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By virtue of their direct cytotoxicity to transformed and virus infected cells, Natural Killer (NK) cells play crucial roles in immunity. NK cells modulate and coordinate innate and adaptive responses through the release of chemokines and cytokines. Although NK cells are endowed only with germ-line encoded receptors, evidence has been accumulating, that subsets of NK cells can bestow adoptively transferable, long-lasting and antigen-specific immune responses to certain haptens and viruses. Growing evidence suggests that adaptive immune responses lie on a spectrum. Rechallenge of cells, canonically belonging to the innate immune system, can result in enhanced responsiveness - a process termed 'trained immunity' and thought to be maintained by epigenetic and metabolic reprogramming. In previous work, our lab studied the role of NK cell responses to rhesus cytomegalovirus (rhCMV) in a genetic barcoding model. We found that new clones arose in the CD16 + NK compartment after primary rhCMV infection. There was rapid clearance without the emergence of new clones in subsequent rechallenge with rhCMV. In this study we used 3'-end single cell RNA-seq (3'-scRNA-seq) with CITE-seq to profile NK and T cells from an initially CMV-naïve rhesus macaque (RM) at four time points before and after primary and secondary infections with rhCMV. We immunophenotypically sorted NK and T cells from peripheral Blood (PB) samples at 'baseline', 30 days after initial rhCMV infection ('primary infection'), ca. 500 days after initial rhCMV infection ('steady state') and 10 days after rmCMV reinfection ('secondary infection'). Alongside the PB samples at 'steady state' and after 'secondary infection', we also sorted NK and T cells from lymph nodes (LN). We applied CD16 and CD56 CITE-seq antibodies to NK cells from all samples; NK cells from the 'steady state' and 'secondary infection' samples were also labeled with CX3CL1 CITE-seq antibodies. We multiplexed NK and T cells from each time point in 4:1 ratios before preparing 3'-scRNA-seq libraries. We used scanpy and scvi-tools as well as custom python code to demultiplex NK from T cells, harmonize 3'-scRNA-seq with CITE-seq data and integrate the 6 different samples. We used scvelo and cellrank to compute RNA velocities and infer trajectories, respectively. We obtained a total of 35,523 high-quality cells. We identified 20 clusters of NK and T cells, on the basis of community detection via the Leiden algorithm. All clusters contained cells from both tissue sources. The 4 clusters characterized by expression of CD56 exhibit higher expression of KLRC1 (protein: NKG2A), IL7R and the transcription factors LEF1 and MYC. The 8 clusters of CD16 + cells are distinguished by high expression of the transcription factors ZEB2 and TBX21/T-BET, cytotoxicity markers, GZMB and PRF1, and activating receptors, KLRC2 (protein: NKG2C), KLRC3 (protein: NKG2E) and NCR3 (protein: NKp30). An adaptive population of NK cells is identified on the basis of high KLRC2 and low FCER1G expression. We analyzed changes in the proportions of cells in each cluster of the time course of CMV infection using a binomial generalized linear model. Clusters associated with proliferation and acute inflammation were increased in proportion after primary rhCMV infection; the proportion of the adaptive population did not significantly change during the acute phase of primary infection but increased markedly by the later 'steady state' samples. RNA velocity and inferred developmental trajectories suggest transitions between the adaptive, proliferating CD16 + and mature effector subsets; the predominant path into the adaptive population occurring from the proliferating CD16 + subset after primary infection. There is a notable paucity of inferred transitions between the CD56 + and CD16 +subpopulations under all the experimental conditions we observed. We have characterized the single cell transcriptional states and dynamics of RM NK cells in response to rhCMV infection. We focus on a subset transcriptionally resembling a previously identified subset with adaptive function and find it arises from a proliferating population of effector cells after primary infection. This may be analogous to the dedifferentiation of effector CD8 T cells into memory T cells proposed by Youngblood et al. Confirmatory experiments to analyze the reconstitution of the CD16 + compartment after treatment with a depleting antibody are on-going. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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.

Apoptotic membrane microparticles (MMPs) derived from dying cells of multiple cell origins are highly immunostimulatory and are indicative of global immune activation and cell death in a variety of diseases. In this study, we developed a flow cytometric bead assay to quantify annexin-V(+) apoptotic (MMPs) in plasma from humans and rhesus macaques. With a combination of flow cytometry and pan-fluorescent beads, MMPs were enumerated in plasma specimens by adding a constant ratio of beads to initial fluid volumes and then calculating MMP/mL based on MMP-to-bead ratios. Using this straightforward assay, we found that circulating MMP quantifications were highly reproducible and similar in number between normal rhesus macaques and humans subjects. However, MMPs increased two- to threefold during HIV and simian immunodeficiency virus (SIV) infections and were positively associated with T cell immune activation. Collectively, we present a rapid bead-based assay for both humans and macaque models to quantify MMPs that could be an instigator and predictor of immune activation, which is a primary source of HIV/SIV disease.

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.

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.

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 MHClow 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.

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.

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.

Despite its importance in shaping adaptive immune responses, viral clearance, and immune-based inflammation, tissue-specific innate immunity remains poorly characterized for hepatitis C virus (HCV) infection due to the lack of access to acutely infected tissues. In this study, we evaluated the impact of natural killer (NK) cells and myeloid (mDCs) and plasmacytoid (pDCs) dendritic cells on control of virus replication and virus-induced pathology caused by another, more rapidly resolving hepacivirus, GB virus B (GBV-B), in infections of common marmosets. High plasma and liver viral loads and robust hepatitis characterized acute GBV-B infection, and while viremia was generally cleared by 2 to 3 months postinfection, hepatitis and liver fibrosis persisted after clearance. Coinciding with peak viral loads and liver pathology, the levels of NK cells, mDCs, and pDCs in the liver increased up to 3-fold. Although no obvious numerical changes in peripheral innate cells occurred, circulating NK cells exhibited increased perforin and Ki67 expression levels and increased surface expression of CXCR3. These data suggested that increased NK cell arming and proliferation as well as tissue trafficking may be associated with influx into the liver during acute infection. Indeed, NK cell frequencies in the liver positively correlated with plasma ( R = 0.698; P = 0.015) and liver ( R = 0.567; P = 0.057) viral loads. Finally, soluble factors associated with NK cells and DCs, including gamma interferon (IFN-γ) and RANTES, were increased in acute infection and also were associated with viral loads and hepatitis. Collectively, the findings showed that mobilization of local and circulating innate immune responses was linked to acute virus-induced hepatitis, and potentially to resolution of GBV-B infection, and our results may provide insight into similar mechanisms in HCV infection. IMPORTANCE Hepatitis C virus (HCV) infection has created a global health crisis, and despite new effective antivirals, it is still a leading cause of liver disease and death worldwide. Recent evidence suggests that innate immunity may be a potential therapeutic target for HCV, but it may also be a correlate of increased disease. Due to a lack of access to human tissues with acute HCV infection, in this study we evaluated the role of innate immunity in resolving infection with a hepacivirus, GBV-B, in common marmosets. Collectively, our data suggest that NK cell and DC mobilization in acute hepacivirus infection can dampen virus replication but also regulate acute and chronic liver damage. How these two opposing effects on the host may be modulated in future therapeutic and vaccine approaches warrants further study.

