PATHOGENIC MECHANISMS IN HIV DISEASE

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The purpose of this project is to characterize the factors that regulate the maintenance of the T cell pool in adult humans and to identify how these parameters are affected by HIV infection. The insights gleaned from these studies are used in the development of therapeutic strategies. The resulting clinical trials are then used as tools for obtaining additional insights into the pathophysiologic mechanisms present in patients with HIV infection.An early observation in studies of the immune systems of patients with HIV infection was that these patients had a profound defect in the ability of their peripheral blood mononuclear cells to respond to stimulation with remote recall antigens such as tetanus toxoid. While defects in blast transformation to mitogens such as pokeweed mitogen or alloantigens could be reversed by culturing larger numbers of CD4 T cells, this type of correction for CD4 T cell numbers had no effect on responses to tetanus. In an effort to better understand this phenomena a detailed analysis of changes in T cell subsets as defined by CD45 isoforms and T cell receptor genotypes was carried out on longitudnal samples obtained from patients with various stages of HIV infection before and after therapy with combination antiretroviral regimens. These studies demonstrated that the CD4 declines seen in the setting of progressive HIV infection were associated with a preferential loss of CD45RO- (?na?ve?) cells and progressive skewing of the T cell receptor (TCR) repertoire as characterized by measurements of size diversity across the CDR3 region of the beta-chain of the T cell receptor. Of note, despite pronounced increases in total CD4 T cell counts, these changes were not immediately reversible following initiation of antiretroviral therapy strongly suggesting that the increases in CD4 T cells are due to peripheral expansion of preexisting T cells rather than stem cell differentiation through a thymic environment. This degree of TCR skewing in the setting of a marked decrease in the overall size of the CD4 T cell pool suggests that some elements of the T cell repertoire may be permanently lost in the absence of significant amounts of stem cell differentiation. The rapid resolution of many of the AIDS-defining illnesses following the initiation of antiretroviral therapy suggests that those elements of the T cell repertoire responsible for host defense against these pathogens are present, however unable to maintain adequate host defense in the setting of high levels of virus. To determine whether or not one could demonstrate the predicted loss of T cell diversity following effective antiretroviral therapy, patients with varying degrees of CD4 T cell recovery were immunized with either the recall antigen tetanus toxoid or the neoantigen bacteriophage PhiX174. Approximately two-thirds of the patients demonstrated normal responses to tetanus toxoid following booster immunization. As predicted, however, immune responses to the neoantigen were significantly less in the patients with treated HIV infection compared to healthy controls. Taken together these data suggest that the earlier finding of lack of antigen-specific responses in patients with HIV infection are most likely due to a decrease in the number of antigen- specific clones below a certain critical level.In a related series of studies we were interested in determining the survival of CD4 T lymphocytes in patients with HIV infection utilizing the identical twin model. Our initial work in this area involved the adoptive transfer of lymphocytes and bone marrow from a healthy donor to a syngeneic twin with HIV infection. In this setting lymphocyte counts were found to increase very transiently with levels back to baseline within 4-6 weeks. In an effort to more accurately track the survival of the newly infused cells, the cells from the healthy donor were marked with a bacteria-derived neomycin resistance gene. To our surprise and despite the confounding variable of immune-elimination of the transduced cells, the persistence of marked cells could be noted long after the time that total lymphocyte counts returned to normal. In addition there appeared to be a strong correlation between the viral burden and the fraction of marked cells persisting at 6 months in either peripheral blood or lymphoid tissue. Consistent with the data on the T cell repertoire, these data again support the importance of peripheral expansion as a means of maintaining the CD4 T cell pool in the adult human.Having two independent sets of data leading to a similar conclusion, we have now focused our efforts in this area on obtaining a more precise look at the in vivo turnover of the lymphocyte pool in adult humans in general and in particular in the setting of HIV infection. Utilizing in vitro labeling of PBMCs with 5-bromo-2?-deoxyuridine (BrdU) and in vivo labeling with either BrdU or 2H-glucose we have begun to derive some insight as to the magnitude of peripheral lymphocyte turnover in the adult human. In preliminary data, approximately 0.1% of the peripheral blood CD4 T cells have been found to enter S-phase over a 4-hour period of time. Viewed another way, these data suggest that the entire CD4 pool has the potential to turnover every 167 days. The rate of cell turnover increases approximately 3-fold in the setting of untreated HIV infection, decreases to close to the levels of healthy controls with anti-retroviral therapy and can be transiently increased with in vivo administration of IL-2. Both CD45 RO+ (?memory) and CD45 RO- (?naive?) CD4 T cells were found to be in S-phase. Analysis of CD4 T lymphocytes from lymphoid tissue from patients with HIV infection suggest the turnover rates of T cells in lymphoid tissue are approximately twice the turnover rates seen in peripheral blood. These numbers are very consistent with the model outlined above and provide direct evidence that peripheral expansion of exiting T cells is adequate to account for the changes in CD4 T cell numbers seen following the initiation of antiretroviral therapy. In addition they provide a firm theoretic background for the use of IL-2 as an adjunct to antiretrovirals in the treatment of patients with HIV infection.