Prameet M. Sheth

Tim-3 expression defines a novel population of dysfunctional T cells with highly elevated frequencies in progressive HIV-1 infection

Progressive loss of T cell functionality is a hallmark of chronic infection with human immunodeficiency virus 1 (HIV-1). We have identified a novel population of dysfunctional T cells marked by surface expression of the glycoprotein Tim-3. The frequency of this population was increased in HIV-1–infected individuals to a mean of 49.4 ± SD 12.9% of CD8+ T cells expressing Tim-3 in HIV-1–infected chronic progressors versus 28.5 ± 6.8% in HIV-1–uninfected individuals. Levels of Tim-3 expression on T cells from HIV-1–infected inviduals correlated positively with HIV-1 viral load and CD38 expression and inversely with CD4+ T cell count. In progressive HIV-1 infection, Tim-3 expression was up-regulated on HIV-1–specific CD8+ T cells. Tim-3–expressing T cells failed to produce cytokine or proliferate in response to antigen and exhibited impaired Stat5, Erk1/2, and p38 signaling. Blocking the Tim-3 signaling pathway restored proliferation and enhanced cytokine production in HIV-1–specific T cells. Thus, Tim-3 represents a novel target for the therapeutic reversal of HIV-1–associated T cell dysfunction.

Perforin Expression Directly Ex Vivo by HIV-Specific CD8+ T-Cells Is a Correlate of HIV Elite Control

Many immune correlates of CD8+ T-cell-mediated control of HIV replication, including polyfunctionality, proliferative ability, and inhibitory receptor expression, have been discovered. However, no functional correlates using ex vivo cells have been identified with the known ability to cause the direct elimination of HIV-infected cells. We have recently discovered the ability of human CD8+ T-cells to rapidly upregulate perforin—an essential molecule for cell-mediated cytotoxicity—following antigen-specific stimulation. Here, we examined perforin expression capability in a large cross-sectional cohort of chronically HIV-infected individuals with varying levels of viral load: elite controllers (n = 35), viremic controllers (n = 29), chronic progressors (n = 27), and viremic nonprogressors (n = 6). Using polychromatic flow cytometry and standard intracellular cytokine staining assays, we measured perforin upregulation, cytokine production, and degranulation following stimulation with overlapping peptide pools encompassing all proteins of HIV. We observed that HIV-specific CD8+ T-cells from elite controllers consistently display an enhanced ability to express perforin directly ex vivo compared to all other groups. This ability is not restricted to protective HLA-B haplotypes, does not require proliferation or the addition of exogenous factors, is not restored by HAART, and primarily originates from effector CD8+ T-cells with otherwise limited functional capability. Notably, we found an inverse relationship between HIV-specific perforin expression and viral load. Thus, the capability of HIV-specific CD8+ T-cells to rapidly express perforin defines a novel correlate of control in HIV infection.

Rebound of plasma viremia following cessation of antiretroviral therapy despite profoundly low levels of HIV reservoir: implications for eradication.

Objectives:Sustained suppression of plasma viremia in HIV-infected individuals is attainable with antiretroviral therapy (ART); however, eradication of virus that would allow discontinuation of ART has been hampered by the persistence of HIV reservoirs. It is of great interest to identify individuals who had received ART for prolonged periods of time with extremely low or undetectable HIV reservoirs and monitor plasma viremia following discontinuation of therapy. Methods:We measured the size of HIV reservoirs in CD4+ T cells of individuals on long-term ART and monitored plasma viremia following cessation of ART in one individual with an exceptionally low viral burden after a decade of therapy. Results:We demonstrated undetectable levels of HIV DNA in the blood of eight of 45 infected individuals on long-term ART. Among those eight individuals, the frequency of cells carrying infectious virus was significantly lower in those who initiated ART during the early versus the chronic phase of infection. One individual with undetectable HIV DNA in both blood and tissue and a profoundly low level of infectious virus experienced plasma viral rebound 50 days following discontinuation of ART. Conclusions:Our data suggest that a significant reduction in the size of viral reservoirs may be achievable in selected individuals who initiate standard ART early in infection. However, given re-emergence of plasma viremia in an individual with an extraordinarily low viral burden, therapeutic strategies aimed at specifically targeting these extremely rare HIV-infected cells with novel interventions may be necessary in order to achieve eradication of virus.

