Immune Responses in Controllers of HIV Infection.

A Critical Role for Type I IFN–dependent NK Cell Activation in Innate Immune Elimination of Adenoviral Vectors In Vivo

Recent evidence suggests that natural killer (NK) cells play a crucial role in the HIV pathogenesis. Long-term nonprogressor (LTNP) and HIV controllers are rare HIV-infected patients who control viral replication and show delayed disease progression. They represent fascinating models of natural protection against disease progression and for studying the immunological response to the virus. We have conducted an extensive analysis of the phenotypic and functional properties of CD56, CD56 and CD56/CD16 NK cell subsets from LTNP and HIV-controllers, and compared them with HIV progressors and healthy donors. Hierarchical clustering analysis of NK phenotypic markers revealed that LTNP and HIV controllers, exhibit peculiar phenotypic features, associated with high levels of interferon-g, activation markers, and cytolytic activity in CD3CD56 NK cells against K562 target cells. More importantly, cytolytic activity against autologous CD4 T cells is abrogated after treatment with anti-NKp44L mAb, in LTNP and HIV progressors, suggesting a key role of NKp44L. In contrast, in HIV controllers and healthy donors, NKp44L expression on CD4 T cells and autologous NK lysis were both poorly detected. These results show that NK cells from LTNP and HIV controllers display phenotypic and functional features and suggest a consistent continuous involvement of the innate immune response in the failure to control viral replication. Collectively, these data may have important implication in the design of new anti-HIV therapeutical strategies based on the particular functional activity of NK cells.

Repeated exposure to HIV does not necessarily result in infection and HIV infection does not inevitably lead to the development of the AIDS. Multiple immunological and genetic features can confer resistance to HIV acquisition and progression at different steps in viral infection; a full understanding of these mechanisms could result in the development of novel therapeutic and vaccine approaches for HIV infection. In this review, we focus on the genetic mechanisms associated with resistance to HIV infection and to the progression to AIDS.

Research into the effects of host genetics on HIV disease susceptibility and progression, and on the efficacy and toxicity of drug treatment, occupies a particular position in the landscape of HIV research...

A rare subset of human immunodeficiency virus (HIV)-infected individuals maintains undetectable HIV RNA levels without therapy ("elite controllers"). To clarify the role of T-cell responses in mediating virus control, we compared HLA class I polymorphisms and HIV-specific T-cell responses among a large cohort of elite controllers (HIV-RNA < 75 copies/ml), "viremic" controllers (low-level viremia without therapy), "noncontrollers" (high-level viremia), and "antiretroviral therapy suppressed" individuals (undetectable HIV-RNA levels on antiretroviral therapy). The proportion of CD4(+) and CD8(+) T cells that produce gamma interferon (IFN-gamma) and interleukin-2 (IL-2) in response to Gag and Pol peptides was highest in the elite and viremic controllers (P < 0.0001). Forty percent of the elite controllers were HLA-B*57 compared to twenty-three percent of viremic controllers and nine percent of noncontrollers (P < 0.001). Other HLA class I alleles more common in elite controllers included HLA-B*13, HLA-B*58, and HLA-B*81 (P < 0.05 for each). Within elite and viremic controller groups, those with protective class I alleles had higher frequencies of Gag-specific CD8(+) T cells than those without these alleles (P = 0.01). Noncontrollers, with or without protective alleles, had low-level CD8(+) responses. Thus, certain HLA class I alleles are enriched in HIV controllers and are associated with strong Gag-specific CD8(+)IFN-gamma(+)IL-2(+) T cells. However, the absence of evidence of T cell-mediated control in many controllers suggests the presence of alternative mechanisms for viral control in these individuals. Defining mechanisms for virus control in "non-T-cell controllers" might lead to insights into preventing HIV transmission or preventing virus replication.

To evaluate the impact of chikungunya virus (CHIKV) infection on the quality of the HIV-specific CD8 T-cell (CTL) response in an HIV elite controller. Three blood samples were obtained from an elite controller at 27 days (EC-CHIKV, Sample 1, S1), 41 days (S2) and 1 year (S3) after CHIKV infection. Additionally, samples from another nine elite controllers and nine viremic chronics were obtained. CD4 T-cell counts, viral load and immune activation were recorded. Natural killer (NK) cells and HIV-specific CTL quality were evaluated. Data were analyzed using nonparametric statistics. A male HIV elite controller was confirmed for CHIKV infection. At S1, he presented 211 cells/μl CD4 T-cell count, a HIV viral load blip (145 copies/ml) and high T-cell activation. NK cell percentage and activation were higher at S2. All parameters were recovered by S3. CTLs at S1 were exclusively monofunctional with a high proportion (>80%) of degranulating CTLs. By S3, CTL polyfunctionality was more similar to that of a typical elite controller. The distribution of CTL memory subsets also displayed altered profiles. The results showed that the phenotype and function of HIV-specific CTLs were modified in temporal association with an HIV viral load blip that followed CHIKV infection. This might have helped to control the transient HIV rebound. Additionally, NK cells could have been involved in this control. These results provide useful information to help understand how elite controllers maintain their status, control HIV infection and alert about the negative impact to the immune function of HIV-infected individuals living in CHIKV endemic areas.

