Paul U. Cameron

Conjugates of dendritic cells and memory T lymphocytes from skin facilitate productive infection with HIV-1

Experimentally, a productive infection with HIV-1 requires that virus be administered to T cells that are activated by mitogens. We describe a productive milieu for HIV-1 within the confines of normal skin that does not require standard stimuli. The milieu consists of dendritic cells and T cells that emigrate from skin and produce distinctive stable, nonproliferating conjugates. These conjugates, upon exposure to each of seven different HIV-1 isolates, begin to release high levels of virus progeny within 4 days. Numerous infected syncytia, comprised of both dendritic and T cells, rapidly develop. We propose that conjugates of dendritic cells and T cells, as found in the external linings of organs involved in sexual transmission of HIV-1, represent an important site for the productive phase of HIV-1 infection. Because the affected T cells carry the memory phenotype, this site additionally provides a mechanism for the chronic depletion of CD4+ memory cells in HIV-1 disease.

Diversity of receptors binding HIV on dendritic cell subsets.

The ability of HIV-1 to use dendritic cells (DCs) for transport and to transfer virus to activated T cells in the lymph node may be crucial in early HIV-1 pathogenesis. We have characterized primary DCs for the receptors involved in viral envelope attachment and observed that C-type lectin receptor (CLR) binding was predominant in skin DCs, whereas binding to emigrating and tonsil DCs was CD4-dependent. No one CLR was solely responsible for envelope binding on all skin DC subsets. DC-SIGN (DC-specific ICAM-3–grabbing nonintegrin) was only expressed by CD14+CDlalo dermal DCs. The mannose receptor was expressed by CD1ahi and CD14+CDlalo dermal DCs, and langerin was expressed by Langerhans cells. The diversity of CLRs able to bind HIV-1 in skin DCs may reflect their ability to bind a range of microbial glycoproteins.

Predisposition to nevirapine hypersensitivity associated with HLA-DRB1*0101 and abrogated by low CD4 T-cell counts

Genetic (human leukocyte antigen), disease-related and demographic risk factors for nevirapine reactions were examined in a nevirapine-exposed cohort. Cases involving combinations of hepatitis, fever or rash were associated with an interaction between HLA-DRB1*0101 and the percentage of CD4, whereas no associations were detected for isolated rash. These data suggest that HLA-DRB1*0101 and the CD4 status may determine susceptibility to nevirapine hypersensitivity, consistent with a CD4 T-cell-dependent immune response to nevirapine-specific antigens.

The CD16+ Monocyte Subset Is More Permissive to Infection and Preferentially Harbors HIV-1 In Vivo

HIV-1 persists in peripheral blood monocytes in individuals receiving highly active antiretroviral therapy (HAART) with viral suppression, despite these cells being poorly susceptible to infection in vitro. Because very few monocytes harbor HIV-1 in vivo, we considered whether a subset of monocytes might be more permissive to infection. We show that a minor CD16+ monocyte subset preferentially harbors HIV-1 in infected individuals on HAART when compared with the majority of monocytes (CD14highCD16−). We confirmed this by in vitro experiments showing that CD16+ monocytes were more susceptible to CCR5-using strains of HIV-1, a finding that is associated with higher CCR5 expression on these cells. CD16+ monocytes were also more permissive to infection with a vesicular stomatitis virus G protein-pseudotyped reporter strain of HIV-1 than the majority of monocytes, suggesting that they are better able to support HIV-1 replication after entry. Consistent with this observation, high molecular mass complexes of apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G) were observed in CD16+ monocytes that were similar to those observed in highly permissive T cells. In contrast, CD14highCD16− monocytes contained low molecular mass active APOBEC3G, suggesting this is a mechanism of resistance to HIV-1 infection in these cells. Collectively, these data show that CD16+ monocytes are preferentially susceptible to HIV-1 entry, more permissive for replication, and constitute a continuing source of viral persistence during HAART.