Given the critical role of mucosal surfaces in susceptibility to infection, it is imperative that effective mucosal responses are induced when developing efficacious vaccines and prevention strategies for infection. Modulating the microbiota in the gastrointestinal (GI) tract through the use of probiotics (PBio) is a safe and well-tolerated approach to enhance mucosal and overall health. We assessed the longitudinal impact of daily treatment with the VSL#3 probiotic on cellular and humoral immunity and inflammation in healthy macaques. PBio therapy resulted in significantly increased frequencies of B cells expressing IgA in the colon and lymph node (LN), likely because of significantly increased LN T follicular helper cell frequencies and LN follicles. Increased frequencies of IL-23+ APCs in the colon were found post-PBio treatment, which correlated with LN T follicular helper cells. Finally, VSL#3 significantly downmodulated the response of TLR2-, TLR3-, TLR4-, and TLR9-expressing HEK293 cells to stimulation with Pam3CSK4, polyinosinic-polycytidylic acid, LPS, and ODN2006, respectively. These data provide a mechanism for the beneficial impact of PBio on mucosal health and implicates the use of PBio therapy in the context of vaccination or preventative approaches to enhance protection from mucosal infection by improving immune defenses at the mucosal portal of entry.

Novel influenza vaccine approaches to achieve cross-protection against a broad range of antigenically distinct strains are needed. Beyond their ability to eliminate infected cells without the need for prior sensitization, natural killer (NK) cells have been shown to mediate virus-specific recall responses. Notably, exposure to influenza antigens induces protective influenza-specific memory NK cells in mice, but evidence of influenza-specific memory NK cells in humans is still lacking. Using intracellular cytokine staining, we found that NK cells from 45% of adults can robustly respond to nucleoprotein (NP) from at least 2 heterosubtypic influenza strains completely independent of T cell help. To further determine if a subset of NK cells can specifically react to NP peptide pools from distinct strains, we clonally expanded individual NK cells (NKCL) and measured H1N1, H2N2 and H3N2 NP-specific killing by 33 NKCL from 6 donors. 63% of NKCL showed positive responses against the 2009 H1N1 pandemic strain (2%–57% specific lysis). Strikingly, half of H1N1 NP-specific NKCL also displayed cytotoxicity against either H2N2 or H3N2 (9%–100% specific lysis) or against both H2N2 and H3N2 strains (5%–11% specific lysis). Potent killing by NKCLs was associated with high cell surface expression of the activating NKG2C receptor, a ligand for HLA-E. Accordingly, we identified an HLA-E-restricted NP epitope that triggers activation of influenza-specific NK cells. Collectively, these data provide the first mechanistic evidence of influenza-specific memory NK cells in humans and suggest influenza-specific responses mediated by NK cells may have the potential to be harnessed to enhance influenza vaccine-induced cross-protection.

Granulocytes mediate immune protection by phagocytosis, NETs, degranulation, antibody mediated effector functions and recruitment of innate and adaptive cells against pathogens. However, limited research describing granulocytes in HIV infection and in mucosal tissues exists. In particular, the role of granulocytes in the rhesus macaque (RM) model of HIV infection and vaccine/treatment studies have been understudied. Our goal was to characterize granulocyte subsets in systemic, mucosal and lymphoid tissues of naïve and infected RM. Mononuclear cells from jejunum, colon, cervix, vagina, lymph nodes, spleen, liver, and whole blood of naïve, SIV+, and SHIV+ RM were characterized using flow cytometry and Imagestream. Phenotype of granulocyte subsets were identified as- CD45+CD66+CD49d+ eosinophils, CD45+CD66+CD14+ neutrophils, and CD45+CD123+FcRE+ basophils. Polynuclear and mononuclear visualization with DAPI staining and surface marker images by Imagestream analysis further confirmed granulocytic phenotypes. Flow cytometry data showed that differential expression of FcRgs (CD64, CD32 and CD16) can be helpful in distinguishing granulocyte subsets while FcRE was only expressed by basophils. Interestingly, frequencies of granulocyte populations were generally higher in mucosal compared to lymphoid tissues in both naïve and infected animals. Eosinophils were particularly enriched within jejunal tissue, with a median of 45% of CD45+ cells. High frequencies of granulocytes in mucosal tissues indicate their importance in gut and reproductive tract immunity. Overall, further investigation is warranted to understand the significance of granulocytes in tissue homeostasis and infection.

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.

Natural killer (NK) cells are primary immune effector cells with both innate and potentially adaptive functions against viral infections, but commonly become exhausted or dysfunctional during chronic diseases such as human immunodeficiency virus (HIV). Chimpanzees are the closest genetic relatives of humans and have been previously used in immunology, behavior and disease models. Due to their similarities to humans, a better understanding of chimpanzee immunology, particularly innate immune cells, can lend insight into the evolution of human immunology, as well as response to disease. However, the phenotype of NK cells has been poorly defined. In order to define NK cell phenotypes, we unbiasedly quantified NK cell markers among mononuclear cells in both naive and HIV-infected chimpanzees by flow cytometry. We identified NKG2D and NKp46 as the most dominant stable NK cells markers using multidimensional data reduction analyses. Other traditional NK cell markers such as CD8α, CD16 and perforin fluctuated during infection, while some such as CD56, NKG2A and NKp30 were generally unaltered by HIV infection, but did not delineate the full NK cell repertoire. Taken together, these data indicate that phenotypic dysregulation may not be pronounced during HIV infection of chimpanzees, but traditional NK cell phenotyping used for both humans and other non-human primate species may need to be revised to accurately identify chimpanzee NK cells.

HIV and pathogenic SIV infection are characterized by mucosal dysfunction including epithelial barrier damage, loss of Th17 cells, neutrophil infiltration, and microbial translocation with accompanying inflammation. However, it is unclear how and when these contributing factors occur relative to one another. In order to determine whether any of these features initiates the cycle of damage, we longitudinally evaluated the kinetics of mucosal and systemic T-cell activation, microbial translocation, and Th17 cell and neutrophil frequencies following intrarectal SIV infection of rhesus macaques. We additionally assessed the colon proteome to elucidate molecular pathways altered early after infection. We demonstrate increased T-cell activation (HLA-DR+) beginning 3–14 days post-SIV challenge, reduced peripheral zonulin 3–14 days post-SIV, and evidence of microbial translocation 14 days post-SIV. The onset of mucosal dysfunction preceded peripheral and mucosal Th17 depletion, which occurred 14–28 days post-SIV, and gut neutrophil accumulation was not observed. Proteins involved in epithelial structure were downregulated 3 days post-SIV followed by an upregulation of immune proteins 14 days post-SIV. These data demonstrate that immune perturbations such as Th17 loss and neutrophil infiltration occur after alterations to epithelial structural protein pathways, suggesting that epithelial damage occurs prior to widespread immune dysfunction.

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.

