Brief Report: Increased Expression of the Type I Interferon Receptor on CD4+ T Lymphocytes in HIV-1-Infected Individuals.

BACKGROUND: Type I interferons (IFN1s; eg, interferon-alpha and interferon-beta) are potent cytokines that inhibit the replication of human immunodeficiency virus-1 (HIV-1) and other viruses. The antiviral and immunoregulatory activities of IFN1 are mediated through ligand-receptor interactions with the IFN1 receptor complex (IFNAR). Variation in the cell-surface density of IFNAR could play a role in HIV-1 pathogenesis. METHODS: In this cross-sectional study of fresh whole blood, we used flow cytometry to evaluate the expression of IFNAR2 on lymphocyte subsets from HIV-1-infected (n = 33) and HIV-1-uninfected (n = 22) individuals. RESULTS: In comparison with healthy blood bank donors, we observed that the HIV-1-infected individuals, particularly those having advanced to disease, exhibited the increased expression of IFNAR2 on CD4 T cells (relative fluorescence intensity 6.9 vs. 9.0; P = 0.027). The CD4:CD4 T-cell IFNAR2 expression-level ratio provides an internally standardized measure of this alteration. The observed increased expression of IFNAR2 was largely restricted to CD4 T cells that expressed the chemokine receptor CXCR4 and lacked the expression of CCR5. CONCLUSIONS: HIV-1-infected individuals exhibit an increased expression of the IFN1 receptor on CD4 T cells. The level of IFNAR2 expression seems to increase with disease progression. These findings provide insight for the immunologic alterations associated with HIV-1 infection and possibly new therapeutic approaches.

Plasmacytoid dendritic cell number and responses to Toll-like receptor 7 and 9 agonists vary in HIV Type 1-infected individuals in relation to clinical state.

In HIV-1 infection, plasmacytoid dendritic cell (PDC) numbers and function are decreased. No detailed comparisons of PDC responses to various stimuli in HIV-1-infected patients are available. Using for the first time purified PDCs, we compared PDC responses [interferon (IFN)-α production/cell] to various stimuli in a large number (n=48) of HIV-1-infected patients and healthy volunteers (n=19). Toll-like receptor (TLR)7- and TLR9-induced expression of PDC surface activation and maturation markers was also compared in the two populations. We have confirmed that PDC number coincides with CD4(+) T cell counts and clinical state. Notably, we have shown that a direct association of PDC function in terms of IFN-α production/cell exists with PDC numbers and CD4(+) cell counts when PDCs are exposed to a TLR9 ligand and HIV-infected cells, but not with a TLR7 ligand. Moreover, in the HIV-infected subjects but not the healthy controls, the magnitude of IFN-α release per PDC in response to the TLR7 ligand is significantly (p<0.01) lower than that to the TLR9 ligand. However, in both study populations, the TLR7 stimulation in comparison to TLR9 stimulation induced higher expression of PDC surface activation and maturation markers and significantly (p<0.05) decreased the expression of BDCA-2, a negative regulator of interferon. Furthermore, the cross-ligation of BDCA-2 significantly (p<0.05) inhibited TLR9- but not TLR7-induced IFN-α production by PDCs from both clinical groups. These findings suggest that differences exist in TLR7- and TLR9-induced IFN-α production by PDCs in HIV-infected individuals that are not directly related to BDCA-2 down-modulation.

Transfection optimization for primary human CD8+ cells.

Electroporation, a non-virus-mediated gene transfection method, has traditionally had poor outcomes with low gene transfection efficiency and poor cellular viability, particularly in primary human lymphocytes. Herein we have optimized the electroporation conditions for primary CD8+ cells resulting in a maximum rate of 81.3%, and a mean transfection efficiency of 59.6%. After removal of dead cells, the viability of transfected primary CD8+ cells was greater than 90%, similar to untransfected controls. Using this procedure, primary human CD8+ cells transfected with an interferon α8 plasmid produced fluids that inhibited HIV-1 replication by > 95%. This transfection protocol is useful for transfection of other primary blood cells, such as CD4+ T cells, and for studying the function of genes in primary human blood cells instead of cell lines. The transfection procedure also has potential application in gene therapy clinical trials to treat diseases utilizing transfected primary human cells.

Natural suppression of human immunodeficiency virus type 1 replication is mediated by transitional memory CD8+ T cells.

