Costin Tomescu

Surface Downregulation of Major Histocompatibility Complex Class I, PE-CAM, and ICAM-1 following De Novo Infection of Endothelial Cells with Kaposi's Sarcoma-Associated Herpesvirus

ABSTRACT 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. Kaposis 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 (pDMVEC) infected through coculture with induced primary effusion lymphoma cells and telomerase-immortalized DMVEC infected directly with cell-free virus. Employing a panel of antibodies against several endothelial cell surface proteins, we show that de novo infection with KSHV leads to the downregulation of MHC class I, CD31 (PE-CAM), and CD54 (ICAM-I) but not CD58 (LFA-3) or CD95 (Fas). Furthermore, flow cytometry with a fluorescently labeled monoclonal antibody to the latency-associated nuclear antigen (LANA) revealed that downregulation occurred predominantly on KSHV-infected (LANA-positive) cells. Although the vast majority of infected cells displayed this downregulation, less than 1% expressed either immediate-early or late lytic proteins detectable by immunofluorescence. Together, these results suggest that downregulation of immunomodulatory proteins on the surface of target cells may represent a constitutive mode of immune evasion employed by KSHV following de novo infection.

Baseline Viral Load and Immune Activation Determine the Extent of Reconstitution of Innate Immune Effectors in HIV-1-Infected Subjects Undergoing Antiretroviral Treatment

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.

Evidence for the innate immune response as a correlate of protection in human immunodeficiency virus (HIV)-1 highly exposed seronegative subjects (HESN)

The 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 high‐risk cohorts of HESN subjects, including intravenous drug users, HIV‐1 discordant couples and perinatally exposed infants. Inheritance of protective NK KIR3DL1high 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 anti‐viral factors such as β‐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.

Inability of plasmacytoid dendritic cells to directly lyse HIV-infected autologous CD4+ T cells despite induction of tumor necrosis factor-related apoptosis-inducing ligand.

ABSTRACT The function of plasmacytoid dendritic cells (PDC) in chronic human immunodeficiency virus type 1 (HIV-1) infection remains controversial with regard to its potential for sustained alpha interferon (IFN-α) production and induction of PDC-dependent tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-mediated cytotoxicity of HIV-infected cells. We address these areas by a study of chronically HIV-1-infected subjects followed through antiretroviral therapy (ART) interruption and by testing PDC cytolytic function against autologous HIV-infected CD4+ T cells. Rebound in viremia induced by therapy interruption showed a positive association between TRAIL and viral load or T-cell activation, but comparable levels of plasma IFN-α/β were found in viremic ART-treated and control subjects. While PDC from HIV-infected subjects expressed less interferon regulator factor 7 (IRF-7) and produced significantly less IFN-α upon Toll-like receptor 7/9 (TLR7/9) engagement than controls, membrane TRAIL expression in PDC from HIV+ subjects was increased. Moreover, no significant increase in death receptor 5 (DR5) expression was seen in CD4+ T cells from viremic HIV+ subjects compared to controls or following in vitro infection/exposure to infectious and noninfectious virus or exogenous IFN-α, respectively. Although activated PDC killed the DR5-expressing HIV-infected Sup-T1 cell line, PDC did not lyse primary autologous HIV+ CD4+ T cells yet could provide accessory help for NK cells in killing HIV-infected autologous CD4+ T cells. Taken together, our data show a lack of sustained high levels of soluble IFN-α in chronic HIV-1 infection in vivo and document a lack of direct PDC cytolytic activity against autologous infected or uninfected CD4+ T cells.

NK Cell Lysis of HIV-1-Infected Autologous CD4 Primary T Cells: Requirement for IFN-Mediated NK Activation by Plasmacytoid Dendritic Cells

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.

Impact of protective killer inhibitory receptor/human leukocyte antigen genotypes on natural killer cell and T-cell function in HIV-1-infected controllers.

