Cell Interactions in Three Dimensional Tissue Culture

Child Health And Human Development

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Abstract

Critical events of HIV disease occur in lymphoid tissue, where infection leads to T cell depletion and immunodeficiency. Therefore, we study HIV pathogenesis in human lymphoid tissue ex vivo. Using HIV constructs deleted of individual accessory genes or their combinations, we found that nef, vpr, and vpu are relevant for efficient viral infection and for CD4 T cell depletion in HIV-1-infected human lymphoid tissues. However, as we found, even non-infectious virions are immunosuppressive, indicating that defective virions in vivo that constitute the majority of viral particles may contribute to immunodeficiency. Also, we demonstrated using an HIV construct that requires doxycycline for replication, that neither a non-productive tissue infection, nor tissue exposure to high virion concentrations lead to CD4 T cell depletion unless productive infection is triggered. 1. Effect of accessory genes on HIV-1 replication and CD4+ T cell depletion in human lymphoid tissue ex vivo The relevance of the accessory vpr, vpu, and nef genes for HIV-1 replication in human lymphoid tissue, the major site of viral replication in vivo, is largely unknown. We used this system to investigate the role of accessory genes. A single deletion of any of the accessory genes nef, vpr, and vpu dramatically decreased viral replication in ex vivo?infected human lymphoid tissue . Combined deletion of vpr and vpu impaired HIV-1 replication even more severely and additional deletion of either vpr or vpu further attenuated replication of the nef-deleted HIV-1 variant. However, only the combined deletion of all three accessory genes completely disrupted HIV-1 replication. The deletion mutants caused less CD4+ T cell depletion than the wild-type parental virus and there was a strong correlation between the number of productively infected CD4+ T cells and the level of their depletion. Thus, although molecular mechanisms for low viral infectivity and virus production may be different for different mutants, they all result in a lower number of infected cells and hence less CD4+ T cell depletion in infected tissues. In conclusion, our data suggest that, similarly to Nef, Vpr and Vpu are relevant for efficient viral infection and for CD4 T cell depletion in HIV-1-infected individuals. 2. Immunosuppressive activity of non-infectious HIV-1 virions in human lymphoid tissue ex vivo The vast majority of HIV-1 virions in HIV-1-infected patients are not infectious. Therefore, cells are more likely to interact with noninfectious particles than with their infectious counterparts, raising the question of the pathogenesis of non-infectious virions in human lymphoid tissue. To answer this question we used HIV-1 virions rendered non-infectious by treatment with aldrithiol-2 (AT-2). These AT-2-treated virions retain both the structure and function of the viral envelope glycoproteins, but are not infectious, and in human tissues ex vivo, do not induce CD4+ T cell loss. However, exposure of cultures to AT-2 inactivated X4-LAV.04 resulted in inhibition of antibody responses to tetanus toxois (TT) and/or pokweed mitogene (PWM). In contrast, no inhibition of anti-TT IgG or total IgG was observed when stimulated tissues were exposed to comparable or higher amounts of AT-2 inactivated R5-SF162 virions. Furthermore, we found that virion-free medium conditioned by tissues exposed to AT-2-inactivated X4LAV.04 inhibited anti-TT IgG and total IgG responses of fresh cultures indicating that AT-2-inactivated X4-LAV.04 virus induced the secretion of an immunosuppressive factor. This factor(s) was inactivated by heat, but was resistant to freezing and thawing and the activity . of media conditioned by tissue exposed to AT-2-inactivated LAV.04 was associated with the fraction containing molecules of MW greater than 50 kDa. We conclude that medium conditioned by tissue incubated with AT-2 inactivated X4 HIV-1 contains soluble factor(s) that are responsible for suppression of B cell responses. Since the majority of the virions circulating in vivo are not detectably infectious, one can speculate that of these defective HIV-1 virions may suppress immune responses. 3. Reactivation of HIV-1 replication and CD4 depletion T lymphocytes and in human lymphoid tissue infected ex vivo with doxycycline-dependent HIV-1. Is believed that CD4+ T cells which do not produce HIV-1, but which reside in infected tissues are killed along with HIV-infected CD4+ T cells. To address this problem we used human lymphoid tissues infected ex vivo with a newly developed virus construct, HIV-rtTA, that can infect cells and establish an integrated provirus in the absence of doxycycline but whose transcription and replication are dependent on the presence of the antibiotic doxycycline (HIV-rtTA). We established that without adding doxycycline, no productive infection was detected in inoculated tissue blocks whereas in the presence of doxycycline, a vigorous productive infection was detected in inoculated tissues. Also, we demonstrated that HIV replication in latently infected cells could be turned on in HIV-rtTA-inoculated tissues, which were kept several days without doxycycline. In the productively infected tissues (that is, those inoculated with HIV-rtTA and cultured in the constant presence of doxycycline), more than 60% of CD4+ T cells were depleted after 12 days of culture. In contrast, no significant CD4+ T cell depletion was observed in matched tissues that were similarly inoculated but cultured in the absence of doxycycline (and thus without viral replication). Moreover, when the amount of virus present in latently infected cultures was matched to that in productively infected (doxycycline-treated) tissues by multiple inoculation of tissue (13 times over the 9 days) there was no significant depletion of CD4+ T cells in the absence of doxycycline. In conclusion, HIV is sufficient to deplete tissue of productively infected CD4+ T cells but is not sufficient to cause a significant death of uninfected or latently infected CD4+ T cells in the context of human lymphoid tissue, and thus additional factors seem to contribute to bystander cell death in vivo. Since unlike in vivo, ex vivo infected tissues do not respond to HIV infection by general immunostimulation, such an immunostimulation may be the main additional factor for CD4+ T cell depletion in HIV-infected individuals. Nevertheless in spite of general stimulation some of the infected CD4+ T cells in these individuals continue to harbor a replication-competent HIV but do not constantly produce virus. These cells constitute a major obstacle for anti-HIV therapy. To design strategies to purge latent HIV reservoir, we investigated mechanisms by which a non-tumor-promoting phorbol ester, prostratin stimulates cells. Prostratin-induced expression of activation markers CD25 and CD69 but inhibited cell cycling. HIV-1 uptake was reduced in prostratin-stimulated CD4 T cells. Also, prostratin reduced completion of reverse transcription of the viral genome in lymphatic tissue, but facilitated integration of reverse transcribed HIV-1 genome in non-dividing CD4+ T cells and expression of already integrated HIV-1, including latent forms. Thus, while prostratin stimulation restricts susceptibility of primary resting CD4 T cells to HIV infection, it efficiently reactivates HIV-1 from pre- and post-integration latency in resting CD4+ T cells. This dual role makes prostratin an excellent candidate for the elimination of persistent HIV infection from latent reservoirs.