microcultures with allogeneic PBMC bearing serologically identified Class I and/or Class II MHC-encoded alloantigens, 2) determine how cell surface phenotype relates to lymphocyte function when isolated CD3+, CD4+ or CD8+ PBMC from healthy humans are stimulated in limiting dilution microcultures with allogeneic PBMC bearing serologically identified Class I and/or Class II MHC disparities, 3) determine how the ability of human PBMC to generate detectable proliferative and cytotoxic responses to allogenic cells in mixed lymphocyte cultures (MLCs) relates to the frequencies of alloantigen-reactive CTL and/or HTL present in the PBMC population. Having established baseline values, we will perform similar tests on the pre-operative and post-operative PBMC populations of selected surgery patients. These studies represent the first quantitative analyses of antigen- specific T cell subpopulations in human peripheral blood. As such, they may provide a better understanding of the antigenic hierarchy empirically associated with MHC alloantigens. Furthermore, these experiments should produce decisive information regarding the controversial relationship between T cell function and serologically defined T cell surface phenotype. The comparison of CTL and HTL frequency with MLC reactivity will allow our limiting dilution analysis data to be correlated with other published analyses of in vivo and in vitro alloreactivity. Finally, these studies will establish whether healthy humans and pre-operative or post-operative patients differ in the size or reactivity of their T cell functional subpopulations.
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