The persistence of HIV reservoirs, including latently infected, resting CD4+ T cells, is the major obstacle to cure HIV infection. CD32a expression was recently reported to m ark CD4+ T cells harboring a replication-competent HIV reservoir during antiretroviral therapy (ART) suppression. We aimed to determine whether CD32 expression marks HIV latently or transcriptionally active infected CD4+ T cells. Using peripheral blood and lymphoid tissue of ART-treated HIV+ or SIV+ subjects, we found that most of the circulating memory CD32+ CD4+ T cells expressed markers of activation, including CD69, HLA-DR, CD25, CD38, and Ki67, and bore a TH2 phenotype as defined by CXCR3, CCR4, and CCR6. CD32 expression did not selectively enrich for HIV- or SIV-infected CD4+ T cells in peripheral blood or lymphoid tissue; isolated CD32+ resting CD4+ T cells accounted for less than 3% of the total H IV DNA in CD4+ T cells. Cell-associated HIV DNA and RNA loads in CD4+ T cells positively correlated with the frequency of CD32+ CD69+ CD4+ T cells but not with CD32 expression on resting CD4+ T cells. Using RNA fluorescence in situ hybridization, CD32 coexpression with HIV RNA or p24 was detected after in vitro HIV infection (peripheral blood mononuclear cell and tissue) and in vivo within lymph node tissue from HIV-infected individuals. Together, these results indicate that CD32 is not a marker of resting CD4+ T cells or of enriched HIV DNA-positive cells after ART; rather, CD32 is predominately expressed on a subset of activated CD4+ T cells enriched for transcriptionally active HIV after long-term ART.

Under selective pressure from host cytotoxic T lymphocytes, many viruses have evolved to downregulate major histocompatibility complex (MHC) class I and/or T-cell costimulatory molecules from the surface of infected cells. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes two proteins, MIR-1 and MIR-2, that serve this function during lytic replication. In vivo, however, KSHV exists in a predominantly latent state, with less than 5% of infected cells expressing discernible lytic gene products. Thus, mechanisms of immune evasion that depend on genes expressed only during lytic replication are unlikely to be active in most KSHV-infected cells. As a result, we searched for evidence of similar defensive strategies extant during latency, employing culture systems that strongly favor latent KSHV infection. We measured cell surface levels of immunomodulatory proteins on both primary dermal microvascular endothelial cells (

We analyzed dendritic cell (DC) and NK cell compartments in relation to CD4 recovery in 21 HIV-infected subjects followed to <50 copies/ml once starting antiretroviral therapy (ART) and observed for 52 wk of sustained suppression. Although CD4 counts increased in all subjects in response to ART, we observed a restoration of functional plasmacytoid DC (PDC) after 52 wk of sustained suppression under ART (from 1850 cells/ml to 4550 cells/ml) to levels comparable to controls (5120 cells/ml) only in subjects with a low baseline viral load, which also rapidly suppressed to <50 copies/ml upon ≤60 days from ART initiation. Recovery of PDC at week 52 correlates with level of CD95 expression on CD8 T cells and PDC frequency following first ART suppression. NK cytotoxic activity increased rapidly upon viral suppression (VS) and correlated with PDC function at week 52. However, restoration of total NK cells was incomplete even after 52 wk on ART (73 cells/μl vs 122 cells/μl in controls). Direct reconstitution experiments indicate that NK cytotoxic activity against virally infected target cells requires DC/NK cooperation, and can be recovered upon sustained VS and recovery of functional PDC (but not myeloid DC) from ART-suppressed subjects. Our data indicate that viremic HIV-infected subjects may have different levels of reconstitution of DC and NK-mediated function following ART, with subjects with lower initial viremia and the greatest reduction of baseline immune activation at VS achieving the greatest level of innate effector cell reconstitution.

In vivo, several mechanisms have been postulated to protect HIV-1-infected cells from NK surveillance. In vitro, previous research indicates HIV-1-infected autologous CD4+ primary T cells are resistant to NK lysis. We hypothesized that NK lysis of HIV-1-infected target cells would be augmented by the presence of accessory cells and/or accessory cell factors. In this study, we show that stimulation of plasmacytoid dendritic cells (PDC) with the TLR9 agonist, CpG ODN 2216, triggered NK lysis of HIV-1-infected autologous CD4+ primary T cells. PDC-stimulated NK lysis was dependent upon MHC class I (MHC-I) down-regulation on infected cells, and primary HIV-1 isolates that exhibited enhanced MHC-I down-regulation were more susceptible to NK-mediated lysis. PDC-stimulated NK lysis of HIV-1-infected autologous CD4+ primary T cells was blocked by neutralizing Abs to type 1 IFN and was perforin/granzyme dependent. Overall, our data suggest that HIV-infected cells are not innately resistant to NK lysis, and that exogenous NK stimulation derived from PDC can trigger NK cytotoxicity against HIV-1-infected autologous CD4+ primary T cells.

SummaryThe description of highly exposed individuals who remain seronegative (HESN) despite repeated exposure to human immunodeficiency virus (HIV)-1 has heightened interest in identifying potential mechanisms of HIV-1 resistance. HIV-specific humoral and T cell-mediated responses have been identified routinely in HESN subjects, although it remains unknown if these responses are a definitive cause of protection or merely a marker for exposure. Approximately half of HESN lack any detectible HIV-specific adaptive immune responses, suggesting that other mechanisms of protection from HIV-1 infection also probably exist. In support of the innate immune response as a mechanism of resistance, increased natural killer (NK) cell activity has been correlated with protection from infection in several highrisk cohorts of HESN subjects, including intravenous drug users, HIV-1 discordant couples and perinatally exposed infants. Inheritance of protective NK KIR3DL1 high and KIR3DS1 receptor alleles have also been observed to be over-represented in a high-risk cohort of HESN intravenous drug users and HESN partners of HIV-1-infected subjects. Other intrinsic mechanisms of innate immune protection correlated with resistance in HESN subjects include heightened dendritic cell responses and increased secretion of antiviral factors such as b-chemokines, small anti-viral factors and defensins. This review will highlight the most current evidence in HESN subjects supporting the role of epithelial microenvironment and the innate immune system in sustaining resistance against HIV-1 infection. We will argue that as a front-line defence the innate immune response determines the threshold of infectivity that HIV-1 must overcome to establish a productive infection.