HIV replication is suppressed in vitro by a CD8(+) cell noncytotoxic antiviral response (CNAR). This activity directly correlates with an asymptomatic clinical state. The objective of this study was to identify the phenotype of CD8(+) cell subsets having strong CNAR activity. CD8(+) cell subset frequencies and CNAR levels were measured for human immunodeficiency virus (HIV)-uninfected individuals and three groups of HIV type 1 (HIV-1)-infected individuals: asymptomatic individuals with low-level viremia (vHIV), antiretroviral-drug-treated subjects with undetectable virus levels (TxHIV), and therapy-naïve aviremic elite controllers (EC). CD8(+) cells from the vHIV individuals exhibited the highest HIV-suppressing activity and had elevated frequencies of CD45RA(-) CD27(+) and PD-1(+) (CD279(+)) cells. Functional assessments of CD8(+) cells sorted into distinct subsets established that maximal CNAR activity was mediated by CD45RA(-) CCR7(-) CD27(+) and PD-1(+) CD8(+) cells. T cell receptor (TCR) repertoire profiles of CD8(+) cell subsets having strong CNAR activity exhibited increased perturbations in comparison to those of inactive subsets. Together, these studies suggest that CNAR is driven by HIV replication and that this antiviral activity is associated with oligoclonally expanded activated CD8(+) cells expressing PD-1 and having a transitional memory cell phenotype. The findings better describe the identity of CD8(+) cells showing CNAR and should facilitate the evaluation of this important immune response in studies of HIV pathogenesis, resistance to infection, and vaccine development.

Similar changes in plasmacytoid dendritic cell and CD4 T-cell counts during primary HIV-1 infection and treatment.

OBJECTIVES: Reduced dendritic cell (DC) frequencies and functions in individuals with longstanding HIV-1 infection are predictive of opportunistic infections and AIDS. To investigate possible early alterations in DC levels after HIV infection, we prospectively examined plasmacytoid dendritic cell (pDC) and myeloid dendritic cell (mDC) frequencies and plasma IFN-alpha levels in patients undergoing primary HIV-1 infection (PHI). METHODS: Peripheral blood DC frequencies and absolute counts were determined by flow cytometry. Plasma IFN-alpha levels were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: In comparison to uninfected subjects, pDC, but not mDC, levels were reduced (P < 0.001) in subjects with PHI, especially in those with high viral loads or low CD4 T-cell counts. During 24-48 weeks of observation, untreated subjects experienced slight declines in pDC and CD4 T-cell levels. In contrast, subjects initiating early antiretroviral therapy (ART) exhibited increases (P < 0.001) in pDC and CD4 T-cell counts. No effect of treatment on mDC counts was observed. Circulating plasma IFN-alpha was undetectable by ELISA regardless of the duration of HIV-1 infection. CONCLUSION: PHI is characterized by a reduction in pDC and CD4 T-cell counts that correlates with the magnitude of virus replication and is not evidenced by the mDC count or plasma IFN-alpha level. Early ART appears to have similar restorative effects on pDC and CD4 T-cell counts.

Variations in plasmacytoid dendritic cell (PDC) and myeloid dendritic cell (MDC) levels in HIV-infected subjects on and off antiretroviral therapy.

Plasmacytoid and myeloid dendritic cells are reduced in AIDS patients. The number of these circulating cells was assessed cross-sectionally and longitudinally in 27 uninfected and 72 HIV-infected subjects on and off antiretroviral therapy. The plasmacytoid dendritic cell numbers were significantly reduced in the HIV-infected subjects compared to controls (p < 0.001). This reduction correlated directly with CD4+ cell counts (p < 0.001) and inversely with viral load (p < 0.001). These associations were found to a lesser degree for the myeloid dendritic cells. Intra-assay variability of these dendritic cell counts was < 10%. Antiretroviral therapy significantly increased plasmacytoid dendritic cell (p < 0.001) and CD4+ cell (p = 0.05) counts at 8 months by 76.9% and 19%, respectively. The plasmacytoid dendritic cell levels responded more readily to viral load increases and decreases than CD4+ cells. Circulating plasmacytoid dendritic cells may provide important additional information about immune function in HIV-infected subjects receiving or not receiving antiretroviral therapy.

HIV-infected cells are major inducers of plasmacytoid dendritic cell interferon production, maturation, and migration.

Plasmacytoid dendritic cells (PDC), natural type-1 interferon (IFN) producing cells, could play a role in the innate anti-HIV immune response. Previous reports indicated that PDC IFN production is induced by HIV. Our results show a more robust IFN induction when purified PDC (>95%) were exposed to HIV-infected cells. This effect was not observed with non-viable cells, DNA, and RNA extracted from infected cells, and viral proteins. The response was blocked by anti-CD4 and neutralizing anti-gp120 antibodies as well as soluble CD4. IFN induction by HIV-infected cells was also prevented by low-dose chloroquine, which inhibits endosomal acidification. PDC IFN release resulted in reduced HIV production by infected CD4+ cells, supporting an anti-HIV activity of PDC. Stimulated CD4+ cells induced PDC activation and maturation; markers for PDC migration (CCR7) were enhanced by HIV-infected CD4+ cells only. This latter finding could explain the decline in circulating PDC in HIV-infected individuals.

Differential effects of R5 and X4 human immunodeficiency virus type 1 infection on CD4+ cell proliferation and activation.