Activation of HIV Transcription with Short-Course Vorinostat in HIV-Infected Patients on Suppressive Antiretroviral Therapy

Human immunodeficiency virus (HIV) persistence in latently infected resting memory CD4+ T-cells is the major barrier to HIV cure. Cellular histone deacetylases (HDACs) are important in maintaining HIV latency and histone deacetylase inhibitors (HDACi) may reverse latency by activating HIV transcription from latently infected CD4+ T-cells. We performed a single arm, open label, proof-of-concept study in which vorinostat, a pan-HDACi, was administered 400 mg orally once daily for 14 days to 20 HIV-infected individuals on suppressive antiretroviral therapy (ART). The primary endpoint was change in cell associated unspliced (CA-US) HIV RNA in total CD4+ T-cells from blood at day 14. The study is registered at ClinicalTrials.gov (NCT01365065). Vorinostat was safe and well tolerated and there were no dose modifications or study drug discontinuations. CA-US HIV RNA in blood increased significantly in 18/20 patients (90%) with a median fold change from baseline to peak value of 7.4 (IQR 3.4, 9.1). CA-US RNA was significantly elevated 8 hours post drug and remained elevated 70 days after last dose. Significant early changes in expression of genes associated with chromatin remodeling and activation of HIV transcription correlated with the magnitude of increased CA-US HIV RNA. There were no statistically significant changes in plasma HIV RNA, concentration of HIV DNA, integrated DNA, inducible virus in CD4+ T-cells or markers of T-cell activation. Vorinostat induced a significant and sustained increase in HIV transcription from latency in the majority of HIV-infected patients. However, additional interventions will be needed to efficiently induce virus production and ultimately eliminate latently infected cells. Trial Registration ClinicalTrials.gov NCT01365065

Establishment of HIV-1 latency in resting CD4+ T cells depends on chemokine-induced changes in the actin cytoskeleton

Eradication of HIV-1 with highly active antiretroviral therapy (HAART) is not possible due to the persistence of long-lived, latently infected resting memory CD4+ T cells. We now show that HIV-1 latency can be established in resting CD4+ T cells infected with HIV-1 after exposure to ligands for CCR7 (CCL19), CXCR3 (CXCL9 and CXCL10), and CCR6 (CCL20) but not in unactivated CD4+ T cells. The mechanism did not involve cell activation or significant changes in gene expression, but was associated with rapid dephosphorylation of cofilin and changes in filamentous actin. Incubation with chemokine before infection led to efficient HIV-1 nuclear localization and integration and this was inhibited by the actin stabilizer jasplakinolide. We propose a unique pathway for establishment of latency by direct HIV-1 infection of resting CD4+ T cells during normal chemokine-directed recirculation of CD4+ T cells between blood and tissue.

The Intracellular Granzyme B Inhibitor, Proteinase Inhibitor 9, Is Up-Regulated During Accessory Cell Maturation and Effector Cell Degranulation, and Its Overexpression Enhances CTL Potency

Granzyme B (grB) is a serine proteinase released by cytotoxic lymphocytes (CLs) to kill abnormal cells. GrB-mediated apoptotic pathways are conserved in nucleated cells; hence, CLs require mechanisms to protect against ectopic or misdirected grB. The nucleocytoplasmic serpin, proteinase inhibitor 9 (PI-9), is a potent inhibitor of grB that protects cells from grB-mediated apoptosis in model systems. Here we show that PI-9 is present in CD4+ cells, CD8+ T cells, NK cells, and at lower levels in B cells and myeloid cells. PI-9 is up-regulated in response to grB production and degranulation, and associates with grB-containing granules in activated CTLs and NK cells. Intracellular complexes of PI-9 and grB are evident in NK cells, and overexpression of PI-9 enhances CTL potency, suggesting that cytoplasmic grB, which may threaten CL viability, is rapidly inactivated by PI-9. Because dendritic cells (DCs) acquire characteristics similar to those of target cells to activate naive CD8+ T cells and therefore may also require protection against grB, we investigated the expression of PI-9 in DCs. PI-9 is evident in thymic DCs (CD3−, CD4+, CD8−, CD45+), tonsillar DCs, and DC subsets purified from peripheral blood (CD16+ monocytes and CD123+ plasmacytoid DCs). Furthermore, PI-9 is expressed in monocyte-derived DCs and is up-regulated upon TNF-α-induced maturation of monocyte-derived DCs. In conclusion, the presence and subcellular localization of PI-9 in leukocytes and DCs are consistent with a protective role against ectopic or misdirected grB during an immune response.

