Multiplex interrogation of the NK cell signalome reveals global downregulation of CD16 signaling during lentivirus infection through an IL-18/ADAM17-dependent mechanism.

Despite their importance, natural killer (NK) cell responses are frequently dysfunctional during human immunodeficiency virus-1 (HIV-1) and simian immunodeficiency virus (SIV) infections, even irrespective of antiretroviral therapies, with poorly understood underlying mechanisms. NK cell surface receptor modulation in lentivirus infection has been extensively studied, but a deeper interrogation of complex cell signaling is mostly absent, largely due to the absence of any comprehensive NK cell signaling assay. To fill this knowledge gap, we developed a novel multiplex signaling analysis to broadly assess NK cell signaling. Using this assay, we elucidated that NK cells exhibit global signaling reduction from CD16 both in people living with HIV-1 (PLWH) and SIV-infected rhesus macaques. Intriguingly, antiretroviral treatment did not fully restore diminished CD16 signaling in NK cells from PLWH. As a putative mechanism, we demonstrated that NK cells increased surface ADAM17 expression via elevated plasma IL-18 levels during HIV-1 infection, which in turn reduced surface CD16 downregulation. We also illustrated that CD16 expression and signaling can be restored by ADAM17 perturbation. In summary, our multiplex NK cell signaling analysis delineated unique NK cell signaling perturbations specific to lentiviral infections, resulting in their dysfunction. Our analysis also provides mechanisms that will inform the restoration of dysregulated NK cell functions, offering potential insights for the development of new NK cell-based immunotherapeutics for HIV-1 disease.

Natural Killer Cells Regulate Acute SIV Replication, Dissemination, and Inflammation, but Do Not Impact Independent Transmission Events.

Natural killer (NK) cells are potent effector cells of the innate immune system possessing both cytotoxic and immunoregulatory capabilities, which contribute to their crucial role in controlling human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections. However, despite significant evidence for NK cell modulation of HIV disease, their specific contribution to transmission and control of acute infection remains less clear. To elucidate the contribution of NK cells during acute SIV infection, we performed an acute necropsy study, where rhesus macaques (RM) were subjected to preinfection depletion of systemic NK cells using established methods of IL-15 neutralization, followed by subsequent challenge with barcoded SIVmac239X. Our study showed that depletion was highly effective, resulting in near total ablation of all NK cell subsets in blood, liver, oral, and rectal mucosae, and lymph nodes (LN) that persisted through the duration of the study. Meanwhile, frequencies and phenotypes of T cells remained virtually unchanged, indicating that our method of NK cell depletion had minimal off-target effects. Importantly, NK cell-depleted RM demonstrated an early and sustained 1 to 2 log increase in viremia over controls, but sequence analysis suggested no difference in the number of independent transmission events. Acute bulk, central memory (CM), and CCR5+ CD4+ T cell depletion was similar between experimental and control groups, while CD8+ T cell activation was higher in NK cell-depleted RM as measured by Ki67 and PD-1 expression. Using 27-plex Luminex analyses, we also found modestly increased inflammatory cytokines in NK cell-depleted RM compared to control animals. In the effort to determine the impact of NK cells on HIV/SIV transmission and acute viremia, future studies will be necessary to better harness these cells for future viral therapies. Collectively, these data suggest NK cells are important modulators of lentivirus dissemination and disease but may not have the capacity to independently eliminate individual transmission events. IMPORTANCE Natural killer (NK) cells as major effector cells of the innate immune system can contribute significantly to human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) control. However, a specific role for NK cells in blocking lentivirus transmission remains incompletely clear. In this study, we depleted NK cells prior to challenge with a barcoded SIV. Importantly, our studied showed systemic NK cell depletion was associated with a significant increase in acute viremia, but did not impact the number of independent transmission events. Collectively, these data suggest NK cells are critical modulators of early lentivirus replication but may not regulate individual transmission events at mucosal portals of entry.

Systemic and mucosal mobilization of granulocyte subsets during lentiviral infection.

