Lymphocyte numbers are regulated both by responses to conventional exogenous antigens, by stimulation by endogenous peptide/MHC complexes and by the action of a series of cytokines. This multifaceted regulation permits individuals to maintain a broad repertoire of specificities, allowing responses against a vast array of foreign substances and, at the same time, providing a pattern of memory based on the immunization history of the individual. The study of the process of lymphocyte dynamics that underlies this regulation requires a multidisciplinary approach, aimed both at the molecular underpinnings of the processes through which lymphocytes survive and proliferate and a systemics/ computational biology approach to appreciate the overall mechanisms governing total numbers of lymphocytes of distinct phenotype and distinct specificity. Emphasis has been placed on four aspects of this problem: the priming of naive CD4 T cells, the dynamics of lymphocyte memory, the mechanisms underlying CD4 T cell depletion in HIV infection, and the process of homeostatic proliferation and death. Unit scientists have shown that primary responses are highly dependent upon the number of precursor cells that can respond to antigenic challenge. Using both real time PCR and flow cytometric analysis to measure the response when TCR transgenic cells are transferred to intact recipients, it has been shown that the factor of expansion (FE) of the antigen-stimulated CD4 T cells is highly dependent upon the number of specific precursors. When the frequency of precursors in the recipient is 3 or less, FE is 1500, at 300 cells it is 200 and at 30,000 it is 20. Limitation in expansion does not result from a smaller fraction of cells responding but rather, at least in part, from diminished proliferative rates of responding cells. Diminution in FE as number of precursors increase cannot be accounted for by Fas, TNF or IFNg-mediated cell death nor can it be due to limitation in numbers of dendritic cells or in amounts of antigen as increasing either DC number or amount of antigen does not alter the non-linearity of FE and precursor number. Furthermore, the effect is not altered by supplementation with IL-1, IL-2, IL-7 or IL-15. The relative frequency of regulatory T cells, either derived from the responding cells or from the host, is not altered by precursor frequency and the difference in FE occurs even when responding cells are unable to develop into regulatory T cells. The effect is highly antigen specific in that large numbers of cells of one specificity do not effect the rate of expansion of small numbers of cells of another specificity. We have concluded that there is a powerful physiologic regulatory process based on the magnitude of response within a set of responding cells that acts to feedback on cells to diminish their proliferative rate and possibly to alter their decisions regarding growth and differentiation. In the course of analyzing the control of FE on the part of both nave and memory cells, it was observed that the most potent stimulant of FE was the cytokine IL-1. When expansion of CD4 TCR transgenic T cells in a syngeneic host in response to antigen was measured, it was found that implanting a mini-osmotic pump that delivered 10 micrograms of IL-1 over a 7 day period caused a ten fold enhancement in FE when compared to that seen using conventional adjuvants such as LPS. This was equally true for naive and memory cells and was not mediated by other cytokines. The effect could only be partially explained by enhanced proliferation so that greater survival was also implicated. The use of recipients that were IL-1 receptor knockouts and IL-1 receptor-sufficient donors of TCR transgenic T cells showed that IL-1 could act directly on the responding T cells. Anti-IL-1 antibody diminished the adjuvant effect of LPS indicating that at least a portion of the effect of this conventional adjuvant was due to endogenous production of IL-1. Initial analysis of genes activated and suppressed in cells responding to antigen in the presence of LPS suggest avenues for further analysis that may lead to a mechanistic understanding of the IL-1 effect. The very robust effect of IL-1 suggests it may have a role in certain immunization strategies. Analysis of the steady state proliferation of memory CD4 T cells reveals that it is similar in conventional and germfree mice. The similarity of the prolifertative rate in conventional and germfree mice suggests that this is not driven by intestinal microflora or conventional antigens and that it may represent self-reactivity. Analysis of recently divided cells by extensive sequencing of Vb2-CDR3- Jb1.1 CD4 T cells as well as quiescent CD44 bright cells has revealed no difference in receptor complexity and a degree of complexity suggesting only a very limited number of recent divisions among the Ki67 bright cells. This implies that division is largely stochastic and probably dominantly driven by cytokines rather than by peptide/ MHC complexes, whether of exogenous or endogenous origin. Working with colleagues at the Oregon Regional Primate Center, we have analyzed lymphocyte dynamics in SIV-infected macaques and have obtained evidence for CD4 T cell populations that decay at very distinct rates in the period after their burst-like expansion. Laboratory of Immunology scientists and our colleagues have shown the critical role that aberrant immune activation in lentiviral infection plays in the decay of CD4 T cell number and in the degradation of immunocompetence in SIV-infected macaques and HIV-infected humans. The ongoing activation of the immune system caused directly or indirectly by infection appears to be of particular importance in the loss of potential effector cells during the chronic phase of the infection. Working with Professor G. Bacherov of the Russian Academy of Sciences has allowed the of use computational methods to analyze lymphocyte dynamics. The models that have been constructed conform extremely well to the behavior of cells primed at small or large precursor frequency and give further insight into the mechanism underlying the remarkable precursor number effect.
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