Natural killer (NK) cells respond rapidly as a first line of defense against infectious pathogens. In addition, NK cells may provide a “rheostat” function and have been shown to reduce the magnitude of antigen-specific T cell responses following infection to avoid immunopathology. However, it remains unknown whether NK cells similarly modulate vaccine-elicited T cell responses following virus challenge. We used the lymphocytic choriomeningitis virus (LCMV) clone 13 infection model to address whether NK cells regulate T cell responses in adenovirus vector-vaccinated mice following challenge. As expected, NK cell depletion in unvaccinated mice resulted in increased virus-specific CD4 + and CD8 + T cell responses and immunopathology following LCMV challenge. In contrast, NK cell depletion had minimal to no impact on antigen-specific T cell responses in mice that were vaccinated with an adenovirus serotype 5 (Ad5)-GP vector prior to LCMV challenge. Moreover, NK cell depletion in vaccinated mice prior to challenge did not result in immunopathology and did not compromise protective efficacy. These data suggest that adenovirus vaccine-elicited T cells may be less sensitive to NK cell rheostat regulation than T cells primed by LCMV infection. IMPORTANCE Recent data have shown that NK cell depletion leads to enhanced virus-elicited T cell responses that can result in severe immunopathology following LCMV infection in mice. In this study, we observed that NK cells exerted minimal to no impact on vaccine-elicited T cells following LCMV challenge, suggesting that adenovirus vaccine-elicited T cells may be less subject to NK cell regulation. These data contribute to our understanding of NK cell regulatory functions and T cell-based vaccines.

Collectively, viral hepatitis remains a global epidemic causing an estimated 1.4 million deaths per year, of which hepatitis C virus (HCV) is the leading cause (1). Despite the availability of new antivirals that are capable of effective cure, the number of HCV infections and re-infections continue to rise worldwide. In addition to progressive liver disease and hepatocellular carcinoma (HCC), HCV is associated with metabolic disorders and co-morbidities including obesity, insulin resistance, type 2 diabetes mellitus, cardiovascular disease, mixed cryoglobulinemia among others (2-4)—all adding to the morbidity and mortality associated with this disease. HCV continues to be a challenge to control, due, at least in part, to the lack of a tangible animal model. Chimpanzees are susceptible to HCV and played a major role in understanding the natural history of the disease. But with limitations on the use of chimpanzees in biomedical research and an attenuated disease course, a tangible animal model could provide information on several gaps in HCV knowledge such as viral pathogenesis and persistence, immune correlates of protection, and importantly vaccine development and testing.

Disruption of the mucosal epithelium during lentivirus infections permits translocation of microbial products into circulation, causing immune activation and driving disease. Although the liver directly filters blood from the intestine and is the first line of defense against gut-derived antigens, the effects of microbial products on the liver are unclear. In livers of normal macaques, minute levels of bacterial products were detectable, but increased 20-fold in simian immunodeficiency virus (SIV)–infected animals. Increased microbial products in the liver induced production of the chemoattractant CXCL16 by myeloid dendritic cells (mDCs), causing subsequent recruitment of hypercytotoxic natural killer (NK) cells expressing the CXCL16 receptor, CXCR6. Microbial accumulation, mDC activation, and cytotoxic NK cell frequencies were significantly correlated with markers of liver damage, and SIV-infected animals consistently had evidence of hepatitis and fibrosis. Collectively, these data indicate that SIV-associated accumulation of microbial products in the liver initiates a cascade of innate immune activation, resulting in liver damage.

Classically, natural killer (NK) cells have been defined by nonspecific innate killing of virus-infected and tumor cells. However, burgeoning evidence suggests that the functional repertoire of NK cells is far more diverse than has been previously appreciated, thus raising the possibility that there may be unexpected functional specialization and even adaptive capabilities among NK cell subpopulations. Some of the first evidence that NK cells respond in an antigen-specific fashion came from experiments revealing that subpopulations of murine NK cells were able to respond to a specific murine cytomegalovirus (MCMV) protein and that in the absence of T and B cells, murine NK cells also mediated adaptive immune responses to a secondary challenge with specific haptens. These data have been followed by demonstrations of NK cell memory of viruses and viral antigens in mice and primates. Herein, we discuss different forms of NK cell antigen specificity and how these responses may be tuned to specific viral pathogens, and we provide assessment of the current literature that may explain molecular mechanisms of the novel phenomenon of NK cell memory.

International audience; Natural killer (NK) cells play an essential role in antiviral immunity, but knowledge of their function in secondary lymphoid organs is incomplete. Lymph node follicles constitute a major viral reservoir during infections with HIV-1 and simian immunodeficiency virus of macaques (SIVmac). In contrast, during nonpathogenic infection with SIV from African green monkeys (SIVagm), follicles remain generally virus free. We show that NK cells in secondary lymphoid organs from chronically SIVagm-infected African green monkeys (AGMs) were frequently CXCR5+ and entered and persisted in lymph node follicles throughout the follow-up (240 d post-infection). These follicles were strongly positive for IL-15, which was primarily presented in its membrane-bound form by follicular dendritic cells. NK cell depletion through treatment with anti-IL-15 monoclonal antibody during chronic SIVagm infection resulted in high viral replication rates in follicles and the T cell zone and increased viral DNA in lymph nodes. Our data suggest that, in nonpathogenic SIV infection, NK cells migrate into follicles and play a major role in viral reservoir control in lymph nodes.

We sought design principles for a vaccine to prevent HIV transmission to women by identifying correlates of protection conferred by a highly effective live attenuated SIV vaccine in the rhesus macaque animal model. We show that SIVmac239Δnef vaccination recruits plasma cells and induces ectopic lymphoid follicle formation beneath the mucosal epithelium in the rhesus macaque female reproductive tract. The plasma cells and ectopic follicles produce IgG Abs reactive with viral envelope glycoprotein gp41 trimers, and these Abs are concentrated on the path of virus entry by the neonatal FcR in cervical reserve epithelium and in vaginal epithelium. This local Ab production and delivery system correlated spatially and temporally with the maturation of local protection against high-dose pathogenic SIV vaginal challenge. Thus, designing vaccines to elicit production and concentration of Abs at mucosal frontlines could aid in the development of an effective vaccine to protect women against HIV-1.

NK cell responses to HIV/SIV infection have been well studied in acute and chronic infected patients/monkeys, but little is known about NK cells during viral transmission, particularly in mucosal tissues. In this article, we report a systematic study of NK cell responses to high-dose vaginal exposure to SIVmac251 in the rhesus macaque female reproductive tract (FRT). Small numbers of NK cells were recruited into the FRT mucosa following vaginal inoculation. The influx of mucosal NK cells preceded local virus replication and peaked at 1 wk and, thus, was in an appropriate time frame to control an expanding population of infected cells at the portal of entry. However, NK cells were greatly outnumbered by recruited target cells that fuel local virus expansion and were spatially dissociated from SIV RNA+ cells at the major site of expansion of infected founder populations in the transition zone and adjoining endocervix. The number of NK cells in the FRT mucosa decreased rapidly in the second week, while the number of SIV RNA+ cells in the FRT reached its peak. Mucosal NK cells produced IFN-γ and MIP-1α/CCL3 but lacked several markers of activation and cytotoxicity, and this was correlated with inoculum-induced upregulation of the inhibitory ligand HLA-E and downregulation of the activating receptor CD122/IL-2Rβ. Examination of SIVΔnef-vaccinated monkeys suggested that recruitment of NK cells to the genital mucosa was not involved in vaccine-induced protection from vaginal challenge. In summary, our results suggest that NK cells play, at most, a limited role in defenses in the FRT against vaginal challenge.

