Kiera L. Clayton

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.

HERV-K–specific T cells eliminate diverse HIV-1/2 and SIV primary isolates

The genetic diversity of HIV-1 represents a major challenge in vaccine development. In this study, we establish a rationale for eliminating HIV-1-infected cells by targeting cellular immune responses against stable human endogenous retroviral (HERV) antigens. HERV DNA sequences in the human genome represent the remnants of ancient infectious retroviruses. We show that the infection of CD4+ T cells with HIV-1 resulted in transcription of the HML-2 lineage of HERV type K [HERV-K(HML-2)] and the expression of Gag and Env proteins. HERV-K(HML-2)-specific CD8+ T cells obtained from HIV-1-infected human subjects responded to HIV-1-infected cells in a Vif-dependent manner in vitro. Consistent with the proposed mode of action, a HERV-K(HML-2)-specific CD8+ T cell clone exhibited comprehensive elimination of cells infected with a panel of globally diverse HIV-1, HIV-2, and SIV isolates in vitro. We identified a second T cell response that exhibited cross-reactivity between homologous HIV-1-Pol and HERV-K(HML-2)-Pol determinants, raising the possibility that homology between HIV-1 and HERVs plays a role in shaping, and perhaps enhancing, the T cell response to HIV-1. This justifies the consideration of HERV-K(HML-2)-specific and cross-reactive T cell responses in the natural control of HIV-1 infection and for exploring HERV-K(HML-2)-targeted HIV-1 vaccines and immunotherapeutics.

IL-10-Producing B Cells Are Induced Early in HIV-1 Infection and Suppress HIV-1-Specific T Cell Responses

A rare subset of IL-10-producing B cells, named regulatory B cells (Bregs), suppresses adaptive immune responses and inflammation in mice. In this study, we examined the role of IL-10-producing B cells in HIV-1 infection. Compared to uninfected controls, IL-10-producing B cell frequencies were elevated in both blood and sigmoid colon during the early and chronic phase of untreated HIV-1 infection. Ex vivo IL-10-producing B cell frequency in early HIV-1 infection directly correlated with viral load. IL-10-producing B cells from HIV-1 infected individuals were enriched in CD19+TIM-1+ B cells and were enriched for specificity to trimeric HIV-1 envelope protein. Anti-retroviral therapy was associated with reduced IL-10-producing B cell frequencies. Treatment of B cells from healthy donors with microbial metabolites and Toll-like receptor (TLR) agonists could induce an IL-10 producing phenotype, suggesting that the elevated bacterial translocation characteristic of HIV-1 infection may promote IL-10-producing B cell development. Similar to regulatory B cells found in mice, IL-10-producing B cells from HIV-1-infected individuals suppressed HIV-1-specific T cell responses in vitro, and this suppression is IL-10-dependent. Also, ex vivo IL-10-producing B cell frequency inversely correlated with contemporaneous ex vivo HIV-1-specific T cell responses. Our findings show that IL-10-producing B cells are induced early in HIV-1 infection, can be HIV-1 specific, and are able to inhibit effective anti-HIV-1 T cell responses. HIV-1 may dysregulate B cells toward Bregs as an immune evasion strategy.

TIGIT Marks Exhausted T Cells, Correlates with Disease Progression, and Serves as a Target for Immune Restoration in HIV and SIV Infection.

HIV infection induces phenotypic and functional changes to CD8+ T cells defined by the coordinated upregulation of a series of negative checkpoint receptors that eventually result in T cell exhaustion and failure to control viral replication. We report that effector CD8+ T cells during HIV infection in blood and SIV infection in lymphoid tissue exhibit higher levels of the negative checkpoint receptor TIGIT. Increased frequencies of TIGIT+ and TIGIT+ PD-1+ CD8+ T cells correlated with parameters of HIV and SIV disease progression. TIGIT remained elevated despite viral suppression in those with either pharmacological antiretroviral control or immunologically in elite controllers. HIV and SIV-specific CD8+ T cells were dysfunctional and expressed high levels of TIGIT and PD-1. Ex-vivo single or combinational antibody blockade of TIGIT and/or PD-L1 restored viral-specific CD8+ T cell effector responses. The frequency of TIGIT+ CD4+ T cells correlated with the CD4+ T cell total HIV DNA. These findings identify TIGIT as a novel marker of dysfunctional HIV-specific T cells and suggest TIGIT along with other checkpoint receptors may be novel curative HIV targets to reverse T cell exhaustion.

Antigen-independent induction of Tim-3 expression on human T cells by the common γ-chain cytokines IL-2, IL-7, IL-15, and IL-21 is associated with proliferation and is dependent on the phosphoinositide 3-kinase pathway.