The role of dendritic cell C-type lectin receptors in HIV pathogenesis

Dendritic cells play a major role in HIV pathogenesis. Epithelial dendritic cells appear to be one of the first cells infected after sexual transmission and transfer of the virus to CD4 lymphocytes, simultaneously activating these cells to produce high levels of HIV replication. Such transfer may occur locally in inflamed mucosa or after dendritic cells have matured and migrated to local lymph nodes. Therefore, the mechanism of binding, internalization, infection and transfer of HIV to CD4 lymphocytes is of great interest. Recently, the role of the C‐type lectin DC‐SIGN as a dendritic cell receptor for HIV has been intensively studied with in vitro monocyte‐derived dendritic cells. However, it is clear that other C‐type lectin receptors such as Langerin on Langerhan cells and mannose receptor on dermal dendritic cells are at least equally important for gp120 binding on epithelial dendritic cells. C‐type lectin receptors play a role in virus transfer to T cells, either via de novo infection (“cis transfer”) or without infection (“in trans” or transinfection). Both these processes are important in vitro, and both may have a role in vivo, although the low‐level infection of immature dendritic cells may be more important as it leads to R5 HIV strain selection and persistence of virus within dendritic cells for at least 24 h, sufficient for these cells to transit to lymph nodes. The exact details of these processes are currently the subject of intense study.

Dendritic cells and the replication of HIV-1.

Dendritic cells (DCs) are a distinct lineage of white cells that arise from CD34+ progenitors in the bone marrow. DCs exhibit many specializations that lead to efficient antigen capture and presentation to T cells, both CD4+ helpers and CD8+ killers. In several human tissues, DCs express the CD4 receptor for HIV‐1. Some early reports described the explosive infection of blood‐derived DCs by HIV‐1 and a severe compromise of their presenting function. In contrast, other studies described active HIV‐1 replication when DCs were interacting with CD4+ T cells. This productive infection could begin with a low viral burden in DCs but required that the DCs retain their normal binding and stimulatory function for T cells. In this review we first summarize those features of the DC system that seem pertinent to HIV‐1 infection. Then we consider the current literature on the interaction of HIV‐1 with DCs, from several different tissues, in HIV‐1‐infected patients or following challenge with HIV‐1 in vitro. The literature leads to the hypothesis that HIV‐1 infection is a battleground in which DCs could be leading both of the armies, the aggressor that promotes HIV‐1 replication from relatively small numbers of infected cells and the defender that mediates T cell‐dependent resistance.

During HIV-1 infection most blood dendritic cells are not productively infected and can induce allogeneic CD4+ T cells clonal expansion

We have considered the possibility that antigen‐presenting cells of the dendritic cell lineage may be infected in vivo and spread HIV‐1 at the time dendritic cells initiate the clonal expansion of antigen‐specific T cells. Dendritic cells were isolated from 25 HIV‐1–infected subjects (CDC stages II‐ IV). Fewer dendritic cells were recovered from most infected subjects. Reduced numbers of total non‐T cells were also found in these patients, so that preferential loss of dendritic cells did not occur. Dendritic cell function was assessed by stimulatory capacity for allogeneic CD4+ T cells in the mixed leucocyte reaction (MLR). Potent M LR stimulator activity was retained in the dendritic cell‐enriched populations from HIV‐infected patients. Seven out of nine patients without AIDS (asymptomatic, lymphadenopathy or ARC) and three out of six patients with AIDS had proliferative responses equivalent to those induced by dendritic cells from controls. Dendritic cells from HIV+ subjects were able to initiate the expansion of allogeneic CD4+ T cell clones with cloning efficiency not different from controls and without evidence of cytopathic effect in the expanding CD4+ clones. In situ hybridization of the different mononuclear cell populations with a gag‐specific riboprobe demonstrated positive cells in the T cell fractions of 12 of the 15 patients tested. None of the asymptomatic or ARC patients had riboprobe‐positive cells in the dendritic cell‐enriched populations. Four out of nine patients with AIDS had cells positive for HIV‐1 expression in the dendritic cell‐enriched fraction. However, the positive cells had the nuclear profile of lymphocytes, and by cytofluorography some residual low‐density T cells were present. By limiting dilution and polymerase chain reaction (PCR), CD4+ lymphocytes carried HIV provirus in inocula of 500–5000 cells, while provirus could only be detected in 50000 cells from the dendritic cell‐enriched fraction. The latter signal may be due to the demonstrated levels of T cell contamination. Our data indicate that productive or latent HIV‐1 infection of blood dendritic cells in vivo is rare, certainly no greater than in T lymphocytes, and that in vitro dendritic cell preparations from patients can expand CD4’ T cells efficiently and therefore may be able to expand T cells with immunotherapeutic activity.