NK cells modulate in vivo control of SARS-CoV-2 replication and suppression of lung damage.

Natural killer (NK) cells play a critical role in virus control. However, it has remained largely unclear whether NK cell mobilization in SARS-CoV-2 infections is beneficial or pathologic. To address this deficit, we employed a validated experimental NK cell depletion non-human primate (NHP) model with SARS-CoV-2 Delta variant B.1.617.2 challenge. Viral loads (VL), NK cell numbers, activation, proliferation, and functional measures were evaluated in blood and tissues. In non-depleted (control) animals, infection rapidly induced NK cell expansion, activation, and increased tissue trafficking associated with VL. Strikingly, we report that experimental NK cell depletion leads to higher VL, longer duration of viral shedding, significantly increased levels of pro-inflammatory cytokines in the lungs, and overt lung damage. Overall, we find the first significant and conclusive evidence for NK cell-mediated control of SARS-CoV-2 virus replication and disease pathology. These data indicate that adjunct therapies for infection could largely benefit from NK cell-targeted approaches.

Knockdowns of CD3zeta Chain in Primary NK Cells Illustrate Modulation of Antibody-Dependent Cellular Cytotoxicity Against Human Immunodeficiency Virus-1.

Multifaceted natural killer (NK) cell activities are indispensable for controlling human immunodeficiency virus (HIV)-1 transmission and pathogenesis. Among the diverse functions of NK cells, antibody-dependent cellular cytotoxicity (ADCC) has been shown to predict better HIV-1 protection. ADCC is initiated by the engagement of an Fc γ receptor CD16 with an Fc portion of the antibody, leading to phosphorylation of the CD3 ζ chain (CD3ζ) and Fc receptor γ chain (FcRγ) as well as downstream signaling activation. Though CD3ζ and FcRγ were thought to have overlapping roles in NK cell ADCC, several groups have reported that CD3ζ-mediated signals trigger a more robust ADCC. However, few studies have illustrated the direct contribution of CD3ζ in HIV-1-specific ADCC. To further understand the roles played by CD3ζ in HIV-1-specific ADCC, we developed a CD3ζ knockdown system in primary human NK cells. We observed that HIV-1-specific ADCC was inhibited by CD3ζ perturbation. In summary, we demonstrated that CD3ζ is important for eliciting HIV-1-specific ADCC, and this dynamic can be utilized for NK cell immunotherapeutics against HIV-1 infection and other diseases.

Natural killer-like B cells are a distinct but infrequent innate immune cell subset modulated by SIV infection of rhesus macaques.

Natural killer-like B (NKB) cells are unique innate immune cells expressing both natural killer (NK) and B cell receptors. As first responders to infection, they secrete IL-18 to induce a critical cascade of innate and adaptive immune cell infiltration and activation. However, limited research exists on the role of NKB cells in homeostasis and infection, largely due to incomplete and erroneous evaluations. To fill this knowledge gap, we investigated the expression of signaling and trafficking proteins, and the in situ localization and transcriptome of naïve NKB cells compared to conventionally-defined NK and B cells, as well as modulations of these cells in SIV infection. Intracellular signaling proteins and trafficking markers were expressed differentially on naïve NKB cells, with high expression of CD62L and Syk, and low expression of CD69, α4ß7, FcRg, Zap70, and CD3z, findings which were more similar to B cells than NK cells. CD20+NKG2a/c+ NKB cells were identified in spleen, mesenteric lymph nodes (MLN), colon, jejunum, and liver of naïve rhesus macaques (RM) via tissue imaging, with NKB cell counts concentrated in spleen and MLN. For the first time, single cell RNA sequencing (scRNAseq), including B cell receptor (BCR) sequencing, of sorted NKB cells confirmed that NKB cells are unique. Transcriptomic analysis of naïve splenic NKB cells by scRNAseq showed that NKB cells undergo somatic hypermutation and express Ig receptors, similar to B cells. While only 15% of sorted NKB cells showed transcript expression of both KLRC1 (NKG2A) and MS4A1 (CD20) genes, only 5% of cells expressed KLRC1, MS4A1, and IgH/IgL transcripts. We observed expanded NKB frequencies in RM gut and buccal mucosa as early as 14 and 35 days post-SIV infection, respectively. Further, mucosal and peripheral NKB cells were associated with colorectal cytokine milieu and oral microbiome changes, respectively. Our studies indicate that NKB cells gated on CD3-CD14-CD20+NKG2A/C+ cells were inclusive of transcriptomically conventional B and NK cells in addition to true NKB cells, confounding accurate phenotyping and frequency recordings that could only be resolved using genomic techniques. Although NKB cells were clearly elevated during SIV infection and associated with inflammatory changes during infection, further interrogation is necessary to acurately identify the true phenotype and significance of NKB cells in infection and inflammation.