Beyond their ability to eliminate infected cells without the need for prior sensitization, natural killer (NK) cells have been shown to exhibit adaptive immune functions. In particular, our lab demonstrated that antigen-specific memory NK cells responses against SIV can be induced by infection and vaccination in non-human primates. These data suggested that a subset of NK cells in humans may also mediate immunological memory responses to HIV. To study human HIV-antigen-specific NK cells in more detail, we adapted limiting dilution assays to clonally expand individual peripheral blood NK cells (NKCL) from untreated HIV-infected individuals. Using a calcein acetoxymethyl ester cytotoxicity assay, the ability of each NKCL to lyse MHC-devoid K562 cell lines or autologous B-LCLs pulsed with a pool of MOG- (human self-peptide), HIV Gag- or HIV Env-derived overlapping peptides was assessed. As expected, all tested NKCL could potently lyse K562 cells (median=56% killing) but did not kill B-LCL pulsed with the MOG control. Strikingly, 37.5% of NKCL displayed anti-HIV Gag cytotoxic activity up to 31% specific lysis (range: 1%–31%), while 25% of NKCL displayed anti-HIV Env cytotoxic activity up to 72% specific lysis (range: 1%–72%). Furthermore, pre-treatment with an anti-NKG2C blocking antibody decreased the specific lysis, thereby confirming the importance of NKG2C receptor in the function of antigen-specific NK cells. Thus, we were able to isolate single NK cells displaying potent HIV-specific killing, within the range of robust cytotoxicity normally found against tumor cells. Together these data suggest that HIV-specific responses mediated by NK cells exist in humans and may have the potential to be harnessed for therapeutic modalities.

The objective of this study was to determine the systemic effects of chronic simian immunodeficiency virus (SIV) infection on plasmacytoid dendritic cells (pDCs). pDCs play a critical role in antiviral immunity, but current data are conflicting on whether pDCs inhibit HIV/SIV replication, or, alternatively, contribute to chronic immune activation and disease. Furthermore, previous pDC studies have been complicated by incomplete descriptions of generalized depletion during HIV/SIV infection, and the effects of infection on pDCs outside peripheral blood remain unclear. In scheduled-sacrifice studies of naive and chronically SIV-infected rhesus macaques we evaluated the distribution and functionality of pDCs in multiple tissues using surface and intracellular polychromatic flow cytometry. As previously observed, pDCs were reduced in peripheral blood and spleens, but were also depleted in non-lymphoid organs such as the liver. Interestingly, pDCs accumulated up to fourfold in jejunum, colon and gut-draining lymph nodes, but not in peripheral lymph nodes. Most unexpectedly, SIV infection induced a multi-functional interferon-α, tumour necrosis factor-α, and macrophage inflammatory protein-1β cytokine secretion phenotype, whereas in normal animals these were generally distinct and separate functions. Herein we show a systemic redistribution of pDCs to gut tissues and gut-draining lymph nodes during chronic SIV infection, coupled to a novel multi-functional cytokine-producing phenotype. While pDC accumulation in the mucosa could aid in virus control, over-production of cytokines from these cells could also contribute to the increased immune activation in the gut mucosa commonly associated with progressive lentivirus infections.

An altered intestinal microbiome during chronic human immunodeficiency virus (HIV) infection is associated with mucosal dysfunction, inflammation, and disease progression. We performed a preclinical evaluation of the safety and efficacy of fecal microbiota transplantation (FMT) as a potential therapeutic in HIV-infected individuals. Antiretroviral-treated, chronically simian immunodeficiency virus (SIV)-infected rhesus macaques received antibiotics followed by FMT. The greatest microbiota shift was observed after antibiotic treatment. The bacterial community composition at 2 weeks post-FMT resembled the pre-FMT community structure, although differences in the abundances of minor bacterial populations remained. Immunologically, we observed significant increases in the number of peripheral Th17 and Th22 cells and reduced CD4 + T cell activation in gastrointestinal tissues post-FMT. Importantly, the transplant was well tolerated with no negative clinical side effects. Although this pilot study did not control for the differential contributions of antibiotic treatment and FMT to the observed results, the data suggest that FMT may have beneficial effects that should be further evaluated in larger studies. IMPORTANCE Due to the immunodeficiency and chronic inflammation that occurs during HIV infection, determination of the safety of FMT is crucial to prevent deleterious consequences if it is to be used as a treatment in the future. Here we used the macaque model of HIV infection and performed FMT on six chronically SIV-infected rhesus macaques on antiretroviral treatment. In addition to providing a preclinical demonstration of the safety of FMT in primates infected with a lentivirus, this study provided a unique opportunity to examine the relationships between alterations to the microbiome and immunological parameters. In this study, we found increased numbers of Th17 and Th22 cells as well as decreased activation of CD4 + T cells post-FMT, and these changes correlated most strongly across all sampling time points with lower-abundance taxonomic groups and other taxonomic groups in the colon. Overall, these data provide evidence that changes in the microbiome, particularly in terms of diversity and changes in minor populations, can enhance immunity and do not have adverse consequences.

Defining the correlates of immune protection conferred by SIVΔnef, the most effective vaccine against SIV challenge, could enable the design of a protective vaccine against HIV infection. Here we provide a comprehensive assessment of immune responses that protect against SIV infection through detailed analyses of cellular and humoral immune responses in the blood and tissues of rhesus macaques vaccinated with SIVΔnef and then vaginally challenged with wild-type SIV. Despite the presence of robust cellular immune responses, animals at 5 weeks after vaccination displayed only transient viral suppression of challenge virus, whereas all macaques challenged at weeks 20 and 40 post-SIVΔnef vaccination were protected, as defined by either apparent sterile protection or significant suppression of viremia in infected animals. Multiple parameters of CD8 T cell function temporally correlated with maturation of protection, including polyfunctionality, phenotypic differentiation, and redistribution to gut and lymphoid tissues. Importantly, we also demonstrate the induction of a tissue-resident memory population of SIV-specific CD8 T cells in the vaginal mucosa, which was dependent on ongoing low-level antigenic stimulation. Moreover, we show that vaginal and serum antibody titers inversely correlated with post-challenge peak viral load, and we correlate the accumulation and affinity maturation of the antibody response to the duration of the vaccination period as well as to the SIVΔnef antigenic load. In conclusion, maturation of SIVΔnef-induced CD8 T cell and antibody responses, both propelled by viral persistence in the gut mucosa and secondary lymphoid tissues, results in protective immune responses that are able to interrupt viral transmission at mucosal portals of entry as well as potential sites of viral dissemination., Author Summary Annually, more than two million people worldwide are infected with HIV, the virus that causes AIDS. Rhesus macaques can be infected with SIV, a close relative and ancestor of HIV, resulting in simian AIDS, recapitulating key aspects of human HIV infection. SIVΔnef, a live attenuated form of SIV, protects rhesus macaques from subsequent challenge with pathogenic SIV and is widely viewed as the most effective SIV vaccine. Here we demonstrate that SIVΔnef persistence during the vaccination period drives both cell-mediated and humoral immune response maturation. During the vaccination period, cell-mediated immune responses elicited by SIVΔnef target more conserved regions of the virus rendering immune escape more difficult. Furthermore, the localization of the cell-mediated immune responses is shifted over time from peripheral blood to sites of viral production that are rich in uninfected SIV target cells, thereby positioning cell-mediated immune responses where they are most needed after wild-type SIV challenge. Similarly, SIVΔnef persistence during the vaccination period also leads to the accumulation and maturation of the humoral immune response. Our findings highlight the unique capacity of persistent vaccines to elicit durable and effective immune responses against wild-type SIV challenge.