Viral reservoirs in elite controllers of HIV-1 infection: Implications for HIV cure strategies

Natural killer (NK) cells are essential components of the immune system, and due to their rapid response potential, can have a great impact during early anti-viral immune responses. We have previously shown that interleukin-2-dependent NK and CD4(+) T-cell co-operative immune responses exist in long-term simian immunodeficiency virus (SIV) -infected controlling macaques and can be rescued in SIV-infected non-controlling macaques by a short course of antiretroviral therapy (ART). Given that co-operative responses may play an important role in disease prevention and therapeutic treatment, in the present study we sought to determine if these responses can be enhanced in chronically SIV-infected macaques by vaccination with a single-dose of envelope protein given during ART. To this end, we treated 14 chronically SIV-infected macaques with ART for 11 weeks and gave 10 of these macaques a single intramuscular dose of SIV gp120 at week 9 of treatment. ART significantly decreased plasma and mucosal viral loads, increased the numbers of circulating CD4(+) T cells in all macaques, and increased T-cell-dependent envelope- and gag-specific interferon-γ and tumour necrosis factor-α production by circulatory CD56(+) NK cells. The therapeutic envelope immunization resulted in higher envelope-specific responses compared with those in macaques that received ART only. Functional T-cell responses restored by ART and therapeutic Env immunization were correlated with transiently reduced plasma viraemia levels following ART release. Collectively our results indicate that SIV-specific T-cell-dependent NK cell responses can be efficiently rescued by ART in chronically SIV-infected macaques and that therapeutic immunization may be beneficial in previously vaccinated individuals.

Accessory function for NK1.1+ natural killer cells producing interferon-gamma in xenospecific cytotoxic T lymphocyte differentiation. Whither the natural killer cell.

Natural killer (NK) cells are important in innate immune responses to bacterial and viral pathogens. HIV-1 infection is associated with opportunistic bacterial infections and with microbial translocation, but the nature of the NK cell response to bacteria during HIV-1 infection has not been studied extensively. The objective of this study was to compare NK cell responses to bacteria in HIV-1-infected versus that in uninfected individuals. Multicolor flow cytometry was used to evaluate the ability of blood NK cell subsets (CD56CD16, CD56CD16, and CD56CD16) from treated, virally suppressed, and untreated viremic subjects with chronic HIV-1 infection and uninfected controls, to secrete interferon gamma (IFN-γ) in response to the in vitro stimulation of peripheral blood mononuclear cells with heat-killed commensal Escherichia coli or pathogenic Salmonella typhimurium. All 3 NK cell subsets produced IFN-γ in response to bacteria, but CD56CD16 NK cells were least responsive. Untreated HIV-1-infected donors had increased frequencies of CD56CD16 NK cells and lower overall frequencies of IFN-γ-producing NK cells responding to E. coli and S. typhimurium than did NK cells from uninfected donors. These NK cell defects were not fully restored in antiretroviral therapy-treated donors. Monocytes were necessary for NK cells to respond to bacteria, but the HIV-associated defect was intrinsic to NK cells because the addition of normal monocytes did not restore IFN-γ production in response to bacteria. Functional defects and numeric alterations of NK cell subsets lead to decreased frequencies of bacteria-reactive, IFN-γ-producing NK cells in HIV-1-infected subjects, even those on antiretroviral therapy.

Although highly effective at durably suppressing plasma HIV-1 viremia, combination antiretroviral therapy (ART) treatment regimens do not eradicate the virus, which persists in long-lived CD4+ T cells. This latent viral reservoir serves as a source of plasma viral rebound following treatment interruption, thus requiring lifelong adherence to ART. Additionally, challenges remain related not only to access to therapy but also to a higher prevalence of comorbidities with an inflammatory etiology in treated HIV-1+ individuals, underscoring the need to explore therapeutic alternatives that achieve sustained virologic remission in the absence of ART. Natural killer (NK) cells are uniquely positioned to positively impact antiviral immunity, in part due to the pleiotropic nature of their effector functions, including the acquisition of memory-like features, and, therefore, hold great promise for transforming HIV-1 therapeutic modalities. In addition to defining the ability of NK cells to contribute to HIV-1 control, this review provides a basic immunologic understanding of the impact of HIV-1 infection and ART on the phenotypic and functional character of NK cells. We further delineate the qualities of “memory” NK cell populations, as well as the impact of HCMV on their induction and subsequent expansion in HIV-1 infection. We conclude by highlighting promising avenues for optimizing NK cell responses to improve HIV-1 control and effect a functional cure, including blockade of inhibitory NK receptors, TLR agonists to promote latency reversal and NK cell activation, CAR NK cells, BiKEs/TriKEs, and the role of HIV-1-specific bNAbs in NK cell–mediated ADCC activity against HIV-1-infected cells.