The Climate Change Burden on Immune Health: Are Persons Living with HIV More at Risk?

Climate change poses one of the most significant modern threats to overall human health,especially for vulnerable populations including persons living with HIV (PLWH). In this perspective, we specifically explore the concept of immune resilience in human health and how climate change phenomena - including extreme weather events, food insecurity, pollution, and emerging diseases - may exacerbate immune dysfunction and comorbidities faced by PLWH and hinder access to HIV treatment and prevention services. Multidisciplinary, collaborative efforts are urgently needed to quantify these impacts, develop mitigation strategies, and strengthen policies and funding to bolster immune resilience for PLWH in the face of accelerating climate change.

FcαRI (CD89) is upregulated on subsets of mucosal and circulating NK cells and regulates IgA-class specific signaling and functions.

Immunoglobulin A (IgA) is the predominant mucosal antibody class with both anti- and pro-inflammatory roles1-3. However, the specific role of the IgA receptor cluster of differentiation (CD)89, expressed by a subset of natural killer (NK) cells, is poorly explored. We found that CD89 protein expression on circulating NK cells is infrequent in humans and rhesus macaques, but transcriptomic analysis showed ubiquitous CD89 expression, suggesting an inducible phenotype. Interestingly, CD89+ NK cells were more frequent in cord blood and mucosae, indicating a putative IgA-mediated NK cell function in the mucosae and infant immune system. CD89+ NK cells signaled through upregulated CD3 zeta chain (CD3ζ), spleen tyrosine kinase (Syk), zeta chain-associated protein kinase 70 (ZAP70), and signaling lymphocytic activation molecule family 1 (SLAMF1), but also showed high expression of inhibitory receptors such as killer cell lectin-like receptor subfamily G (KLRG1) and reduced activating NKp46 and NKp30. CD89-based activation or antibody-mediated cellular cytotoxicity with monomeric IgA1 reduced NK cell functions, while antibody-mediated cellular cytotoxicity with combinations of IgG and IgA2 was enhanced compared to IgG alone. These data suggest that functional CD89+ NK cells survey mucosal sites, but CD89 likely serves as regulatory receptor which can be further modulated depending on IgA and IgG subclass. Although the full functional niche of CD89+ NK cells remains unexplored, these intriguing data suggest the CD89 axis could represent a novel immunotherapeutic target in the mucosae or early life.

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.

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

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

Upregulation of the NKG2D ligand ULBP2 by JC polyomavirus infection promotes immune recognition by natural killer cells.

BACKGROUND: JC polyomavirus(JCPyV) causes progressive multifocal leukoencephalopathy(PML), a potentially fatal complication of severe immune suppression with no effective treatment. Natural killer (NK) cells play critical roles in defense against viral infections, yet NK cell response to JCPyV infection remains unexplored. METHODS: NK and T cell responses against the JCPyV VP1 were compared using intracellular cytokine staining (ICS) upon stimulation with peptide pools. A novel flow cytometry-based assay was developed to determine NK cell killing efficiency of JCPyV-infected astrocyte-derived SVG-A cells. Blocking antibodies were used to identify the specific NK cell receptors in immune recognition of JCPyV-infected cells. RESULTS: In about 40% of healthy donors, we detected robust CD107a upregulation and IFN-γ production by NK cells, extending beyond T cell responses. Next, using the NK cell-mediated killing assay, we showed that co-culture of NK cells and JCPyV-infected SVG-A cells leads to a 60% reduction in infection, on average. JCPyV-infected cells had enhanced expression of ULBP2 - a ligand for the activating NK cell receptor NKG2D and addition of NKG2D blocking antibodies decreased NK cell degranulation. CONCLUSION: NKG2D-mediated activation of NK cells plays a key role in controlling JCPyV replication and may be a promising immunotherapeutic target to boost NK cell anti-JCPyV activity.