Multiple studies suggest that plasmacytoid dendritic cells (pDCs) are depleted and dysfunctional during human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) infection, but little is known about pDCs in the gut-the primary site of virus replication. Here, we show that during SIV infection, pDCs were reduced 3--fold in the circulation and significantly upregulated the gut-homing marker α4β7, but were increased 4-fold in rectal biopsies of infected compared to naive macaques. These data revise the understanding of pDC immunobiology during SIV infection, indicating that pDCs are not necessarily depleted, but instead may traffic to and accumulate in the gut mucosa.

Natural killer (NK) cells are classically viewed as effector cells that kill virus-infected and neoplastic cells, but recent studies have identified a rare mucosal NK- cell subpopulation secreting the TH17 cytokine IL-22. Here, we report identification of 2 distinct lineages of mucosal NK cells characterized as NKG2A+NFIL3+RORC– and NKp44+NFIL3+RORC+. NKG2A+ NK cells were systemically distributed, cytotoxic, and secreted IFN-γ, whereas NKp44+ NK cells were mucosae-restricted, noncytotoxic, and produced IL-22 and IL-17. During SIV infection, NKp44+ NK cells became apoptotic, were depleted, and had an altered functional profile characterized by decreased IL-17 secretion; increased IFN-γ secretion; and, surprisingly, increased potential for cytotoxicity. NKp44+ NK cells showed no evidence of direct SIV infection; rather, depletion and altered function were associated with SIV-induced up-regulation of inflammatory mediators in the gut, including indoleamine 2,3-dioxygenase 1. Furthermore, treatment of NKp44+ NK cells with indoleamine 2,3-dioxygenase 1 catabolites in vitro ablated IL-17 production in a dose-dependent manner, whereas other NK-cell functions were unaffected. Thus lentiviral infection both depletes and modifies the functional repertoire of mucosal NK cells involved in the maintenance of gut integrity, a finding that highlights the plasticity of this rare mucosal NK-cell population.

Since the vast majority of infections occur at mucosal surfaces, accurate characterization of mucosal immune cells is critically important for understanding transmission and control of infectious diseases. Standard flow cytometric analysis of cells obtained from mucosal tissues can provide valuable information on the phenotype of mucosal leukocytes and their relative abundance, but does not provide absolute cell counts of mucosal cell populations. We developed a bead-based flow cytometry assay to determine the absolute numbers of multiple mononuclear cell types in colorectal biopsies of rhesus macaques. Using 10-color flow cytometry panels and pan-fluorescent beads, cells were enumerated in biopsy specimens by adding a constant ratio of beads per mg of tissue and then calculating cell numbers/mg of tissue based on cell-to-bead ratios determined at the time of sample acquisition. Testing in duplicate specimens showed the assay to be highly reproducible (Spearman R=0.9476, P0.0001). Using this assay, we report enumeration of total CD45(+) leukocytes, CD4(+) and CD8(+) T cells, B cells, NK cells, CD14(+) monocytes, and myeloid and plasmacytoid dendritic cells in colorectal biopsies, with cell numbers in normal rhesus macaques varying from medians of 4486 cells/mg (T cells) to 3 cells/mg (plasmacytoid dendritic cells). This assay represents a significant advancement in rapid, accurate quantification of mononuclear cell populations in mucosal tissues and could be applied to provide absolute counts of a variety of different cell populations in diverse tissues.

Precise identification of NK-cell populations in humans and nonhuman primates has been confounded by imprecise phenotypic definitions. A common definition used in nonhuman primates, including chimpanzees, is CD3(-) CD8α(+) CD16(+) , and this is the dominant NK-cell phenotype in peripheral blood. However, recent data suggest that in chimpanzees a rare CD8α(-) CD16(+) population also exists. Herein, we present evidence validating the existence of this rare subset in chimpanzee peripheral blood, but also demonstrating that gating on CD3(-) CD8α(-) CD16(+) cells can inadvertently include a large number of CD16(+) myeloid DCs (mDCs). We confirmed the inclusion of mDCs in CD3(-) CD8α(-) CD16(+) gated cells by demonstrating high expression of CD11c, BDCA-1 and HLA-DR, and by the lack of expression of NKp46 and intracellular perforin. We also functionally validated the CD8α(-) NK-cell and mDC populations by mutually exclusive responsiveness to a classical NK-cell stimulus, MHC class I-deficient cells, and a prototypic mDC stimulus, poly I:C, respectively. Overall, these data demonstrate common problems with gating of NK cells that can lead to erroneous conclusions and highlight a critical need for consensus protocols for NK-cell phenotyping.

Fms-like tyrosine kinase 3 ligand (FLT3-L) is critical for the differentiation and self-renewal of CD34 + progenitor cells in primates and has been used therapeutically to mobilize progenitor and dendritic cells in vivo . However, little is known regarding the expansion of progenitor cells outside of peripheral blood, particularly in bone marrow (BM), where progenitor cells primarily reside. Evaluation of FLT3-L-mediated cell mobilization during lentivirus infections, where the numbers of CD34 + progenitor cells are reduced, is limited. We enumerated frequencies and absolute numbers of CD34 + progenitor cells in blood and BM of naive and SIV- or SHIV-infected macaques during and after the administration of FLT3-L. Flow cytometric analyses revealed that, while CD34 + cells increased in the circulation, no expansion was observed in BM. Furthermore, in the BM intracellular Ki67, a marker of cell proliferation, was downregulated in CD34 + progenitor cells but was upregulated significantly in the bulk cell population. Although the exact mechanism(s) remains unclear, these data suggest that CD34 + cell mobilization in blood was the result of cellular emigration from BM and not the proliferation of CD34 + cells already in the periphery. It is possible that the decreased progenitor cell proliferation observed in BM is evidence of a negative regulatory mechanism preventing hyperproliferation and development of neoplastic cells.

Natural killer (NK) cells contribute to control of HIV/SIV infection. We defined macaque NK-cell subsets based on expression of CD56 and CD16 and found their distribution to be highly disparate. CD16+ NK cells predominated in peripheral blood, whereas most mucosal NK cells were CD56+, and lymph nodes contained both CD56+ and CD16−CD56− (double-negative [DN]) subsets. Functional profiles were also distinct among subsets—CD16+ NK cells expressed high levels of cytolytic molecules, and CD56+ NK cells were predominantly cytokine-secreting cells, whereas DN NK possessed both functions. In macaques chronically infected with SIV, circulating CD16+ and DN NK cells were expanded in number and, although markers of cytoxicity increased, cytokine secretion decreased. Notably, CD56+ NK cells in SIV-infected animals up-regulated perforin, granzyme B, and CD107a. In contrast, the lymph node–homing molecules CD62 ligand (CD62L) and C-C chemokine receptor type 7 (CCR7), which are expressed primarily on CD56+ and DN NK cells, were significantly down-regulated on NK cells from infected animals. These data demonstrate that SIV infection drives a shift in NK-cell function characterized by decreased cytokine production, expanded cytotoxicity, and trafficking away from secondary lymphoid organs, suggesting that the NK-cell repertoire is not only heterogeneous but also plastic.