T cell Ig mucin domain-containing molecule 3 (Tim-3) is a glycoprotein found on the surface of a subset of CD8+ and Th1 CD4+ T cells. Elevated expression of Tim-3 on virus-specific T cells during chronic viral infections, such as HIV-1, hepatitis B virus, and hepatitis C virus, positively correlates with viral load. Tim-3+ cytotoxic T cells are dysfunctional and are unable to secrete effector cytokines, such as IFN-γ and TNF-α. In this study, we examined potential inducers of Tim-3 on primary human T cells. Direct HIV-1 infection of CD4+ T cells, or LPS, found to be elevated in HIV-1 infection, did not induce Tim-3 on T cells. Tim-3 was induced by the common γ-chain (γc) cytokines IL-2, IL-7, IL-15, and IL-21 but not IL-4, in an Ag-independent manner and was upregulated on primary T cells in response to TCR/CD28 costimulation, as well as γc cytokine stimulation with successive divisions. γc cytokine-induced Tim-3 was found on naive, effector, and memory subsets of T cells. Tim-3+ primary T cells were more prone to apoptosis, particularly upon treatment with galectin-9, a Tim-3 ligand, after cytokine withdrawal. The upregulation of Tim-3 could be blocked by the addition of a PI3K inhibitor, LY 294002. Thus, Tim-3 can be induced via TCR/CD28 costimulation and/or γc cytokines, likely through the PI3K pathway.

Tim-3 Negatively Regulates Cytotoxicity in Exhausted CD8+ T Cells in HIV Infection

Cytotoxic CD8+ T cells (CTLs) contain virus infections through the release of granules containing both perforin and granzymes. T cell ‘exhaustion’ is a hallmark of chronic persistent viral infections including HIV. The inhibitory regulatory molecule, T cell Immunoglobulin and Mucin domain containing 3 (Tim-3) is induced on HIV-specific T cells in chronic progressive infection. These Tim-3 expressing T cells are dysfunctional in terms of their capacities to proliferate or to produce cytokines. In this study, we evaluated the effect of Tim-3 expression on the cytotoxic capabilities of CD8+ T cells in the context of HIV infection. We investigated the cytotoxic capacity of Tim-3 expressing T cells by examining 1) the ability of Tim-3+ CD8+ T cells to make perforin and 2) the direct ability of Tim-3+ CD8+ T cells to kill autologous HIV infected CD4+ target cells. Surprisingly, Tim-3+ CD8+ T cells maintain higher levels of perforin, which was mainly in a granule-associated (stored) conformation, as well as express high levels of T-bet. However, these cells were also defective in their ability to degranulate. Blocking the Tim-3 signalling pathway enhanced the cytotoxic capabilities of HIV specific CD8+ T cells from chronic progressors by increasing; a) their degranulation capacity, b) their ability to release perforin, c) their ability to target activated granzyme B to HIV antigen expressing CD4+ T cells and d) their ability to suppress HIV infection of CD4+ T cells. In this latter effect, blocking the Tim-3 pathway enhances the cytotoxcity of CD8+ T cells from chronic progressors to the level very close to that of T cells from viral controllers. Thus, the Tim-3 receptor, in addition to acting as a terminator for cytokine producing and proliferative functions of CTLs, can also down-regulate the CD8+ T cell cytotoxic function through inhibition of degranulation and perforin and granzyme secretion.

T Cell Ig and Mucin Domain–Containing Protein 3 Is Recruited to the Immune Synapse, Disrupts Stable Synapse Formation, and Associates with Receptor Phosphatases

CD8+ CTLs are adept at killing virally infected cells and cancer cells and releasing cytokines (e.g., IFN-γ) to aid this response. However, during cancer and chronic viral infections, such as with HIV, this CTL response is progressively impaired due to a process called T cell exhaustion. Previous work has shown that the glycoprotein T cell Ig and mucin domain–containing protein 3 (Tim-3) plays a functional role in establishing T cell exhaustion. Tim-3 is highly upregulated on virus and tumor Ag-specific CD8+ T cells, and antagonizing Tim-3 helps restore function of CD8+ T cells. However, very little is known of how Tim-3 signals in CTLs. In this study, we assessed the role of Tim-3 at the immunological synapse as well as its interaction with proximal TCR signaling molecules in primary human CD8+ T cells. Tim-3 was found within CD8+ T cell lipid rafts at the immunological synapse. Blocking Tim-3 resulted in a significantly greater number of stable synapses being formed between Tim-3hiCD8+ T cells and target cells, suggesting that Tim-3 plays a functional role in synapse formation. Further, we confirmed that Tim-3 interacts with Lck, but not the phospho-active form of Lck. Finally, Tim-3 colocalizes with receptor phosphatases CD45 and CD148, an interaction that is enhanced in the presence of the Tim-3 ligand, galectin-9. Thus, Tim-3 interacts with multiple signaling molecules at the immunological synapse, and characterizing these interactions could aid in the development of therapeutics to restore Tim-3–mediated immune dysfunction.