Multiplex Analysis of Cytokines and Chemokines in Persons Aging With or Without HIV.

People with HIV (PWH) on combination antiretroviral therapy (cART) are living longer lives due to modern cART advances and increased routine medical care. The full landscape of aging with HIV is unclear; given that HIV emerged relatively recently in human history and initially had a high mortality rate, there has not been a substantially aged population to evaluate. In this study, we set out to perform high-throughput plasma analyte profiling by multiplex analysis, focusing on various T helper (Th)-related cytokines, chemokines, and proinflammatory and anti-inflammatory cytokines. The primary goals being to provide reference ranges of these analytes for aging PWH cohorts, as well as testing the utility of high-throughput multiplex plasma assays. The cohort used in this study comprised age-matched healthy donors (32.6-73.5 years of age), PWH on cART (26.7-60.2 years of age), and viremic PWH (27.5-59.4 years of age). The patients in each group were then stratified across the age span to examine age-related impacts of these plasma biomarkers. Our results largely indicate feasibility of plasma analyte monitoring by multiplex and demonstrate a high degree of person-to-person variability regardless of age and HIV status. Nonetheless, we find multiple associations with age, duration of known infection, and viral load, all of which appear to be driven by either prolonged HIV disease progression or long-term use of cART.

Multiplex analysis of cytokines and chemokines in persons aging with or without HIV.

People with HIV (PWH) on combined antiretroviral therapy (cART) are living longer lives due to modern cART advances and increased routine medical care. The full landscape of aging with HIV is unclear; given that HIV emerged relatively recently in human history and initially had a high mortality rate, there has not been a substantially aged population to evaluate. In the present study, we set out to perform high throughput plasma analyte profiling by multiplex analysis, focusing on various T helper (Th)-related cytokines, chemokines, and pro- and anti-inflammatory cytokines. The primary goals being to provide reference ranges of these analytes for aging PWH cohorts, as well as testing the utility of high throughput multiplex plasma assays. The cohort used in this study was comprised of age-matched healthy donors (aged 32.6-73.5), PWH on cART (aged 26.7-60.2), and viremic PWH (aged 27.5-59.4). The patients in each group were then stratified across the age span to examine age-related impacts of these plasma biomarkers. Our results largely indicate feasibility of plasma analyte monitoring by multiplex and demonstrate a high degree of person-to-person variability regardless of age and HIV status. Nonetheless, we find multiple associations with age, duration of known infection, and viral load, all of which appear to be driven by either prolonged HIV disease progression or long-term use of cART.

Protocol for identification and computational analysis of human natural killer cells using flow cytometry and R.

Identifying differential protein expression is routinely used to delineate natural killer (NK) cells from various sample cohorts. This protocol describes key steps for NK cell analysis: identifying human NK cells using flow gating, data export from FlowJo, data loading in R, dimensionality reduction and visualization with Uniform Manifold Approximation and Projection, and generalized linear modeling with CyotGLMM. These analyses can help generate potential biomarkers of interest to identify NK cells across aging, treatment groups, and others. For complete details on the use and execution of this protocol, please refer to Kroll et al. (2022).1.

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.

A genetically engineered, stem-cell-derived cellular vaccine.