Granulocytes mediate broad immunoprotection through phagocytosis, extracellular traps, release of cytotoxic granules, antibody effector functions and recruitment of other immune cells against pathogens. However, descriptions of granulocytes in HIV infection and mucosal tissues are limited. Our goal was to characterize granulocyte subsets in systemic, mucosal and lymphoid tissues during lentiviral infection using the rhesus macaque (RM) model. Mononuclear cells from jejunum, colon, cervix, vagina, lymph nodes, spleen, liver and whole blood from experimentally naïve and chronically SHIVsf162p3-infected RM were analysed by microscopy and polychromatic flow cytometry. Granulocytes were identified using phenotypes designed specifically for RM: eosinophils-CD45+  CD66+  CD49d+ ; neutrophils-CD45+  CD66+  CD14+ ; and basophils-CD45+  CD123+  FcRε+ . Nuclear visualization with DAPI staining and surface marker images by ImageStream (cytometry/microscopy) further confirmed granulocytic phenotypes. Flow cytometric data showed that all RM granulocytes expressed CD32 (FcRγII) but did not express CD16 (FcRγIII). Additionally, constitutive expression of CD64 (FcRγI) on neutrophils and FcRε on basophils indicates the differential expression of Fc receptors on granulocyte subsets. Granulocytic subsets in naïve whole blood ranged from 25·4% to 81·5% neutrophils, 0·59% to 13·3% eosinophils and 0·059% to 1·8% basophils. Interestingly, elevated frequencies of circulating neutrophils, colorectal neutrophils and colorectal eosinophils were all observed in chronic lentiviral disease. Conversely, circulating basophils, jejunal eosinophils, vaginal neutrophils and vaginal eosinophils of SHIVsf162p3-infected RM declined in frequency. Overall, our data suggest modulation of granulocytes in chronic lentiviral infection, most notably in the gastrointestinal mucosae where a significant inflammation and disruption occurs in lentivirus-induced disease. Furthermore, granulocytes may migrate to inflamed tissues during infection and could serve as targets of immunotherapeutic intervention.

Simian Immunodeficiency Virus Infection Modulates CD94+ (KLRD1+) NK Cells in Rhesus Macaques.

Recently, we and others have shown that natural killer (NK) cells exhibit memory-like recall responses against cytomegalovirus (CMV) and human immunodeficiency/virus simian immunodeficiency virus (HIV/SIV) infections. Although the mechanism(s) have not been fully delineated, several groups have shown that the activating receptor NKG2C is elevated on NK cells in the context of rhesus CMV (rhCMV) or human CMV (hCMV) infections. CD94, which heterodimerizes with NKG2C is also linked to adaptive NK cell responses. Because nonhuman primates (NHP) play a crucial role in modeling HIV (SIV) infections, it is crucial to be able to assess and characterize the NKG2 family in NHP. Unfortunately, it is not possible to detect CD94 using commercially available antibodies in NHP. Our work, a first for NHP, has focused on developing RNA flow cytometry using mRNA transcripts as proxies distinguishing NKG2C from NKG2A. We have expanded the application of this technology and here we show the first characterization of CD94+ (KLRD1+) NK cells in NHP using multiparametric RNA flow cytometry. Peripheral blood mononuclear cells from naive and matched acutely (n = 4) or chronically (n = 12) SIV-infected rhesus macaques were analyzed by flow cytometry using commercially available antibodies, determining expression of transcripts for NKG2A, NKG2C, and CD94 (KLRC1, KLRC2, and KLRD1, respectively) on NK cells using RNA flow cytometry. Our data show that KLRC1+/- KLRC2+ KLRD1+ NK cells decrease following chronic, but not acute, infection with SIV. This approach will allow us to investigate the kinetics of infection and NK memory formation and will further improve our understanding of basic NK cell biology, especially in the context of SIV infection.IMPORTANCE Nonhuman primates play a crucial role in approximating human biology and many diseases that are difficult, if not impossible, to achieve in other animal models, notably HIV. Current advances in adaptive NK cell research positions us to address fundamental deficiencies in our fight against infection and disease at the earliest moments after infection or substantially earlier in disease progression. We show here that we can identify specific NK cell subpopulations that are modulated following chronic, but not acute, SIV infection. The ability to identify these subsets more precisely will inform therapeutic and vaccine strategies targeting an optimized NK cell response.

Tracking KLRC2 (NKG2C)+ memory-like NK cells in SIV+ and rhCMV+ rhesus macaques.

Natural killer (NK) cells classically typify the nonspecific effector arm of the innate immune system, but have recently been shown to possess memory-like properties against multiple viral infections, most notably CMV. Expression of the activating receptor NKG2C is elevated on human NK cells in response to infection with CMV as well as HIV, and may delineate cells with memory and memory-like functions. A better understanding of how NKG2C+ NK cells specifically respond to these pathogens could be significantly advanced using nonhuman primate (NHP) models but, to date, it has not been possible to distinguish NKG2C from its inhibitory counterpart, NKG2A, in NHP because of unfaithful antibody cross-reactivity. Using novel RNA-based flow cytometry, we identify for the first time true memory NKG2C+ NK cells in NHP by gene expression (KLRC2), and show that these cells have elevated frequencies and diversify their functional repertoire specifically in response to rhCMV and SIV infections.

