Antigen-specific memory NK cell responses against HIV and influenza use the NKG2/HLA-E axis.

Multiple studies have broadened the roles of natural killer (NK) cells functioning as purely innate lymphocytes by demonstrating that they are capable of putative antigen-specific immunological memory against multiple infectious agents including HIV-1 and influenza. However, the mechanisms underlying antigen specificity remain unknown. Here, we demonstrate that antigen-specific human NK cell memory develops upon exposure to both HIV and influenza, unified by a conserved and epitope-specific targetable mechanism largely dependent on the activating CD94/NKG2C receptor and its ligand HLA-E. We validated the permanent acquisition of antigen specificity by individual memory NK cells by single-cell cloning. We identified elevated expression of KLRG1, α4ß7, and NKG2C as biomarkers of antigen-specific NK cell memory through complex immunophenotyping. Last, we uncovered individual HLA-E-restricted peptides that may constitute the dominant NK cell response in HIV-1- and influenza-infected persons in vivo. Our findings clarify the mechanisms contributing to antigen-specific memory NK cell responses and suggest that they could be potentially targeted therapeutically for vaccines or other therapeutic interventions.

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.

Increased IL-6 expression precedes reliable viral detection in the rhesus macaque brain during acute SIV infection.

Knowledge of immune activation in the brain during acute HIV infection is crucial for the prevention and treatment of HIV-associated neurological disorders. We determined regional brain (basal ganglia, thalamus, and frontal cortex) immune and virological profiles at 7 and 14 days post infection (dpi) with SIVmac239 in rhesus macaques. The basal ganglia and thalamus had detectable viruses earlier (7 dpi) than the frontal cortex (14 dpi) and contained higher quantities of viruses than the latter. Increased immune activation of astrocytes and significant infiltration of macrophages in the thalamus at 14 dpi coincided with elevated plasma viral load, and SIV colocalized only within macrophages. RNA signatures of proinflammatory responses, including IL-6, were detected at 7 dpi in microglia and interestingly, preceded reliable detection of virus in tissues and were maintained in the chronically infected macaques. Countering the proinflammatory response, the antiinflammatory response was not detected until increased TGF-ß expression was found in perivascular macrophages at 14 dpi. But this response was not detected in chronic infection. Our data provide evidence that the interplay of acute proinflammatory and antiinflammatory responses in the brain likely contributed to the overt neuroinflammation, where the immune activation preceded reliable viral detection.

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.

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.

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.

Functional Perturbation of Mucosal Group 3 Innate Lymphoid and Natural Killer Cells in Simian-Human Immunodeficiency Virus/Simian Immunodeficiency Virus-Infected Infant Rhesus Macaques.

Mother-to-child transmission of human immunodeficiency virus type 1 (HIV-1) via breastfeeding is responsible for nearly half of new infections of children with HIV. Although innate lymphoid cells (ILC) and natural killer (NK) cells are found throughout the oral mucosae, the effects of HIV/simian-human immunodeficiency virus (SHIV) in these tissues are largely unknown. To better understand the mechanics of postnatal transmission, we performed a comprehensive study of simian immunodeficiency virus (SIV)/SHIV-infected infant rhesus macaques (RM) and tracked changes in frequency, trafficking, and function of group 3 ILC (ILC3) and NK cells using polychromatic flow cytometry and cell stimulation assays in colon, tonsil, and oral lymph node samples. Infection led to a 3-fold depletion of ILC3 in the colon and an increase in the levels of NK cells in tonsils and oral lymph nodes. ILC3 and NK cells exhibited alterations in their trafficking repertoires as a result of infection, with increased expression of CD103 in colon NK cells and curtailment of CXCR3, and a significant decrease in α4ß7 expression in colon ILC3. SPICE analyses revealed that ILC3 and NK cells displayed distinct functional profiles by tissue in naive samples. Infection perturbed these profiles, with a nearly total loss of interleukin-22 (IL-22) production in the tonsil and colon; an increase in the levels of CD107a, gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α) from ILC3; and an increase in the levels of CD107a, macrophage inflammatory protein 1 beta (MIP-1ß), and TNF-α from NK cells. Collectively, these data reveal that lentivirus infection alters the frequencies, receptor repertoires, and functions of innate cells in the oral and gut mucosa of infants. Further study will be required to delineate the full extent of the effect that these changes have on oral and gut homeostasis, SHIV/SIV pathogenesis, and oral opportunistic disease.IMPORTANCE Vertical transmission of HIV from mother to child accounts for many of the new cases seen worldwide. There is currently no vaccine to mitigate this transmission, and there has been limited research on the effects that lentiviral infection has on the innate immune system in oral tissues of infected children. To fill this knowledge gap, our laboratory studied infant rhesus macaques to evaluate how acute SIV/SHIV infections impacted ILC3 and NK cells, which are immune cells critical for mucosal homeostasis and antimicrobial defense. Our data revealed that SIV/SHIV infection led to a depletion of ILC3 and an increase of NK cells and to a functional shift from a homeostatic to a multifunctional proinflammatory state. Taking the results together, we describe how lentiviral infection perturbs the oral and gastrointestinal mucosae of infant macaques through alterations of resident innate immune cells giving rise to chronic inflammation and potentially exacerbating morbidity and mortality in children living with HIV.

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.

Hepatic immunopathology during occult hepacivirus re-infection.

Despite drug advances for Hepatitis C virus (HCV), re-infections remain prevalent in high-risk populations. Unfortunately, the role of preexisting viral immunity and how it modulates re-infection is unclear. GBV-B infection of common marmosets is a useful model to study tissue immune responses in hepacivirus infections, and in this study we re-challenged 4 animals after clearance of primary viremia. Although only low-to-absent viremia was observed following re-challenge, GBV-B viral RNA was detectable in liver, confirming re-infection. Microscopic hepatic lesions indicated severe-to-mild lymphocyte infiltration and fibrosis in 3 out of 4 animals. Further, GBV-B-specific T cells were elevated in animals with moderate-to-severe hepatopathology, and up to 3-fold increases in myeloid dendritic and activated natural killer cells were observed after infection. Our data indicate that occult hepacivirus re-infections occur and that new liver pathology is possible even in the presence of anti-hepacivirus T cells and in the absence of high viremia.