Stable complex formation between HIV Rev and the nucleosome assembly protein, NAP1, affects Rev function

The Rev protein of HIV-1 is essential for HIV-1 proliferation due to its role in exporting viral RNA from the nucleus. We used a modified version of tandem affinity purification (TAP) tagging to identify proteins interacting with HIV-1 Rev in human cells and discovered a prominent interaction between Rev and nucleosome assembly protein 1 (Nap1). This interaction was also observed by specific retention of Nap1 from human cell lysates on a Rev affinity column. Nap1 was found to bind Rev through the Rev arginine-rich domain and altered the oligomerization state of Rev in vitro. Overexpression of Nap1 stimulated the ability of Rev to export RNA, reduced the nucleolar localization of Rev, and affected Rev nuclear import rates. The results suggest that Nap-1 may influence Rev function by increasing the availability of Rev.

Soluble T Cell Immunoglobulin Mucin Domain 3 Is Shed from CD8+ T Cells by the Sheddase ADAM10, Is Increased in Plasma during Untreated HIV Infection, and Correlates with HIV Disease Progression

ABSTRACT Chronic HIV infection results in a loss of HIV-specific CD8+ T cell effector function, termed “exhaustion,” which is mediated, in part, by the membrane coinhibitory receptor T cell immunoglobulin mucin domain-3 (Tim-3). Like many other receptors, a soluble form of this protein has been described in human blood plasma. However, soluble Tim-3 (sTim-3) is poorly characterized, and its role in HIV disease is unknown. Here, we show that Tim-3 is shed from the surface of responding CD8+ T cells by the matrix metalloproteinase ADAM10, producing a soluble form of the coinhibitory receptor. Despite previous reports in the mouse model, no alternatively spliced, soluble form of Tim-3 was observed in humans. Shed sTim-3 was found in human plasma and was significantly elevated during early and chronic untreated HIV infection, but it was not found differentially modulated in highly active antiretroviral therapy (HAART)-treated HIV-infected subjects or in elite controllers compared to HIV-uninfected subjects. Plasma sTim-3 levels were positively correlated with HIV load and negatively correlated with CD4 counts. Thus, plasma sTim-3 shedding correlated with HIV disease progression. Despite these correlations, we found that shedding Tim-3 did not improve the function of CD8+ T cells in terms of gamma interferon production or prevent their apoptosis through galectin-9. Further characterization studies of sTim-3 function are needed to understand the contribution of sTim-3 in HIV disease pathogenesis, with implications for novel therapeutic interventions. IMPORTANCE Despite the overall success of HAART in slowing the progression to AIDS in HIV-infected subjects, chronic immune activation and T cell exhaustion contribute to the eventual deterioration of the immune system. Understanding these processes will aid in the development of interventions and therapeutics to be used in combination with HAART to slow or reverse this deterioration. Here, we show that a soluble form of T cell exhaustion associated coinhibitory molecule 3, sTim-3, is shed from the surface of T cells. Furthermore, sTim-3 is elevated in the plasma of treatment-naive subjects with acute or chronic HIV infection and is associated with markers of disease progression. This is the first study to characterize sTim-3 in human plasma, its source, and mechanism of production. While it is still unclear whether sTim-3 contributes to HIV pathogenesis, sTim-3 may represent a new correlate of HIV disease progression.

Expansion of Dysfunctional Tim-3–Expressing Effector Memory CD8 + T Cells during Simian Immunodeficiency Virus Infection in Rhesus Macaques

The T cell Ig- and mucin domain–containing molecule-3 (Tim-3) negative immune checkpoint receptor demarcates functionally exhausted CD8+ T cells arising from chronic stimulation in viral infections like HIV. Tim-3 blockade leads to improved antiviral CD8+ T cell responses in vitro and, therefore, represents a novel intervention strategy to restore T cell function in vivo and protect from disease progression. However, the Tim-3 pathway in the physiologically relevant rhesus macaque SIV model of AIDS remains uncharacterized. We report that Tim-3+CD8+ T cell frequencies are significantly increased in lymph nodes, but not in peripheral blood, in SIV-infected animals. Tim-3+PD-1+CD8+ T cells are similarly increased during SIV infection and positively correlate with SIV plasma viremia. Tim-3 expression was found primarily on effector memory CD8+ T cells in all tissues examined. Tim-3+CD8+ T cells have lower Ki-67 content and minimal cytokine responses to SIV compared with Tim-3−CD8+ T cells. During acute-phase SIV replication, Tim-3 expression peaked on SIV-specific CD8+ T cells by 2 wk postinfection and then rapidly diminished, irrespective of mutational escape of cognate Ag, suggesting non-TCR–driven mechanisms for Tim-3 expression. Thus, rhesus Tim-3 in SIV infection partially mimics human Tim-3 in HIV infection and may serve as a novel model for targeted studies focused on rejuvenating HIV-specific CD8+ T cell responses.