Balance between short and long isoforms of cFLIP regulates Fas-mediated apoptosis in vivo.

cFLIP, an inhibitor of apoptosis, is a crucial regulator of cellular death by apoptosis and necroptosis; its importance in development is exemplified by the embryonic lethality in cFLIP-deficient animals. A homolog of caspase 8 (CASP8), cFLIP exists in two main isoforms: cFLIPL (long) and cFLIPR (short). Although both splice variants regulate death receptor (DR)-induced apoptosis by CASP8, the specific role of each isoform is poorly understood. Here, we report a previously unidentified model of resistance to Fas receptor-mediated liver failure in the wild-derived MSM strain, compared with susceptibility in C57BL/6 (B6) mice. Linkage analysis in F2 intercross (B6 x MSM) progeny identified several MSM loci controlling resistance to Fas-mediated death, including the caspase 8- and FADD-like apoptosis regulator (Cflar) locus encoding cFLIP. Furthermore, we identified a 21-bp insertion in the 3' UTR of the fifth exon of Cflar in MSM that influences differential splicing of cFLIP mRNA. Intriguingly, we observed that MSM liver cells predominantly express the FLIPL variant, in contrast to B6 liver cells, which have higher levels of cFLIPR. In keeping with this finding, genome-wide RNA sequencing revealed a relative abundance of FLIPL transcripts in MSM hepatocytes whereas B6 liver cells had significantly more FLIPR mRNA. Importantly, we show that, in the MSM liver, CASP8 is present exclusively as its cleaved p43 product, bound to cFLIPL. Because of partial enzymatic activity of the heterodimer, it might prevent necroptosis. On the other hand, it prevents cleavage of CASP8 to p10/20 necessary for cleavage of caspase 3 and, thus, apoptosis induction. Therefore, MSM hepatocytes are predisposed for protection from DR-mediated cell death.

Indirect activation of rhesus macaque (Macaca mulatta) NK cells in oral and mucosal draining lymph nodes.

The oral mucosae and draining lymph nodes are primary entry points for invading pathogens, particularly during immunosuppressive HIV/SIV infections. Innate immunity against oral stimuli, including natural killer (NK) cells, is understudied. Herein, we demonstrate functional NK cell responses to pathogen-associated molecular patterns (PAMPs) of potential oral pathogens in rhesus macaques.

The distribution of insertionally polymorphic endogenous retroviruses in breast cancer patients and cancer-free controls.

BACKGROUND: Integration of retroviral DNA into a germ cell can result in a provirus that is transmitted vertically to the host's offspring. In humans, such endogenous retroviruses (HERVs) comprise >8% of the genome. The HERV-K(HML-2) proviruses consist of ~90 elements related to mouse mammary tumor virus, which causes breast cancer in mice. A subset of HERV-K(HML-2) proviruses has some or all genes intact, and even encodes functional proteins, though a replication competent copy has yet to be observed. More than 10% of HML-2 proviruses are human-specific, having integrated subsequent to the Homo-Pan divergence, and, of these, 11 are currently known to be polymorphic in integration site with variable frequencies among individuals. Increased expression of the most recent HML-2 proviruses has been observed in tissues and cell lines from several types of cancer, including breast cancer, for which expression may provide a meaningful marker of the disease. RESULTS: In this study, we performed a case-control analysis to investigate the possible relationship between the genome-wide presence of individual polymorphic HML-2 proviruses with the occurrence of breast cancer. For this purpose, we screened 50 genomic DNA samples from individuals diagnosed with breast cancer or without history of the disease (n = 25 per group) utilizing a combination of locus-specific PCR screening, in silico analysis of HML-2 content within the reference human genome sequence, and high-resolution genomic hybridization in semi-dried agarose. By implementing this strategy, we were able to analyze the distribution of both annotated and previously undescribed polymorphic HML-2 proviruses within our sample set, and to assess their possible association with disease outcome. CONCLUSIONS: In a case-control analysis of 50 humans with regard to breast cancer diagnosis, we found no significant difference in the prevalence of proviruses between groups, suggesting common polymorphic HML-2 proviruses are not associated with breast cancer. Our findings indicate a higher level of putatively novel HML-2 sites within the population, providing support for additional recent insertion events, implying ongoing, yet rare, activities. These findings do not rule out either the possibility of involvement of such proviruses in a subset of breast cancers, or their possible utility as tissue-specific markers of disease.