The latent HIV-1 reservoir represents a major barrier to achieving a long-term antiretroviral therapy (ART)-free remission or cure for HIV-1. Natural Killer (NK) cells are innate immune cells that play a critical role in controlling viral infections and have been shown to be involved in preventing HIV-1 infection and, in those who are infected, delaying time to progression to AIDS. However, their role in limiting HIV-1 persistence on long term ART is still uncharacterized. To identify associations between markers of HIV-1 persistence and the NK cell receptor-ligand repertoire, we used twin mass cytometry panels to characterize the peripheral blood NK receptor-ligand repertoire in individuals with long-term antiretroviral suppression enrolled in the AIDS Clinical Trial Group A5321 study. At the time of testing, participants had been on ART for a median of 7 years, with virological suppression <50 copies/mL since at most 48 weeks on ART. We found that the NK cell receptor and ligand repertoires did not change across three longitudinal samples over one year—a median of 25 weeks and 50 weeks after the initial sampling. To determine the features of the receptor-ligand repertoire that associate with markers of HIV-1 persistence, we performed a LASSO normalized regression. This analysis revealed that the NK cell ligands CD58, HLA-B, and CRACC, as well as the killer cell immunoglobulin-like receptors (KIRs) KIR2DL1, KIR2DL3, and KIR2DS4 were robustly predictive of markers of HIV-1 persistence, as measured by total HIV-1 cell-associated DNA, HIV-1 cell-associated RNA, and single copy HIV-RNA assays. To characterize the roles of cell populations defined by multiple markers, we augmented the LASSO analysis with FlowSOM clustering. This analysis found that a less mature NK cell phenotype (CD16+CD56dimCD57-LILRB1-NKG2C-) was associated with lower HIV-1 cell associated DNA. Finally, we found that surface expression of HLA-Bw6 measured by CyTOF was associated with lower HIV-1 persistence. Genetic analysis revealed that this was driven by lower HIV-1 persistence in HLA-Bw4/6 heterozygotes. These findings suggest that there may be a role for NK cells in controlling HIV-1 persistence in individuals on long-term ART, which must be corroborated by future studies.

BackgroundDespite advances in treatment, glioblastoma (GBM) remains an incurable primary brain tumor with a median survival of only 15 months, highlighting the need for new therapeutic approaches. Natural killer (NK)...

Potently Cytotoxic Natural Killer Cell Potential Initially Emerges from Erythro-Myeloid Progenitors during Mammalian Development

Despite over-expression of epidermal growth factor receptor (EGFR) in 80-90% of head and neck cancers (HNC), only ~20% of HNC patients are clinically responsive the EGFR-specific therapeutic monoclonal antibody (mAb) cetuximab. In addition to blocking EGFR signaling that leads to tumor cell survival and proliferation, increasing evidence suggests that innate and adaptive immunity likely plays a significant role in patient response to cetuximab. Natural killer (NK) cells recognize the Fc-region of EGFR-bound cetuximab via their activating Fcγ receptor IIIa (FcγRIIIa, also known as CD16) and mediate tumor cell lysis by antibody dependent cellular cytotoxicity (ADCC). NK cells are increasingly recognized as key mediators of crosstalk bridging the innate and adaptive arms of the immune response to human malignancies. We have previously shown that in addition to being direct effectors of ADCC against cetuximab-coated HNC cells, NK cells are instrumental in indirectly promoting an anti-tumor adaptive immune response via secretion of immunomodulatory cytokines such as IFNγ, which is known to enhance dendritic cell (DC) antigen processing and presentation to CD8+ T cells. Multiple studies have consistently demonstrated a relative decrease in the CD56dimCD16bright NK cell subset, traditionally considered to be primarily cytotoxic effectors, and an increase in the immunoregulatory CD56brightCD16dim NK cell subset in the peripheral blood of patients with various malignancies. However, given that the immunoregulatory CD56brightCD16dim NK cells are generally considered to be the more prolific cytokine producing subset, it was unclear how this redistribution of NK cell subsets might affect the dynamics of cytokine-mediated NK:DC crosstalk in the presence of mAb-coated tumor cells. Using primary NK cells and monocyte-derived DC isolated from PBMC of healthy donors, we therefore sought to better elucidate the patterns and kinetics of IFNγ secretion by the two NK cell subsets in response to cetuximab-coated HNC cells in the presence of DC. In this context, we found that while CD56brightCD16dim NK cells express more of the activating NKG2D receptor, rapid (by 4hrs) IFNγ secretion by CD56dimCD16bright NK cells is a CD16-dependent process. CD56brightCD16dim NK cells only produced IFNγ following stimulation for at least 12 hours with a combination of IL-12 and IL-15. IL-12/IL-15 stimulation also induced IFNγ secretion by CD56dimCD16bright NK cells, though to a lesser extent as compared to the CD56brightCD16dim subset. Taken together, these data contribute significantly to a mechanistic understanding of how NK cells function to bridge innate and adaptive anti-tumor responses in the context of mAb cancer therapies. Understanding this link is of broad relevance to the optimization of current therapeutic mAbs and will help devise novel ways to improve clinical responses. Citation Format: Christopher A. Lord, Raghvendra M. Srivastava, Robert L. Ferris. Patterns and kinetics of interferon-γ secretion by natural killer cell subsets co-cultured with cetuximab-coated head and neck cancer cells and dendritic cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A41.