Objective:Both protective T-cell genotypes and natural killer (NK) cell genotypes have been associated with delayed progression to AIDS and shown to be co-inherited in HIV-1-infected individuals who limit viral replication in absence of antiretroviral therapy (‘controllers’). However, a comparative analysis of the genotype and function of the innate and adaptive immune compartments in HIV-1-infected controller individuals has been understudied to date. Design:Here, we simultaneously tested NK and T-cell function in controllers to investigate the mechanism(s) that might account for host immune control over viral replication. Methods:We measured CD8 T-cell responses against HIV-1 utilizing overlapping 15-mer peptides spanning the HIV-1 consensus clade B Gag protein and tested NK cell degranulation and cytokine secretion against tumor target cells following interferon-&agr; (IFN&agr;) stimulation. Results:Among a cohort of 37 controllers, the presence of protective major histocompatibility complex class I human leukocyte antigen (HLA) alleles (such as HLA-B*57) was not correlated with HIV-specific CD8 responses. In contrast, the inheritance of a protective killer inhibitory receptor KIR3DL1*h/*y receptor genotype along with the corresponding HLA-Bw4*80I ligand was associated with significantly heightened target cell-induced NK degranulation and cytokine secretion following IFN&agr; stimulation (P = 0.0201, n = 13). Interestingly, we observed a significant inverse association between the IFN&agr; stimulated NK response to K562 cells and the HIV-specific CD8 T-cell response to Gag among elite controllers (rho = −0.8321, P = 0.0010, n = 12). Conclusion:Together, these results suggest that heightened NK responses can be evidenced independently of HIV-specific T-cell responses in HIV-1-infected elite controllers.

Increased plasmacytoid dendritic cell maturation and natural killer cell activation in HIV-1 exposed, uninfected intravenous drug users.

Background:Increased natural killer (NK) activation has been associated with resistance to HIV-1 infection in several cohorts of HIV-1 exposed, uninfected individuals. Inheritance of protective NK receptor alleles (KIR3DS1 and KIR3DL1high) has also been observed in a subset of HIV-1 exposed, uninfected individuals. However, the exact mechanism contributing to NK activation in HIV-1 exposed, uninfected intravenous drug users (EU-IDU) remains to be elucidated. Objective:We investigated the role of both host genotype and pathogen-induced dendritic cell modulation of NK activation during high-risk activity in a cohort of 15 EU-IDU individuals and 15 control, uninfected donors from Philadelphia. Design:We assessed the activation status of NK cells and dendritic cells by flow cytometry and utilized functional assays of NK-DC cross-talk to characterize the innate immune compartment in EU-IDU individuals. Results:As previously reported, NK cell activation (CD69) and/or degranulation (CD107a) was significantly increased in EU-IDU individuals compared with control uninfected donors (P = 0.0056, n = 13). Genotypic analysis indicated that the frequency of protective KIR (KIR3DS1) and HLA-Bw4*80I ligands was not enriched in our cohort of EU-IDU individuals. Rather, plasmacytoid dendritic cells (PDC) from EU-IDU exhibited heightened maturation (CD83) compared with control uninfected donors (P = 0.0011, n = 12). When stimulated in vitro, both PDCs and NK cells from EU-IDU individuals maintained strong effector cell function and did not exhibit signs of exhaustion. Conclusion:Increased maturation of PDCs is associated with heightened NK activation in EU-IDU individuals suggesting that both members of the innate compartment may contribute to resistance from HIV-1 infection in EU-IDU.

Lysis of HIV-1-infected autologous CD4+ primary T cells by interferon-alpha-activated NK cells requires NKp46 and NKG2D.