Increased HIV-specific CD8 + T-cell cytotoxic potential in HIV elite controllers is associated with T-bet expression

Recent data suggest that CD8+ T-cell effector activity is an important component in the control of HIV replication in elite controllers (ECs). One critical element of CD8+ T-cell effector function and differentiation is the T-box transcription factor T-bet. In the present study, we assessed T-bet expression, together with the effector proteins perforin, granzyme A (Grz A), granzyme B (Grz B), and granulysin, in HIV-specific CD8+ T cells from ECs (n = 20), chronically infected progressors (CPs; n = 18), and highly active antiretroviral therapy (HAART)-suppressed individuals (n = 19). Compared with the other cohort groups, HIV-specific CD8+ T cells among ECs demonstrated a superior ability to express perforin and Grz B, but with no detectable difference in the levels of Grz A or granulysin. We also observed higher levels of T-bet in HIV-specific CD8+ T cells from ECs, with an ensuing positive correlation between T-bet and levels of both perforin and Grz B. Moreover, HIV-specific CD8+ T cells in ECs up-regulated T-bet to a greater extent than CPs after in vitro expansion, with concomitant up-regulation of perforin and Grz B. These results suggest that T-bet may play an important role in driving effector function, and its modulation may lead to enhanced effector activity against HIV.

Persistent HIV RNA shedding in semen despite effective antiretroviral therapy.

Effective antiretroviral therapy (ART) may reduce HIV sexual transmission by lowering genital HIV levels. A prospective study of men starting ART (n = 25) demonstrated rapid, substantial reductions in semen HIV RNA. However, despite an undetectable blood viral load, isolated semen HIV shedding was detected at more than one visit in 12 of 25 (48%) participants, with semen HIV RNA levels exceeding 5000 copies/ml in four of 25 (16%). Isolates were drug-sensitive, and this phenomenon was not associated with semen drug levels or regimen.

Sigmoid Th17 populations, the HIV latent reservoir, and microbial translocation in men on long-term antiretroviral therapy.

Objective:Th17 cells play an important role in mucosal defence and repair and are highly susceptible to infection by HIV. Antiretroviral therapy (ART) suppresses HIV viremia and can restore CD4+ numbers in the blood and gastrointestinal mucosa, but the resolution of systemic inflammation and gut microbial translocation is often incomplete. We hypothesized that this might relate to persistent dysregulation of gut CD4+ Th17 subsets. Methods:Blood and sigmoid biopsies were collected from HIV-uninfected men, chronically HIV-infected, ART-naive men, and men on effective ART for more than 4 years. Sigmoid provirus levels were assayed blind to participant status, as were CD4+ Th17 subsets, systemic markers of microbial translocation, and cellular immune activation. Results:There was minimal CD4+ Th17 dysregulation in the blood until later stage HIV infection, but gastrointestinal Th17 depletion was apparent much earlier, along with increased plasma markers of microbial translocation. Plasma lipopolysaccharide (LPS) remained elevated despite overall normalization of sigmoid Th17 populations on long-term ART, although there was considerable interindividual variability in Th17 reconstitution. An inverse correlation was observed between plasma LPS levels and gut Th17 frequencies, and higher plasma LPS levels correlated with an increased gut HIV proviral reservoir. Conclusion:Sigmoid Th17 populations were preferentially depleted during HIV infection. Despite overall CD4+ T-cell reconstitution, sigmoid Th17 frequencies after long-term ART were heterogeneous and higher frequencies were correlated with reduced microbial translocation.

HCV-specific T cells in HCV/HIV co-infection show elevated frequencies of dual Tim-3/PD-1 expression that correlate with liver disease progression.

Co‐infection of HCV with HIV has been associated with more rapid progression of HCV‐related disease. HCV‐specific T‐cell immune responses, which are essential for disease control, are attenuated in co‐infection with HIV. T‐cell exhaustion has recently been implicated in the deficient control of chronic viral infections. In the current study, we investigated the role of programmed death‐1 (PD‐1) and T‐cell immunoglobulin and mucin domain‐containing molecule‐3 (Tim‐3) expression in T‐cell exhaustion during HCV/HIV co‐infection. We show that in HCV/HIV co‐infection, both total and HCV‐specific T cells co‐express Tim‐3 and PD‐1 in significantly higher frequencies, compared with HCV mono‐infection. Co‐expression of these two markers on HCV‐specific CD8+ T cells positively correlated with a clinical parameter of liver disease progression. HCV‐specific CD8+ T cells showed greater frequencies of Tim‐3/PD‐1 co‐expression than HIV‐specific CD8+ T cells, which may indicate a greater degree of exhaustion in the former. Blocking Tim‐3 or PD‐1 pathways restored both HIV‐ and HCV‐specific CD8+ T‐cell expansion in the blood of co‐infected individuals. These data demonstrate that co‐expression of Tim‐3 and PD‐1 may play a significant role in HCV‐specific T‐cell dysfunction, especially in the setting of HIV co‐infection.

