Critical events of many human infectious diseases occur in tissues where cells contact neighboring cells and are immersed in a rich heterogeneous microenvironment. Microbes, viruses in particular, may cause disease by disrupting cell-cell interactions. We study the pathogenesis of microbes, in particular HIV-1, in a system of human lymphoid tissue ex vivo. In this model, we have investigated the contribution of non-activated cells to the viral load, whether cell activation status determines cell death in lymphoid tissues, and whether uninfected (bystander) CD4+ T cells are depleted in HIV-1?infected tissues. Also, we continue to study the pathogenesis of HIV-1 co-pathogens in the context of human tissues, and we report below on our study of the pathogenesis of human herpesvirus 6 (HHV-6). 1. Viral production and depletion of activated and non-activated cells in HIV-infected tissues ex vivo First, we investigated whether T cell activation patterns in tissue determine the efficiency of viral infection, and we evaluated the contributions of activated and non-activated lymphocytes to T cell depletion. As reported for tissues in vivo, both activated and non-activated T cells are productively infected ex vivo. An activated T cell is only twice as likely as a non-activated T cell to be productively infected. However, the contribution of the latter pool to total viral production seems to be small in spite of its larger size. Thus, the number of activated cells in ex vivo tissue is a determinant of viral load. Although the proportion of activated cells is increased among productively infected T cells, infection itself did not change the pattern of activation marker expression in bystander uninfected T cells. Furthermore, the presence of infected T cells is not sufficient to induce massive death of bystander CD4+ T cells. This was proven in experiments in which we inoculated tissue with infectious HIV-1 and, after productive tissue infection was established, added Nevirapine, a non-nucleotide reverse transcriptase inhibitor. Nevirapine did not prevent already infected cells from continuing to produce virus, but protected uninfected cells from becoming productively infected. Thus, in these tissues, one pool of CD4+ T cells was infected prior to Nevirapine application and continued to produce virus, and the other pool of CD4+ T cells, residing in the same tissue, remained uninfected. The latter cells are by definition bystanders. We compared the levels of apoptosis and depletion of CD4+ T cells in control HIV-1?infected and Nevirapine-treated tissues. Tissue infection increased the expression of an apoptotic marker in productively infected T cells, and activated T cells predominantly became apoptotic. Consequently, productively infected T cells are depleted from the tissue. However, the depletion of uninfected (p24-) CD4+ T cells stopped after Nevirapine was applied, and CD4+ T cell numbers remained stable. In conclusion, our results indicate that HIV-1 infection in ex vivo human lymphoid tissues does not affect the activation status of bystander T cells and that the majority of CD4+ T cells, which are not productively infected but which reside among productively infected cells in HIV-infected lymphoid tissues, are not depleted. It seems that neither productive infection alone nor activation alone is sufficient to induce T cell apoptosis. Only a combination of these two factors is sufficient for T cell apoptosis. Therefore, general immunostimulation may be a critical factor for CD4+ T cell depletion in HIV-infected individuals. 2. Pathogenic effects of human herpesvirus 6 in human lymphoid tissue ex vivo HHV-6 is a potentially immunosuppressive agent that has been suggested to act as a cofactor in the progression of HIV disease. We have found that HHV-6 inhibits HIV-1 infection in coinfected human tissues ex vivo. We have further characterized the cellular tropism and pathogenic mechanisms of HHV-6 by asking (i) whether the lymphoid tissue system can efficiently support the growth of HHV-6 of both subgroups (A and B) in the absence of exogenous stimuli; (ii) which cell subsets are primarily targeted by HHV-6 A and which by B; (iii) whether naive and memory T cells show a difference in susceptibility to HHV-6 infection; (iv) whether the infection is cytopathic for tissue-residing lymphocytes and whether it alters the expression of functionally important cell surface molecules such as the viral receptors CD46 , CD3, and CD4; and (v) whether HHV-6 selectively modulates the expression of chemokines. Both subgroup-A and subgroup-B HHV-6 isolates replicate in an ex vivo system without exogenous stimulation. The vast majority of viral antigen?positive cells were CD2+, with very low productive infection in other cell types. The mean proportions of productively infected cells among CD4+ T cells and CD8+ T cells were similar; nevertheless, since CD4+ T cells represent the predominant T cell subpopulation in human lymphoid tissue, the majority of the infected cells expressed CD4+. Infection of CD4+ T cells occurred with a similar efficiency with the two viral subgroups, whereas CD8+ T cells were efficiently infected only by HHV-6 A. Analysis of the relative levels of HHV-6 infection in the non-naive (CD45RA-CD62L+, CD45RA-CD62L,- or CD45RA+CD62L-) and the naive (CD45RA+CD62L) T cell subsets revealed a preferential infection of non-naive cells by both the A and B HHV-6 variants. To evaluate the cytopathic effects of HHV-6 in human lymphoid tissue ex vivo, we measured the absolute numbers of cells recovered from pooled tissue fragments. HHV-6 A depleted CD4+ and CD8+ T cells with similar efficiency, while HHV-6 B depleted CD4+ T cells with a significantly greater efficiency. These data indicate that in the context of human lymphoid tissue HHV-6 is cytopathic and that cell depletion primarily involves the cells in which HHV-6 is replicating, without significant depletion of bystander cells. HHV-6 induces a dramatic and generalized downmodulation of its receptor, CD46, in lymphoid tissue, both in CD2+ T cells expressing HHV-6 antigens and in HHV-6?antigen negative T cells. These data demonstrate that HHV-6, particularly of subgroup A, induces a marked loss of CD46 in human lymphoid tissue, which may result in an increased susceptibility to tissue damage secondary to spontaneous activation of the complement cascade. The CD3 antigen was found to be dramatically downmodulated on the surface of cells infected with either HHV-6 A or HHV-6 B, while no significant reduction of CD3 was observed in uninfected cells. Unlike CD3 expression, CD4 antigen expression was doubled on the surface membranes of cells isolated from tissues infected with HHV-6. This increased surface expression of CD4 was observed only on the surfaces of infected cells. HHV-6 infection upregulates the production of RANTES, but not of other cytokines or chemokines, in lymphoid tissue. The amount of secreted RANTES correlated with the level of HHV-6 replication. This increased secretion of RANTES in HHV-6?infected tissues may profoundly influence the physiology of the immune responses, as well as, more specifically, the replication of different HIV-1 biological variants. In conclusion, the dramatic effects that we documented in the lymphoid tissue infected ex vivo with HHV-6 of both the A and B subgroups provide compelling evidence, in a physiologically relevant system, that HHV-6 can efficiently infect both memory and naive T cells, causing downmodulation of physiologically relevant cell surface receptors in both infected and bystander cells and thereby potentially causing immune dysregulation and immunosuppression.
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