Ongoing antigenic stimulation appears to be an important prerequisite for the persistent expression of programmed death 1 (PD-1), an inhibitory TCR coreceptor of the CD28 family. Although recent publications have emphasized the utility of PD-1 as a marker for dysfunctional T cells in chronic viral infections, its dependence on antigenic stimulation potentially renders it a sensitive indicator of low-level viral replication. To explore the antigenic threshold for the maintenance of PD-1 expression on virus-specific T cells, we compared PD-1 expression on virus-specific and memory T cell populations in controlled and uncontrolled SIV and HIV-1 infection. In both controlled live attenuated SIV infection in rhesus macaques and HIV-1 infection in elite controllers, elevated levels of PD-1 expression were observed on SIV- and HIV-1–specific CD8+ T cells. However, in contrast to chronic wild-type SIV infection and uncontrolled HIV-1 infection, controlled SIV/HIV-1 infection did not result in increased expression of PD-1 on total memory T cells. PD-1 expression on SIV-specific CD8+ T cells rapidly decreased after the emergence of CTL escape in cognate epitopes, but was maintained in the setting of low or undetectable levels of plasma viremia in live attenuated SIV-infected macaques. After inoculation of naive macaques with a single-cycle SIV, PD-1 expression on SIV-specific CD8+ T cells initially increased, but was rapidly downregulated. These results demonstrate that PD-1 can serve as a sensitive indicator of persistent, low-level virus replication and that generalized PD-1 expression on T lymphocytes is a distinguishing characteristic of uncontrolled lentiviral infections.

Plasmacytoid dendritic cells (pDCs), one of two types of bone marrow (BM)-derived blood DCs, play an important role in linking innate and adaptive immune responses. However, little is known about the nature of pDCs that reside in the BM. Because the simian immunodeficiency virus-macaque model closely mimics human immunodeficiency virus disease in humans, with both infections inducing a decrease in pDCs, we characterized and compared pDCs in the BM with those in peripheral blood (PB) of healthy pig-tailed macaques. The results revealed that pDCs from both compartments had the same CD123++HLA-DR+Lin−phenotype and were similar in size. Although BM-derived pDCs (BM-pDCs) were 3-fold greater in frequency and 10-fold greater in number, they had lower cell surface expression of both HLA-DR and the costimulatory molecule CD86 than did PB-pDCs. Both BM- and PB-pDCs responded ex vivo to synthetic CpG oligodeoxynucleotides and inactivated influenza virus by upregulating HLA-DR and CD86 and secreting cytokines; however, stimulated BM-pDCs secreted less alpha interferon and tumor necrosis factor alpha per cell than did PB-pDCs. These results suggest that while BM-pDCs appear to be phenotypically less mature than PB-pDCs, they do respond to pathogens. Thus, during acute infections, these cells could initiate immune responses either in the BM or after rapidly migrating from the BM into the periphery. A better characterization of pDCs in blood and tissues will be beneficial for future studies of macaques that focus on either pathogenesis or vaccine development.

Blood plasmacytoid dendritic cells (pDCs) contribute to both innate and adaptive immune responses by secreting high levels of IFN-α following acute bacterial and viral infections and indirectly by augmenting cell-mediated immunity. Cross-sectional studies have shown that the number of circulating pDCs in HIV patients, compared to that in uninfected individuals, is reduced. However, since the time of infection is usually unknown in HIV-infected patients, pDC–virus interactions that occur immediately after virus exposure are poorly understood. The current study investigated pDC dynamics during acute and chronic infections of macaques with either SIVmac239 or the pathogenic SIV–HIV chimera, SHIV-89.6P, as models for HIV infection. In three rhesus and three pig-tailed macaques infected intravenously with SIVmac239, the percentages of pDCs in blood declined 2- to 6-fold during the first 6 weeks after infection and remained depressed throughout the disease course. Surprisingly, no consistent, comparable decline in peripheral blood pDCs was observed in six macaques infected with SHIV-89.6P. In this latter group, percentages of pDCs did not correlate with CD4+ T cells, but there was an inverse relationship with viral load. In addition, when compared to naïve controls, the percentages of pDCs were reduced in spleens and peripheral lymph nodes of SIVmac239- but not SHIV-89.6P-infected animals that had progressed to AIDS. Proviral DNA was detected during the acute phase in pDCs isolated from macaques infected with either virus. These results imply that, even though macaque pDCs can be infected by both SIVmac239 and SHIV-89.6P, the subsequent effects on in vivo pathogenesis differ. The underlying mechanism(s) for these differences is unclear, but the selection of SIV or SHIV as a challenge virus might influence the outcome of some studies, such as those evaluating vaccines or the therapeutic efficacy of drugs.

Natural killer (NK) cells are generally considered as the effector arm of nonspecific innate immunity, providing rapid recognition and lysis of virus-infected and tumor cells, but can also play significant regulatory roles in pregnancy, cooperate with T and B cell responses, and even exhibit independent adaptive memory. However, much like other lymphocyte populations, recent burgeoning evidence suggests that in chronic conditions NK cells can become functionally exhausted. T cell exhaustion is generally induced by constant exposure to persistent antigens resulting in a loss of effector functions and an overall decline in resistance to disease, and NK cell exhaustion appears to share many similar features. One of the best-studied examples of NK cell dysregulation is in HIV infection and in SIV-infected nonhuman primate models. Indeed, recent work from our laboratory found that NK cells in lymph nodes of SIV-infected rhesus macaques are continuously activated, likely by low-level ongoing virus replication [1]. This continuous activation resulted in in situ differentiation followed by functional anergy, as indicated by a failure to lyse target cells and upregulated Tim-3 expression. These data helped clarify empirical observations in both HIV and SIV infections of impaired cytotoxic function, altered cytokine production and impaired antibody-dependent cell-mediated cytotoxicity (ADCC) [2]. Indirect effects of HIV/SIV include the consistent bombardment of NK cells with target cells expressing dysregulated HLA and systemic cytokines, resulting in net exhaustion. Interestingly, similar indicators of upregulated Tim-3, PD-1 and lytic failure have all been found in CMV, Hepatitis C and Hepatitis B infections. Together, these data indicate NK cell exhaustion in chronic stages of viral disease may be directly related to a loss of control of virus replication and/or reactivation of latent virus reservoirs. In addition to chronic viral infections, NK cell exhaustion has also been observed in several human cancers and animal tumor models. Much like NK cells in SIV infection and previous descriptions of exhausted T cells, common features of NK cells in cancer and cancer models are high expression of inhibitory receptors including Tim-3, downregulated activating receptors, and decreased expression of transcription factors, EOMES and T-bet [3–5]. Furthermore, mouse models have demonstrated that NK cell dysfunction due to inefficient phosphorylation in activation signaling pathways arises only against MHC-deficient tumors, not against tumors that maintain MHC expression [6]. Indeed, just repeated exposure of NK cells in the presence of tumors can reduce cytotoxic activity [3]. While NK cell exposure to tumors can reduce cytotoxic activity, excess proliferation also contributes to NK cell dysfunction, as even homeostatic proliferation may induce an exhausted state in the absence of a tumor. Exhaustion of NK cells is also highly specific, as NK cell defects are far more severe in tumor-associated NK cells than in peripheral NK cells from the same individual or animal [3, 5, 6]. The clinical relevance of decreased NK cell function is also evident in disease progression, a prime example being breast cancer where NK cells with reduced function and high expression of inhibitory receptors are associated with more aggressive and invasive tumors [5]. NK cell exhaustion in chronic diseases appears to have many conserved features, including upregulation of inhibitory receptors and excess proliferation due to virus or cytokine-induced activation. Furthermore, NK cell recognition is highly dependent on ‘self’ and ‘missing-self’ discrimination through MHC binding, and repeated NK cell interaction with target cells where MHC is lacking, either by viral or oncogenic etiology, is a prominent cause of exhaustion. Although in its infancy, therapeutic restoration of NK cell numbers and functions are being developed. Preliminary success reverting NK cell exhaustion by therapeutic overexpression of EOMES [3], blocking of Tim-3 [4], or cytokine stimulation by IL-12, IL-18 or an IL-2 derivatives [6] have improved NK cell anti-tumor responses. Exogenous IL-12, IL-15 and/or IFN-α have also proven beneficial in reactivating NK cell function in nonhuman primate models [7]. Additionally, nonpathogenic hosts of SIV infection do not appear to exhibit evidence of NK cell exhaustion despite high virus replication [8], and these models could inform future strategies to augment NK cell function. In summary, these data highlight the concept that functional exhaustion of NK cells is associated with loss of control of chronic viral infections and inadequate elimination of neoplastic cells, but current strategies for augmentation of impaired NK cell function are underdeveloped and should be considered for future therapies.