Disproportionately High Semen Shedding of HIV Is Associated with Compartmentalized Cytomegalovirus Reactivation

Semen transmission of human immunodeficiency virus (HIV) drives the global pandemic. HIV loads are generally lower in semen than in blood, but semen loads may be disproportionately high in a subgroup of men. HIV loads in semen exceeded those in blood in 9 (35%) of 26 of antiretroviral therapy-naive men, and disproportionately high shedding was strongly associated with compartmentalized semen cytomegalovirus (CMV) reactivation (odds ratio [OR], 10.5; P<.01). Overall, 17 of 26 participants were shedding CMV in semen. Semen levels of HIV and CMV were closely correlated (r=0.5; P<.01), independently of blood HIV load and CD4(+) T cell count. Prevention of CMV reactivation warrants further study as a possible strategy to reduce semen shedding of HIV.

Coinfection with Herpes Simplex Virus Type 2 Is Associated with Reduced HIV-Specific T Cell Responses and Systemic Immune Activation

BACKGROUND Chronic coinfection with herpes simplex virus type 2 (HSV-2) and human immunodeficiency virus (HIV) has been associated with an increased HIV viral load and more rapid disease progression, perhaps related to HSV-2-associated alterations in host immunity. METHODS Studies were nested within (1) a cross-sectional study of men coinfected with HIV and HSV-2 and (2) women not infected with HIV, both before and after HSV-2 acquisition. HSV-2 infection status was determined by ELISA. HIV-specific CD8(+) T cell epitopes were mapped, and proliferation of HIV-specific cells was also assessed. Systemic inflammatory and regulatory T cell populations were assayed by flow cytometry. RESULTS The breadth of both the HIV-specific CD8(+) T cell interferon-gamma and proliferative responses was reduced in participants coinfected with HIV and HSV-2, independent of the HIV plasma viral load and CD4(+) T cell count, and the magnitude of the responses was also reduced. HSV-2 infection in this group was associated with increased T cell CD38 expression but not with differences in the proportion of CD4(+) FoxP3(+) regulatory T cells. However, in women not infected with HIV, acquisition of HSV-2 was associated with an increase in the proportion of regulatory T cells. CONCLUSIONS HSV-2 coinfection was associated with reduced HIV-specific T cell responses and systemic inflammation. The immune effects of HSV-2 may underlie the negative impact that this coinfection has on the clinical course of HIV infection.

HIV-Specific CD8+ Lymphocytes in Semen Are Not Associated with Reduced HIV Shedding

Sexual contact with HIV-infected semen is a major driving force behind the global HIV pandemic. Little is known regarding the immune correlates of virus shedding in this compartment, although HIV-1-specific CD8+ T cells are present in semen. We collected blood and semen from 27 chronically HIV-infected, therapy-naive men without common sexually transmitted infections or urethral inflammation and measured HIV-1 RNA viral load and cytokine/chemokine levels in both compartments. HIV-1 RNA levels were 10-fold higher in blood than semen, but discordantly high semen shedding was associated with higher semen levels of the proinflammatory cytokines IL-6, IL-8, IL-12, and IFN-γ. Virus-specific CD8+ T cell epitopes were mapped in blood by IFN-γ ELISPOT, using an overlapping HIV-1 clade B peptide matrix, and blood and semen CD8+ T cell responses were then assayed ex vivo using intracellular IFN-γ staining. HIV-specific CD8+ responses were detected in 70% of semen samples, and their frequency was similar to or higher than blood. There was no correlation between the presence of virus-specific CD8+ T cells in semen and levels of HIV-1 RNA shedding. Among participants with detectable CD8+ IFN-γ semen responses, their relative frequency was not associated with reduced HIV-1 RNA shedding, and their absolute number was correlated with higher levels of HIV-1 RNA semen shedding (r = 0.6; p = 0.03) and of several proinflammatory cytokines. Neither the presence nor the frequency of semen HIV-specific CD8+ T cell IFN-γ responses in semen correlated with reduced levels of HIV RNA in semen.