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Rhesus macaques (RMs) are a common pre-clinical model used to test HIV vaccine efficacy and passive immunization strategies. Yet, it remains unclear to what extent the Fc-Fc receptor (FcR) interactions impacting antiviral activities of antibodies in RMs recapitulate those in humans. Here, we evaluated the FcR-related functionality of natural killer cells (NKs) from peripheral blood of uninfected humans and RMs to identify intra- and inter-species variation. NKs were screened for FcγRIIIa (human) and FcγRIII (RM) genotypes (FcγRIII(a)), receptor signaling, and antibody-dependent cellular cytotoxicity (ADCC), the latter mediated by a cocktail of monoclonal IgG1 antibodies with human or RM Fc. FcγRIII(a) genetic polymorphisms alone did not explain differences in NK effector functionality in either species cohort. Using the same parameters, hierarchical clustering separated each species into two clusters. Importantly, in principal components analyses, ADCC magnitude, NK contribution to ADCC, FcγRIII(a) cell-surface expression, and frequency of phosphorylated CD3ζ NK cells all contributed similarly to the first principal component within each species, demonstrating the importance of measuring multiple facets of NK cell function. Although ADCC potency was similar between species, we detected significant differences in frequencies of NK cells and pCD3ζ+ cells, level of cell-surface FcγRIII(a) expression, and NK-mediated ADCC (P<0.001), indicating that a combination of Fc-FcR parameters contribute to overall inter-species functional differences. These data strongly support the importance of multi-parameter analyses of Fc-FcR NK-mediated functions when evaluating efficacy of passive and active immunizations in pre- and clinical trials and identifying correlates of protection. The results also suggest that pre-screening animals for multiple FcR-mediated NK function would ensure even distribution of animals among treatment groups in future preclinical trials., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Tuyishime, Spreng, Hueber, Nohara, Goodman, Chan, Barfield, Beck, Jha, Asdell, Wiehe, He, Easterhoff, Conley, Hoxie, Gurley, Jones, Adhikary, Villinger, Thomas, Denny, Moody, Tomaras, Pollara, Reeves and Ferrari.)

Natural killer (NK) cells represent a critical defense against viral infections and cancers. NK cells require integration of activating and inhibitory NK cell receptors to detect target cells and the balance of these NK cell inputs defines the global NK cell response. The sensitivity of the response is largely defined by interactions between self-major histocompatibility complex class I (MHC-I) molecules and specific inhibitory NK cell receptors, so-called NK cell education. Thus, NK cell education is a crucial process to generate tuned effector NK cell responses in different diseases. In this review, we discuss the relationship between NK cell education and physiologic factors (type of self-MHC-I, self-MHC-I allelic variants, variant of the self-MHC-I-binding peptides, cytokine effects and inhibitory KIR expression) underlying NK cell education profiles (effector function or metabolism). Additionally, we describe the broad-spectrum of effector educated NK cell functions on different pathologies (such as HIV-1, CMV and tumors, among others)., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Rascle, Woolley, Jost, Manickam and Reeves.)

Although antiretroviral therapy (ART) has drastically changed the lives of people living with human immunodeficiency virus-1 (HIV-1), long-term treatment has been associated with a vast array of comorbidities. Therefore, a cure for HIV-1 remains the best option to globally eradicate HIV-1/acquired immunodeficiency syndrome (AIDS). However, development of strategies to achieve complete eradication of HIV-1 has been extremely challenging. Thus, the control of HIV-1 replication by the host immune system, namely functional cure, has long been studied as an alternative approach for HIV-1 cure. HIV-1 elite controllers (ECs) are rare individuals who naturally maintain undetectable HIV-1 replication levels in the absence of ART and whose immune repertoire might be a desirable blueprint for a functional cure. While the role(s) played by distinct human leukocyte antigen (HLA) expression and CD8+ T cell responses expressing cognate ligands in controlling HIV-1 has been widely characterized in ECs, the innate immune phenotype has been decidedly understudied. Comparably, in animal models such as HIV-1-infected humanized mice and simian Immunodeficiency Virus (SIV)-infected non-human primates (NHP), viremic control is known to be associated with specific major histocompatibility complex (MHC) alleles and CD8+ T cell activity, but the innate immune response remains incompletely characterized. Notably, recent work demonstrating the existence of trained innate immunity may provide new complementary approaches to achieve an HIV-1 cure. Herein, we review the known characteristics of innate immune responses in ECs and available animal models, identify gaps of knowledge regarding responses by adaptive or trained innate immune cells, and speculate on potential strategies to induce EC-like responses in HIV-1 non-controllers., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sugawara, Reeves and Jost.)