Natural killer (NK) cells have traditionally been considered nonspecific components of innate immunity, but recent studies have shown features of antigen-specific memory in mouse NK cells. However, it has remained unclear whether this phenomenon also exists in primates. We found that splenic and hepatic NK cells from SHIV(SF162P3)-infected and SIV(mac251)-infected macaques specifically lysed Gag- and Env-pulsed dendritic cells in an NKG2-dependent fashion, in contrast to NK cells from uninfected macaques. Moreover, splenic and hepatic NK cells from Ad26-vaccinated macaques efficiently lysed antigen-matched but not antigen-mismatched targets 5 years after vaccination. These data demonstrate that robust, durable, antigen-specific NK cell memory can be induced in primates after both infection and vaccination, and this finding could be important for the development of vaccines against HIV-1 and other pathogens.

The onset of protective immunity against pathogenic SIV challenge in SIVΔnef-vaccinated macaques is delayed for 15-20 weeks, a process that is related to qualitative changes in CD8+ T cell responses induced by SIVΔnef. As a novel approach to characterize cell differentiation following vaccination, we used multi-target qPCR to measure transcription factor expression in naïve and memory subsets of CD8++ T cells, and in SIV-specific CD8+ T cells obtained from SIVΔnef-vaccinated or wild type SIVmac239-infected macaques. Unsupervised clustering of expression profiles organized naïve and memory CD8+ T cells into groups concordant with cell surface phenotype. Transcription factor expression patterns in SIV-specific CD8+ T cells in SIVΔnef-vaccinated animals were distinct from those observed in purified CD8+ T cell subsets obtained from naïve animals, and were intermediate to expression profiles of purified central memory and effector memory T cells. Expression of transcription factors elicited by SIVΔnef vaccination also varied over time: cells obtained at later time points, temporally associated with greater protection, appeared more central-memory like than cells obtained at earlier time points, which appeared more effector memory-like. Expression of transcription factors associated with effector differentiation, such as ID2 and RUNX3, were decreased over time, while expression of transcription factors associated with quiescence or memory differentiation, such as TCF7, BCOR and EOMES, increased. CD8+ T cells specific for a more conserved epitope expressed higher levels of TBX21 and BATF, and appeared more effector-like than cells specific for an escaped epitope, consistent with continued activation by replicating vaccine virus. These data suggest transcription factor expression profiling is a novel method that can provide additional data complementary to the analysis of memory cell differentiation based on classical phenotypic markers. Additionally, these data support the hypothesis that ongoing stimulation by SIVΔnef promotes a distinct protective balance of CD8+ T cell differentiation and activation states., Author Summary The live attenuated vaccine SIVΔnef can induce robust CD8+ T cell- mediated protection against infection with pathogenic SIV in macaques. Thus, there is substantial interest in characterizing these immune responses to inform HIV vaccine design. Animals challenged at 15–20 weeks post vaccination exhibit robust protection, whereas animals challenged at 5 weeks post-vaccination manifest little protection. Since the frequency of SIV-specific T cells decreases from week 5 to week 20, it is likely that the quality of the response to challenge changes as virus-specific cells differentiate. We applied a novel approach of transcription factor expression profiling to characterize the differences in SIV-specific cell function and phenotype at more protected and less protected time points. Using unsupervised clustering methods informed by expression profiles assessed in purified CD8+ T cell subsets, we show that SIV-specific cells display expression profiles different than any purified CD8+ T cell subset, and intermediate to sorted effector memory and central memory subsets. SIV-specific cells overall appear more effector memory-like at week 5 post-vaccination, and more central memory-like at week 20 post-vaccination. Distinct profiles of CD8+ T cells specific for different SIV epitopes having different immune escape kinetics suggests maturation is regulated by ongoing low-level replication of vaccine virus.

The live attenuated simian immunodeficiency virus (LASIV) vaccine SIVΔnef is one of the most effective vaccines in inducing protection against wild-type lentiviral challenge, yet little is known about the mechanisms underlying its remarkable protective efficacy. Here, we exploit deep sequencing technology and comprehensive CD8 T cell epitope mapping to deconstruct the CD8 T cell response, to identify the regions of immune pressure and viral escape, and to delineate the effect of epitope escape on the evolution of the CD8 T cell response in SIVΔnef-vaccinated animals. We demonstrate that the initial CD8 T cell response in the acute phase of SIVΔnef infection is mounted predominantly against more variable epitopes, followed by widespread sequence evolution and viral escape. Furthermore, we show that epitope escape expands the CD8 T cell repertoire that targets highly conserved epitopes, defined as anentropic specificity, and generates de novo responses to the escaped epitope variants during the vaccination period. These results correlate SIVΔnef-induced protection with expanded anentropic specificity and increased response depth. Importantly, these findings render SIVΔnef, long the gold standard in HIV/SIV vaccine research, as a proof-of-concept vaccine that highlights the significance of the twin principles of anentropic specificity and repertoire depth in successful vaccine design., Author Summary Annually, more than two million people are infected with HIV, the virus that causes AIDS. Due to the ability of the virus to escape host immune responses, designing a successful HIV vaccine has been elusive. Similar to HIV in humans, rhesus macaques can be infected with SIV, a close relative and ancestor of HIV, resulting in simian AIDS. SIVΔnef, a live attenuated form of SIV, protects rhesus macaques from subsequent challenge with pathogenic SIV and is widely viewed as the most effective SIV vaccine. Here, we demonstrate that after vaccination of macaques with SIVΔnef, the immune response initially targets more variable regions of the virus, which the virus rapidly escapes. However, as the virus escapes, the immune response evolves to target more conserved regions of the virus as well as escape variants. This refocused targeting of conserved regions by the immune response provides a new mechanistic model that contributes to our understanding of how SIVΔnef vaccination protects animals from pathogenic challenge with SIV. Our findings also reinforce the importance of developing HIV vaccines that target conserved regions of the virus as well as their potential variants.