Probiotic supplementation reduces inflammatory profiles but does not prevent oral immune perturbations during SIV infection.

HIV/SIV infections lead to massive loss of mucosal CD4 + T cells and breakdown of the epithelial mucosa resulting in severe microbial dysbiosis and chronic immune activation that ultimately drive disease progression. Moreover, disruption of one of the most understudied mucosal environments, the oral cavity, during HIV-induced immunosuppression results in significant microbial and neoplastic co-morbidities and contributes to and predicts distal disease complications. In this study we evaluated the effects of oral probiotic supplementation (PBX), which can stimulate and augment inflammatory or anti-inflammatory pathways, on early SIV infection of rhesus macaques. Our study revealed that similar to the GI mucosae, oral CD4 + T cells were rapidly depleted, and as one of the first comprehensive analyses of the oral microflora in SIV infection, we also observed significant modulation among two genera, Porphyromonas and Actinobacillus, early after infection. Interestingly, although PBX therapy did not substantially protect against oral dysbiosis or ameliorate cell loss, it did somewhat dampen inflammation and T cell activation. Collectively, these data provide one of the most comprehensive evaluations of SIV-induced changes in oral microbiome and CD4 + T cell populations, and also suggest that oral PBX may have some anti-inflammatory properties in lentivirus infections.

Skipped Over: Tuning Natural Killer Cells Toward HIV Through Alternative Splicing.

Natural killer (NK) cells provide some of the earliest immune responses to infection, but when viruses manipulate or perturb the immune environment to alter NK cell function, this places the host at a disadvantage. Indeed, others and we observe that in the context of HIV/simian immunodeficiency virus (SIV) infection, although NK cells are not infected, they can become dysfunctional over time. Several studies have characterized protein and transcriptional profiles of NK cells during HIV/SIV infection, but none have examined whether the production of alternative transcripts and corresponding isoforms is modulated. This phenomenon occurs broadly in normal biology and in other disease states, and could provide a novel avenue of investigation that may yield better targets to restore or augment NK cell responses to HIV/SIV. Herein, we briefly summarize published and new data that may provide a perspective on how to target NK cell splice variants.

Delineation and Modulation of the Natural Killer Cell Transcriptome in Rhesus Macaques During ZIKV and SIV Infections.

Natural killer (NK) cells are crucial regulators of antiviral and anti-tumor immune responses. Although in humans some NK cell transcriptional programs are relatively well-established, NK cell transcriptional networks in non-human primates (NHP) remain poorly delineated. Here we performed RNA-Seq experiments using purified NK cells from experimentally naïve rhesus macaques, providing the first transcriptional characterization of pure NK cells in any NHP species. This novel NK cell transcriptomic signature (NK RMtsig) overlaps with published human NK signatures, allowing us to identify new key signaling and transcription factor networks underlying NK cell function. Finally, we show that applying NK RMtsig to an unrelated rhesus macaque cohort infected with SIVmac251 or ZIKV can sensitively detect NK cell repertoire perturbations, thus confirming applicability of this approach. In sum, we propose this NHP NK cell signature will serve as a useful resource for future studies involving infection, disease or treatment modalities in NHP.

Characterization of Rhesus Macaque Liver-Resident CD49a+ NK Cells During Retrovirus Infections.

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 Eomeslow T-betlow, 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.

Identification of a secreted fatty acid and retinol-binding protein (Hp-FAR-1) from Heligmosomoides polygyrus.

Hp-FAR-1 is a major, secreted antigen of the parasitic nematode Heligmosomoides polygyrus, a laboratory mouse model frequently used to study the cellular mechanisms of chronic helminth infections. The DNA encoding Hp-FAR-1 was recovered by screening a fourth larval (L4) H. polygyrus cDNA expression library using antibodies raised against L4 stage excretory/secretory (E/S) proteins. Predictions of secondary structure based on the Hp-FAR-1 amino acid sequence indicated that an alpha-helix predominates in Hp-FAR-1, possibly with some coiled-coil conformation, with no beta-structure. Fluorescence-based ligand binding analysis confirmed that the recombinant Hp-FAR-1 (rHp-FAR-1) binds the fluorescent fatty acid analog 11-((5-[dimethylaminoaphthalene-1-sulfonyl)amino)undecanoic acid (DAUDA), and by competition oleic acid. RT-PCR amplification of the hp-far-1 gene indicated that the gene is transcribed in all parasitic stages of the organism's life cycle. The presence of a secreted FAR protein in the well-defined laboratory model of H. polygyrus provides an excellent model for the further study and analysis of the in vivo role of secreted FAR proteins in parasitism, and supports the mounting evidence that secreted FAR proteins play a major role in nematode parasitism.