CD49a + tissue resident NK cells have been implicated in memory-like NK cell responses, but while this population is well-characterized in mice and in humans, they are poorly described in non-human primates (NHP) which are particularly critical for modeling human viral infections. Others and we have shown that memory-like NK cells are enriched in the liver and because of the importance of NHP in modeling HIV infection, understanding the immunobiology of CD49a + NK cells in SIV-infected rhesus macaques is critical to explore the role of this cell type in retroviral infections. In this study mononuclear cells isolated from livers, spleens, and peripheral whole blood were analyzed in acutely and chronically lentivirus-infected and experimentally-naïve Indian rhesus macaques (RM). NK cells were then identified as CD45 + CD14 - CD20 - CD3 - NKG2A/C + cells and characterized using multiparametric flow-cytometry. Our data show that in RM, CD49a + NK cells increase in the liver following retroviral infections [median = 5.2% (naïve) vs. median = 9.48% (SIV+) or median = 16.8% (SHIV+)]. In contrast, there is little change in CD49a + NK frequencies in whole blood or spleens of matched animals. In agreement with human and murine data we also observed that CD49a + NK cells were predominantly Eomes low T-bet low , though these frequencies are elevated in infected animal cohorts. Functionally, our data suggests that infection alters TNF-α, IFN-γ, and CD107a expression in stimulated CD49a + NK cells. Specifically, our analyses found a decrease in CD49a + CD107a + TNFα + IFNγ - NK cells, with a simultaneous increase in CD49a + CD107a + TNFα - IFNγ + NK cells and the non-responsive CD49a + CD107a - TNFα - IFNγ - NK cell population following infection, suggesting both pathogenic and inflammatory changes in the NK cell functional profile. Our data also identified significant global differences in polyfunctionality between CD49a + NK cells in the naïve and chronic (SHIV+) cohorts. Our work provides the first characterization of CD49a + NK cells in tissues from RM. The significant similarities between CD49a + NK cells from RM and what is reported from human samples justifies the importance of studying CD49a + NK cells in this species to support preclinical animal model research., (Copyright © 2020 Ram, Arias, Kroll, Hueber, Manickam, Jones, Smith, Shah, Varner and Reeves.)

Recent functional, gene expression, and epigenetic studies have suggested the presence of a subset of mature natural killer (NK) cells responsible for maintaining NK cell memory. The lack of endogenous clonal markers in NK cells impedes understanding the genesis of these cell populations. In humans, primates, and mice, this phenotype and memory or adaptive functions have been strongly linked to cytomegalovirus or related herpes virus infections. We have used transplantation of lentivirally-barcoded autologous hematopoietic stem and progenitor cells (HSPC) to track clonal hematopoiesis in rhesus macaques and previously reported striking oligoclonal expansions of NK-biased barcoded clones within the CD56 - CD16 + NK cell subpopulation, clonally distinct from ongoing output of myeloid, B cell, T cell, and CD56 + 16 - NK cells from HSPC. These CD56 - CD16 + NK cell clones segregate by expression of specific KIR surface receptors, suggesting clonal expansion in reaction to specific environmental stimuli. We have now used this model to investigate the impact of rhesus CMV(RhCMV) infection on NK clonal dynamics. Following transplantation, RhCMV neg rhesus macaques display less dominant and oligoclonal CD16 + NK cells biased clones compared to RhCMV pos animals, however these populations of cells are still clearly present. Upon RhCMV infection, CD16 + NK cells proliferate, followed by appearance of new groups of expanded NK clones and disappearance of clones present prior to RhCMV infection. A second superinfection with RhCMV resulted in rapid viral clearance without major change in the mature NK cell clonal landscape. Our findings suggest that RhCMV is not the sole driver of clonal expansion and peripheral maintenance of mature NK cells; however, infection of macaques with this herpesvirus does result in selective expansion and persistence of specific NK cell clones, providing further information relevant to adaptive NK cells and the development of NK cell therapies., (Copyright © 2019 Truitt, Yang, Espinoza, Fan, Ram, Moström, Tran, Sprehe, Reeves, Donahue, Kaur, Dunbar and Wu.)