Objective:Autologous HIV-1-infected CD4+ primary T cells (aHIV+CD4) have been shown to be largely resistant to natural killer (NK)-cell-mediated lysis because of viral strategies of immune evasion. We have previously shown that a preactivation of NK cells with plasmacytoid dendritic cells can significantly augment lysis of aHIV+CD4 through a mechanism dependent on interferon-alpha (IFN-&agr;). Design:The goal of the present study is to identify the specific NK-activating receptors involved in NK lysis of aHIV+CD4 following IFN-&agr; activation. Methods:Peripheral blood mononuclear cells (PBMC) were incubated with aHIV+CD4 to induce the secretion of endogenous levels of IFN-&agr; and drive NK activation. We then utilized a standard chromium lysis assay to assess the degree of IFN-&agr;-activated lysis of aHIV+CD4 in the presence or absence of masking antibodies to a panel of NK-activating receptors and co-receptors. Results:Direct recognition of HIV-1-infected, but not uninfected, autologous CD4+ primary T cells by PBMC induced the secretion IFN-&agr; (median 2280 pg/ml, P < 0.001, n = 9) that, in turn, activated NK cells (P < 0.001, n = 12) and significantly increased their cytolytic potential against aHIV+CD4 (P < 0.01, n = 12). The masking of NKp46 (P < 0.01, n = 8) and NKG2D (P < 0.05, n = 8), but not 2B4, NTBA, NKp30 or NKp44, significantly reduced IFN-&agr;-activated lysis of aHIV+CD4. Conclusions:Taken together, these results demonstrate that endogenous levels of IFN-&agr; secreted by plasmacytoid dendritic cells induce NK cells to lyse aHIV+CD4 via the engagement of NKp46 and NKG2D.

Retention of viability, cytotoxicity, and response to IL-2, IL-15, or IFN-α by human NK cells after CD107a degranulation

NK cells represent a critical component of the host innate immune response to viral infection and tumor transformation. Nevertheless, the fate of recently degranulated NK cells subsequent to a primary target cell interaction remains largely unexplored. Here, we investigated the long‐term viability and killing potential of human NK cells following target cell lysis using live‐sorting of CD107a‐degranulated NK cells. We observed that sorted CD107a+ NK cells exhibited continued lytic potential against a wide variety of target cells, including tumor and virally infected target cells. CD107a‐positive‐ and CD107a‐negative‐sorted NK cells displayed similar long‐term viability, killing potential, and response to inflammatory cytokines such as IL‐2, IL‐15, and IFN‐α. Interestingly, we observed that the CD107a signature is remarkably stable over time and that recently degranulated NK cells exhibit an amplification of CD107 expression immediately following a target cell interaction. Together, our data expand previous data showing that NK cells retain the capacity to kill multiple target cells in succession and reveal that NK viability, cytotoxicity, and response to inflammatory cytokines are not altered following a primary target cell interaction. Overall, our data argue for the strength of the NK cell compartment in the continuous surveillance of tumor and virally infected cells in the body and highlight the use of using CD107a expression as a stable marker for NK cytotoxicity.

A Correlate of HIV-1 Control Consisting of Both Innate and Adaptive Immune Parameters Best Predicts Viral Load by Multivariable Analysis in HIV-1 Infected Viremic Controllers and Chronically-Infected Non-Controllers

HIV-1 infected viremic controllers maintain durable viral suppression below 2000 copies viral RNA/ml without anti-retroviral therapy (ART), and the immunological factor(s) associated with host control in presence of low but detectable viral replication are of considerable interest. Here, we utilized a multivariable analysis to identify which innate and adaptive immune parameters best correlated with viral control utilizing a cohort of viremic controllers (median 704 viral RNA/ml) and non-controllers (median 21,932 viral RNA/ml) that were matched for similar CD4+ T cell counts in the absence of ART. We observed that HIV-1 Gag-specific CD8+ T cell responses were preferentially targeted over Pol-specific responses in viremic controllers (p = 0.0137), while Pol-specific responses were positively associated with viral load (rho = 0.7753, p = 0.0001, n = 23). Viremic controllers exhibited significantly higher NK and plasmacytoid dendritic cells (pDC) frequency as well as retained expression of the NK CD16 receptor and strong target cell-induced NK cell IFN-gamma production compared to non-controllers (p<0.05). Despite differences in innate and adaptive immune function however, both viremic controllers (p<0.05) and non-controller subjects (p<0.001) exhibited significantly increased CD8+ T cell activation and spontaneous NK cell degranulation compared to uninfected donors. Overall, we identified that a combination of innate (pDC frequency) and adaptive (Pol-specific CD8+ T cell responses) immune parameters best predicted viral load (R2 = 0.5864, p = 0.0021, n = 17) by a multivariable analysis. Together, this data indicates that preferential Gag-specific over Pol-specific CD8+ T cell responses along with a retention of functional innate subsets best predict host control over viral replication in HIV-1 infected viremic controllers compared to chronically-infected non-controllers.