Adaptive NK cell responses in HIV/SIV infections: A roadmap to cell-based therapeutics?

NK cells play a critical role in antiviral and antitumor responses. Although current NK cell immune therapies have focused primarily on cancer biology, many of these advances can be readily applied to target HIV/simian immunodeficiency virus (SIV)-infected cells. Promising developments include recent reports that CAR NK cells are capable of targeted responses while producing less off-target and toxic side effects than are associated with CAR T cell therapies. Further, CAR NK cells derived from inducible pluripotent stem cells or cell lines may allow for more rapid "off-the-shelf" access. Other work investigating the IL-15 superagonist ALT-803 (now N803) may also provide a recourse for enhancing NK cell responses in the context of the immunosuppressive and inflammatory environment of chronic HIV/SIV infections, leading to enhanced control of viremia. With a broader acceptance of research supporting adaptive functions in NK cells it is likely that novel immunotherapeutics and vaccine modalities will aim to generate virus-specific memory NK cells. In doing so, better targeted NK cell responses against virus-infected cells may usher in a new era of NK cell-tuned immune therapy.

Impact of CMV Infection on Natural Killer Cell Clonal Repertoire in CMV-Naïve Rhesus Macaques.

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, RhCMVneg rhesus macaques display less dominant and oligoclonal CD16+ NK cells biased clones compared to RhCMVpos 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.

Cytokine-Mediated Tissue Injury in Non-human Primate Models of Viral Infections.

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.

CMV Primes Functional Alternative Signaling in Adaptive Δg NK Cells but Is Subverted by Lentivirus Infection in Rhesus Macaques.

Despite burgeoning evidence demonstrating the adaptive properties of natural killer (NK) cells, mechanistic data explaining these phenomena are lacking. Following antibody sensitization, NK cells lacking the Fc receptor (FcR) signaling chain (Δg) acquire adaptive features, including robust proliferation, multifunctionality, rapid killing, and mobilization to sites of virus exposure. Using the rhesus macaque model, we demonstrate the systemic distribution of Δg NK cells expressing memory features, including downregulated Helios and Eomes. Furthermore, we find that Δg NK cells abandon typical γ-chain/Syk in lieu of CD3ζ-Zap70 signaling. FCγRIIIa (CD16) density, mucosal homing, and function are all coupled to this alternate signaling, which in itself requires priming by rhesus cytomegalovirus (rhCMV). Simian immunodeficiency virus (SIV) infections further expand gut-homing adaptive NK cells but result in pathogenic suppression of CD3ζ-Zap70 signaling and function. Herein, we provide a mechanism of virus-dependent alternative signaling that may explain the acquisition of adaptive features by primate NK cells and could be targeted for future vaccine or curative therapies.

Progressive lentivirus infection induces natural killer cell receptor-expressing B cells in the gastrointestinal tract.

OBJECTIVE: Recently, a seemingly novel innate immune cell subset bearing features of natural killer and B cells was identified in mice. So-called NKB cells appear as first responders to infections, but whether this cell population is truly novel or is in fact a subpopulation of B cells and exists in higher primates remains unclear. The objective of this study was to identify NKB cells in primates and study the impact of HIV/SIV infections. DESIGN AND METHODS: NKB cells were quantified in both naive and lentivirus infected rhesus macaques and humans by excluding lineage markers (CD3, CD127) and positive Boolean gating for CD20, NKG2A/C and/or NKp46. Additional phenotypic measures were conducted by RNA-probe and traditional flow cytometry. RESULTS: Circulating cytotoxic NKB cells were found at similar frequencies in humans and rhesus macaques (range, 0.01-0.2% of total lymphocytes). NKB cells were notably enriched in spleen (median, 0.4% of lymphocytes), but were otherwise systemically distributed in tonsil, lymph nodes, colon, and jejunum. Expression of immunoglobulin was highly variable, but heavily favoured IgM and IgA rather than IgG. Interestingly, NKB cell frequencies expanded in PBMC and colon during SIV infection, as did IgG expression, but were generally unaltered in HIV-infected humans. CONCLUSION: These results suggest a cell type expressing both natural killer and B-cell features exists in rhesus macaques and humans and are perturbed by HIV/SIV infection. The full functional niche remains unknown, but the unique phenotype and systemic distribution could make NKB cells unique targets for immunotherapeutics or vaccine strategies.

Innate Lymphoid Cells in HIV/SIV Infections.

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.