Despite efficient suppression of plasma viremia in people living with HIV (PLWH) on cART, evidence of HIV-induced immunosuppression remains, and normally benign and opportunistic pathogens become major sources of co-morbidities, including virus-induced cancers. In fact, cancer remains a primary cause of death even in virally suppressed PLWH. Natural killer (NK) cells provide rapid early responses to HIV infection, contribute substantially to disease modulation and vaccine protection, and are also major therapeutic targets for cancer immunotherapy. However, much like other lymphocyte populations, recent burgeoning evidence suggests that in chronic conditions like HIV, NK cells can become functionally exhausted with impaired cytotoxic function, altered cytokine production and impaired antibody-dependent cell-mediated cytotoxicity. Recent work suggests functional anergy is likely due to low-level ongoing virus replication, increased inflammatory cytokines, or increased presence of MHC low target cells. Indeed, HIV-induced loss of NK cell-mediated control of lytic EBV infection has been specifically shown to cause lymphoma and also increases replication of CMV. In this review, we will discuss current understanding of NK cell modulation of HIV disease, reciprocal exhaustion of NK cells, and how this may impact increased cancer incidences and prospects for NK cell-targeted immunotherapies. Finally, we will review the most recent evidence supporting adaptive functions of NK cells and highlight the potential of adaptive NK cells for cancer immunotherapy.

Natural killer (NK) cells are the major innate effectors primed to eliminate virus-infected and tumor or neoplastic cells. Recent studies also suggest nuances in phenotypic and functional characteristics among NK cell subsets may further permit execution of regulatory and adaptive roles. Animal models, particularly non-human primate (NHP) models, are critical for characterizing NK cell biology in disease and under homeostatic conditions. In HIV infection, NK cells mediate multiple antiviral functions via upregulation of activating receptors, inflammatory cytokine secretion, and antibody dependent cell cytotoxicity through antibody Fc-FcR interaction and others. However, HIV infection can also reciprocally modulate NK cells directly or indirectly, leading to impaired/ineffective NK cell responses. In this review, we will describe multiple aspects of NK cell biology in HIV/SIV infections and their association with viral control and disease progression, and how NHP models were critical in detailing each finding. Further, we will discuss the effect of NK cell depletion in SIV-infected NHP and the characteristics of newly described memory NK cells in NHP models and different mouse strains. Overall, we propose that the role of NK cells in controlling viral infections remains incompletely understood and that NHP models are indispensable in order to efficiently address these deficits.

It is now well-accepted that Fc-mediated effector functions, including antibody-dependent cellular cytotoxicity (ADCC), can contribute to vaccine-elicited protection as well as post-infection control of HIV viremia. This picture was derived using a wide array of ADCC assays, no two of which are strictly comparable, and none of which is qualified at the clinical laboratory level. An earlier comparative study of assay protocols showed that while data from different ADCC assay formats were often correlated, they remained distinct in terms of target cells and the epitopes and antigen(s) available for recognition by antibodies, the effector cells, and the readout of cytotoxicity. This initial study warrants expanded analyses of the relationships among all current assay formats to determine where they detect overlapping activities and where they do not. Here we summarize knowns and unknowns of assaying ADCC against HIV-1.

Viral infections trigger robust secretion of interferons and other antiviral cytokines by infected and bystander cells, which in turn can tune the immune response and may lead to viral clearance or immune suppression. However, aberrant or unrestricted cytokine responses can damage host tissues, leading to organ dysfunction, and even death. To understand the cytokine milieu and immune responses in infected host tissues, non-human primate (NHP) models have emerged as important tools. NHP have been used for decades to study human infections and have played significant roles in the development of vaccines, drug therapies and other immune treatment modalities, aided by an ability to control disease parameters, and unrestricted tissue access. In addition to the genetic and physiological similarities with humans, NHP have conserved immunologic properties with over 90% amino acid similarity for most cytokines. For example, human-like symptomology and acute respiratory syndrome is found in cynomolgus macaques infected with highly pathogenic avian influenza virus, antibody enhanced dengue disease is common in neotropical primates, and in NHP models of viral hepatitis cytokine-induced inflammation induces severe liver damage, fibrosis, and hepatocellular carcinoma recapitulates human disease. To regulate inflammation, anti-cytokine therapy studies in NHP are underway and will provide important insights for future human interventions. This review will provide a comprehensive outline of the cytokine-mediated exacerbation of disease and tissue damage in NHP models of viral infections and therapeutic strategies that can aid in prevention/treatment of the disease syndromes.

Combined antiretroviral therapies (cARTs) efficiently control HIV replication leading to undetectable viremia and drastic increases in lifespan of people living with HIV. However, cART does not cure HIV infection as virus persists in cellular and anatomical reservoirs, from which the virus generally rebounds soon after cART cessation. One major anatomical reservoir are lymph node (LN) follicles, where HIV persists through replication in follicular helper T cells and is also trapped by follicular dendritic cells. Natural hosts of SIV, such as African green monkeys and sooty mangabeys, generally do not progress to disease although displaying persistently high viremia. Strikingly, these hosts mount a strong control of viral replication in LN follicles shortly after peak viremia that lasts throughout infection. Herein, we discuss the potential interplay between viral control in LNs and the resolution of inflammation, which is characteristic for natural hosts. We furthermore detail the differences that exist between non-pathogenic SIV infection in natural hosts and pathogenic HIV/SIV infection in humans and macaques regarding virus target cells and replication dynamics in LNs. Several mechanisms have been proposed to be implicated in the strong control of viral replication in natural host's LNs, such as NK cell-mediated control, that will be reviewed here, together with lessons and limitations of in vivo cell depletion studies that have been performed in natural hosts. Finally, we discuss the impact that these insights on viral dynamics and host responses in LNs of natural hosts have for the development of strategies toward HIV cure.