Human immunodeficiency virus type 1 (HIV-1) isolates can be distinguished by their chemokine coreceptor usage. Non-syncytium-inducing (NSI), macrophage-tropic viruses utilize CCR5 and are called R5 viruses; syncytium-inducing (SI) isolates use CXCR4 and are known as X4 viruses. R5 and X4 HIV isolates are both transmitted but, in most cases, R5 viruses predominate in the blood prior to the development of AIDS-related pathogenesis. The reason for the selective growth of the R5 strain is not known, but could reflect a replication advantage of R5 viruses over X4 viruses in CD4+ cells. To explore this possibility, eight phenotypically distinct viruses were used to infect CD4+ cells and cellular proliferation and activation were evaluated. In unstimulated CD4+ cells, R5 virus isolates increased the level of cell activation compared with X4 virus isolates and uninfected control cells. In CD4+ cells that were stimulated with interleukin 2, both R5 and X4 viruses were found to decrease the level of cell proliferation and reduce the majority of the activation markers studied when compared with uninfected control CD4+ cells from the same donors. However, although equal amounts of CD4+ cells were infected, R5 virus-infected CD4+ cells showed a two- to fourfold increase in cellular proliferation over X4 viruses, as measured by [3H]thymidine incorporation (P=0.001) and nuclear expression of Ki67 (P=0.001). In addition, a larger proportion of CD4+ T cells infected with R5 viruses had significantly higher levels of activation-marker expression (e.g. CD25, CD71 and HLA-DR) than CD4+ T lymphocytes infected with X4 viruses (P<0.02). Taken together, these results indicate that CD4+ cells infected with R5 virus isolates may have a selective advantage over X4 virus-infected CD4+ T cells for survival and, hence, virus spread.

VCAM-1 expression on CD8+ cells correlates with enhanced anti-HIV suppressing activity.

CD8(+) cells from HIV-infected individuals showing the CD8(+) cell noncytotoxic antiviral response unexpectedly revealed mRNA for VCAM-1, a cell surface molecule found on endothelial cells. Uninfected subjects had undetectable levels of VCAM-1 mRNA in their CD8(+) cells. Flow cytometry analysis showed that up to 12% of the CD8(+) cells from HIV-positive individuals expressed VCAM-1 compared with 0.8% of the CD8(+) cells of HIV-negative individuals. Enrichment of the CD8(+)VCAM-1(+) cell population and subsequent coculture with CD4(+) cells acutely infected with HIV-1 showed that the VCAM-1(+)CD8(+) cells were able to suppress viral replication with 50% less input cells than the unseparated CD8(+) cell population. This study demonstrates, for the first time, the expression of VCAM-1 on CD8(+) cells. Moreover, the CD8(+)VCAM-1(+) cells show enhanced CD8(+) cell noncytotoxic antiviral response activity that could have clinical importance in HIV infection.

Low-level HIV infection of plasmacytoid dendritic cells: onset of cytopathic effects and cell death after PDC maturation.

Plasmacytoid dendritic cells (PDC), the natural type-1 interferon (IFN) producing cells, are part of the innate immune defense against human immunodeficiency virus (HIV). PDC numbers are reduced in advanced stages of infection. These cells can be infected in vivo by HIV since highly purified PDC showed evidence of infectious HIV. Moreover, when PDC derived from uninfected donors were exposed to high-titered HIV isolates, productive infection occurred although with low-level replication. Using real-time amplification, PDC and unstimulated CD4+ cells were found equally susceptible to HIV infection; however, HIV replication was considerably limited in the PDC. Virus replication was enhanced after PDC treatment with CD40L and antibodies against IFN-alpha, most likely reflecting the reduction in IFN-alpha activity. On maturation, the infected PDC showed multinuclear cell syncytia formation and death. These findings indicate that PDC can be reservoirs for HIV dissemination and that HIV infection of PDC can contribute to their decline.

Expression patterns of phenotypic markers on lymphocytes from human immunodeficiency virus type 2-infected baboons.

The development of AIDS in HIV-1-infected humans is associated with profound changes in the expression patterns of lymphocyte phenotypic markers associated with increased immune activation and with decreased recall immune responses. In assessing these immunologic changes in an animal model, we characterized the expression patterns of immune activation markers on lymphocyte subsets during the acute, chronic, and end stages of HIV-2 infection in baboons. Using flow cytometry, we identified 21 human-specific monoclonal antibodies that were cross-reactive with baboon lymphocytes; however, expression of only 2 of these markers was altered significantly after HIV-2 infection. We found an increase in baboon class II antigen (as measured by anti-HLA-DR) in the CD4(+) T cell subset within 8 weeks of infection (p = 0.045). Moreover, after 1 year of infection, CD11b was downregulated on CD8(+) T lymphocytes (p = 0.027). This downregulation of CD11b was consistently observed in all of the groups of baboons that were chronically infected with three different HIV-2 isolates. In addition, we found substantial downregulation of the interleukin 2 receptor (CD25) and upregulation of class II antigen on CD8(+) lymphocytes in a baboon with an AIDS-like disease. These and other phenotypic markers of immune activation may facilitate characterization of the immunopathogenesis of AIDS in nonhuman primate animal models.