Hepatitis C virus (HCV) infection has become a global public health burden costing billions of dollars in health care annually. Even with rapidly advancing scientific technologies this disease still poses a significant threat due to a lack of vaccines and affordable treatment options. The immune correlates of protection and predisposing factors toward chronicity remain major obstacles to development of HCV vaccines and immunotherapeutics due, at least in part, to lack of a tangible infection animal model. This review discusses the currently available animal models for HCV disease with a primary focus on GB virus B (GBV-B) infection of New World primates that recapitulates the dual Hepacivirus phenotypes of acute viral clearance and chronic pathologic disease. HCV and GBV-B are also closely phylogenetically related and advances in characterization of the immune systems of New World primates have already led to the use of this model for drug testing and vaccine trials. Herein, we discuss the benefits and caveats of the GBV-B infection model and discuss potential avenues for future development of novel vaccines and immunotherapies.

Lentivirus infections are characterized by a dramatic loss of mucosal CD4(+) T cells, breakdown of the gut mucosa, and subsequent chronic immune activation. Residual immune activation persists even in patients controlling virus replication and remains a significant source of ongoing disease morbidities, but the causes are unclear. Plasmacytoid dendritic cells (pDCs), primary producers of interferon (IFN)-α, have been previously shown to be depleted from peripheral blood of HIV patients and simian immunodeficiency virus (SIV)-infected macaques, and most recently have been shown to accumulate in the gut mucosa. Although previous work has shown that pDC frequencies can be reduced in the circulation of HIV-1 Elite Controllers, it is unknown if gut-homing also occurs. In this new study we found that during progressive HIV-1 infection pDCs were depleted in peripheral blood compared to seronegative controls, and, correlating with plasma viremia, the remaining pDCs upregulated the gut-homing marker, α4β7. Even in HIV-1 Elite Controllers pDCs were significantly reduced in blood and α4β7 expression was still significantly upregulated compared to seronegative controls. Interestingly, pDC trafficking to the gut was associated with increased Ki67 and HLA-DR on circulating CD4(+) and CD8(+) T cells. Overall, these data suggest that gut trafficking of pDCs is independent of virus replication and could be mediated by alternative mechanisms, which in turn could contribute to residual immune activation in HIV-1 Elite Controllers.

Chronic hepatitis C has been associated with metabolic syndrome that includes insulin resistance, hepatic steatosis and obesity. These metabolic aberrations are risk factors for disease severity and treatment outcome in infected patients. Experimental infection of marmosets with GBV-B serves as a tangible, small animal model for human HCV infection, and while virology and pathology are well described, a full investigation of clinical disease and the metabolic milieu is lacking. In this study six marmosets were infected intravenously with GBV-B and changes in hematologic, serum biochemical and plasma metabolic measures were investigated over the duration of infection. Infected animals exhibited signs of lymphocytopenia, but platelet and RBC counts were generally stable or even increased. Although most animals showed a transient decline in blood glucose, infection resulted in several fold increases in plasma insulin, glucagon and glucagon-like peptide 1 (GLP-1). All infected animals experienced transient weight loss within the first 28 days of infection, but also became hypertriglyceridemic and had up to 10-fold increases in adipocytokines such as resistin and plasminogen activator inhibitor 1 (PAI-1). In liver, moderate to severe cytoplasmic changes associated with steatotic changes was observed microscopically at 168 days post infection. Collectively, these results suggest that GBV-B infection is accompanied by hematologic, biochemical and metabolic abnormalities that could lead to obesity, diabetes, thrombosis and atherosclerosis, even after virus has been cleared. Our findings mirror those found in HCV patients, suggesting that metabolic syndrome could be conserved among hepaciviruses, and both mechanistic and interventional studies for treating HCV-induced metabolic complications could be evaluated in this animal model.

Plasmacytoid dendritic cells (pDCs), a primary source of interferon α (IFN-α), provide a first line of innate immune defense against human immunodeficiency virus infection. However, their kinetics and functions during acute infection are poorly understood. In mucosal tissues of normal rhesus macaques, we found CD4(+) pDCs to be the subset responsible for most IFN-α and tumor necrosis factor α (TNF-α) production in response to Toll-like receptor (TLR) 7/8 stimulation, compared with relatively anergic CD4(-) pDCs. During acute simian immunodeficiency virus (SIV) infection, gut homing was imprinted on pDCs in the bone marrow, resulting in a decline in pDCs from circulation and secondary lymphoid tissues. Although the accumulated pDCs in the gut mucosae had robust cytokine responses to TLR7/8 stimulation in vitro, pDC gut migration occurred after infection and detection of SIV in plasma. Our data suggest that innate pDC responses do not control initial SIV seeding and dissemination but instead may contribute to ongoing immune activation in the gut.

Immunization of macaques with attenuated simian immunodeficiency virus (SIV) with deletions in nef (SIVΔnef) is shown to elicit protective immunity to infection by pathogenic SIV, yet the mechanisms that orchestrate protection and prevent pathogenesis remains unknown. We utilized whole-genome transcriptional profiling to reveal molecular signatures of protective immunity in circulating CD8+ T cells of rhesus macaques vaccinated with SIVmac239Δnef and challenged with pathogenic SIVmac251. Our findings suggest that protective immunity to pathogenic SIV infection induced by SIVmac239∆nef is associated with balanced induction of T cell activation and immunoregulatory mechanisms and dampened activation of interferon-induced signaling pathways and cytolytic enzyme production as compared with pathogenic SIVmac251 infection of unvaccinated controls. We provide evidence that protective immunity to SIVmac251 correlates with induction of biomarkers of T cell activation, differentiation, signaling, and adhesion that were down regulated in unvaccinated controls. The study highlights potential immunomodulatory networks associated with protective immunity against the virus.

Natural killer (NK) cells play critical roles in innate immunity and in bridging innate and adaptive immune responses against viral infection. However, the response of NK cells to monkeypox virus (MPXV) infection is not well characterized. In this intravenous challenge study of MPXV infection in rhesus macaques (Macaca mulatta), we analyzed blood and lymph node NK cell changes in absolute cell numbers, cell proliferation, chemokine receptor expression, and cellular functions. Our results showed that the absolute number of total NK cells in the blood increased in response to MPXV infection at a magnitude of 23-fold, manifested by increases in CD56+, CD16+, CD16-CD56- double negative, and CD16+CD56+ double positive NK cell subsets. Similarly, the frequency and NK cell numbers in the lymph nodes also largely increased with the total NK cell number increasing 46.1-fold. NK cells both in the blood and lymph nodes massively proliferated in response to MPXV infection as measured by Ki67 expression. Chemokine receptor analysis revealed reduced expression of CXCR3, CCR7, and CCR6 on NK cells at early time points (days 2 and 4 after virus inoculation), followed by an increased expression of CXCR3 and CCR5 at later time points (days 7-8) of infection. In addition, MPXV infection impaired NK cell degranulation and ablated secretion of interferon-γ and tumor necrosis factor-α. Our data suggest a dynamic model by which NK cells respond to MPXV infection of rhesus macaques. Upon virus infection, NK cells proliferated robustly, resulting in massive increases in NK cell numbers. However, the migrating capacity of NK cells to tissues at early time points might be reduced, and the functions of cytotoxicity and cytokine secretion were largely compromised. Collectively, the data may explain, at least partially, the pathogenesis of MPXV infection in rhesus macaques.