Over the past several years, new populations of innate lymphocytes have been described in mice and primates that are critical for mucosal homeostasis, microbial regulation, and immune defense. Generally conserved from mice to humans, innate lymphoid cells (ILC) have been divided primarily into three subpopulations based on phenotypic and functional repertoires: ILC1 bear similarities to natural killer cells; ILC2 have overlapping functions with TH2 cells; and ILC3 that share many functions with TH17/TH22 cells. ILC are specifically enriched at mucosal surfaces and are possibly one of the earliest responders during viral infections besides being involved in the homeostasis of gut-associated lymphoid tissue and maintenance of gut epithelial barrier integrity. Burgeoning evidence also suggests that there is an early and sustained abrogation of ILC function and numbers during HIV and pathogenic SIV infections, most notably ILC3 in the gastrointestinal tract, which leads to disruption of the mucosal barrier and dysregulation of the local immune system. A better understanding of the direct or indirect mechanisms of loss and dysfunction will be critical to immunotherapeutics aimed at restoring these cells. Herein, we review the current literature on ILC with a particular emphasis on ILC3 and their role(s) in mucosal immunology and the significance of disrupting the ILC niche during HIV and SIV infections.

Human NK (hNK) cells play a key role in mediating host immune responses against various infectious diseases. For practical reasons, the majority of the data on hNK cells has been generated using peripheral blood lymphocytes. In contrast, our knowledge of NK cells in human tissues is limited, and not much is known about developmental pathways of hNK cell subpopulations in vivo. Although research in mice has elucidated a number of fundamental features of NK cell biology, mouse, and hNK cells significantly differ in their subpopulations, functions, and receptor repertoires. Thus, there is a need for a model that is more closely related to humans and yet allows experimental manipulations. Non-human primate models offer numerous opportunities for the study of NK cells, including the study of the role of NK cells after solid organ and stem cell transplantation, as well as in acute viral infection. Macaque NK cells can be depleted in vivo or adoptively transferred in an autologous system. All of these studies are either difficult or unethical to carry out in humans. Here we highlight recent advances in rhesus NK cell research and their parallels in humans. Using high-throughput transcriptional profiling, we demonstrate that the human CD56(bright) and CD56(dim) NK cell subsets have phenotypically and functionally analogous counterparts in rhesus macaques. Thus, the use of non-human primate models offers the potential to substantially advance hNK cell research.

Despite the fact that the majority of human pathogens are transmitted across mucosal surfaces, including the oral mucosae, oral immunity is poorly understood. Furthermore, because the normal flora of the oral cavity is vast and significantly diverse, host immunity must balance a complex system of tolerance and pathogen recognition. Due to the rapid recognition and response to pathogens, the innate immune system, including natural killer (NK) cells, likely plays a critical role in mediating this balance. Because logistical and ethical restraints limit access to significant quantities of human mucosal tissues, non-human primate models offer one of the best opportunities to study mucosal NK cells. In this study we have identified both classical NK cells, as well as innate lymphoid cells (ILCs) in tonsillar and buccal tissues and oral-draining lymph nodes. Identified by mutually exclusive expression of NKG2A and NKp44, NK cells, and ILCs in the oral mucosa are generally phenotypically and functionally analogous to their gut counterparts. NKG2A(+) NK cells were more cytotoxic while NKp44(+) ILCs produced copious amounts of IL-17 and TNF-α. However, in contrast to gut, oral NK cells and ILCs both produced large quantities of IFN-γ and the beta-chemokine, MIP-1β. Also in contrast to what we have previously found in gut tissues of SIV-infected macaques, we found no reduction in NK cells during chronic SIV infection, but rather an expansion of ILCs in oral-draining lymph nodes and tonsils. These data suggest that the lentivirus-induced depletion of the NK cell/ILC compartment in the gut may be absent in the oral mucosa, but the inherent differences and SIV-induced alterations are likely to have significant impact on preventing oral opportunistic infections in lentiviral disease. Furthermore, these data extend our understanding of the oral innate immune system in general and could aid future studies evaluating the regulation of both normal oral flora and limiting transmission of oral mucosal pathogens.