Despite rapid clinical translation of COVID-19 vaccines in response to the global pandemic, an opportunity remains for vaccine technology innovation to address current limitations and meet challenges of inevitable future pandemics. We describe a universal vaccine cell (UVC) genetically engineered to mimic natural physiological immunity induced upon viral infection of host cells. Cells engineered to express the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike as a representative viral antigen induce robust neutralizing antibodies in immunized non-human primates. Similar titers generated in this established non-human primate (NHP) model have translated into protective human neutralizing antibody levels in SARS-CoV-2-vaccinated individuals. Animals vaccinated with ancestral spike antigens and subsequently challenged with SARS-CoV-2 Delta variant in a heterologous challenge have an approximately 3 log decrease in viral subgenomic RNA in the lungs. This cellular vaccine is designed as a scalable cell line with a modular poly-antigenic payload, allowing for rapid, large-scale clinical manufacturing and use in an evolving viral variant environment.

Mucosal-homing natural killer cells are associated with aging in persons living with HIV.

Natural killer (NK) cells are critical modulators of HIV transmission and disease. Recent evidence suggests a loss of NK cell cytotoxicity during aging, yet analysis of NK cell biology and aging in people with HIV (PWH) is lacking. Herein, we perform comprehensive analyses of people aging with and without HIV to determine age-related NK phenotypic changes. Utilizing high-dimensional flow cytometry, we analyze 30 immune-related proteins on peripheral NK cells from healthy donors, PWH with viral suppression, and viremic PWH. NK cell phenotypes are dynamic across aging but change significantly in HIV and on antiretroviral drug therapy (ART). NK cells in healthy aging show increasing ⍺4ß7 and decreasing CCR7 expression and a reverse phenomenon in PWH. These HIV-associated trafficking patterns could be due to NK cell recruitment to HIV reservoir formation in lymphoid tissue or failed mucosal signaling in the HIV-infected gut but appear to be tight delineators of age-related NK cell changes.

Does Neuromodulation Reduce Chronic Pain Patient Emergency Department Utilization?

BACKGROUND: Chronic pain (CP) affects roughly 100 million adults in the United States. These subjects present disproportionately to the emergency department (ED). Neuromodulation (NM) has been shown to reduce ED visits longitudinally in subjects. OBJECTIVE: To compare ED utilization rates between subjects with CP with and without NM. METHODS: Subjects with failed back surgery syndrome, complex regional pain syndrome, or neuropathic pain diagnosis who visited the hospital between January 1, 2019, and December 31, 2019, were included. Subjects were divided into a NM-treated cohort and a non-NM cohort. Demographic information, medications, and pain provider visits were obtained. Pain-related ED visits between 2017 and 2019 were compared. RESULTS: A total of 2516 subjects were identified; 291 (11.6%) previously underwent NM. The non-NM cohort had significantly higher rate of pain-related ED visits compared with the NM cohort (15.1% vs 10.0%, P = .018). Younger age (odds ratio [OR] = 0.888 [0.843-0.935]), shorter distance to the hospital (OR = 0.807 [0.767-0.849]), lower household income (OR = 0.865 [0.831-0.901]), opioid use (OR = 1.375 [1.291-1.465]), nonopioid use (OR = 1.079 [1.033-1.128]), and non-NM therapy (OR = 1.751 [1.283-2.390]) were significant predictors of ED visits. Opioid use was the only significant predictor (OR = 6.124 [1.417-26.473]) associated with ED visits in the NM cohort. CONCLUSION: Subjects who underwent NM had fewer visits to the ED when compared with similar subjects who received conventional treatment. Opioid use prompted increased ED utilization in both cohorts. We posit that NM leads to improvement in pain outcomes, integration with multidisciplinary pain specialists, and reduction in severity and frequency of acute pain exacerbations, thereby limiting health care resource utilization.

Preoperative evaluation of coagulation status in neuromodulation patients.

OBJECTIVE: The incidence of hemorrhage in patients who undergo deep brain stimulation (DBS) and spinal cord stimulation (SCS) is between 0.5% and 2.5%. Coagulation status is one of the factors that can predispose patients to the development of these complications. As a routine part of preoperative assessment, the authors obtain prothrombin time (PT), partial thromboplastin time (PTT), and platelet count. However, insurers often cover only PT/PTT laboratory tests if the patient is receiving warfarin/heparin. The authors aimed to examine their experience with abnormal coagulation parameters in patients who underwent neuromodulation. METHODS: Patients who underwent neuromodulation (SCS, DBS, or intrathecal pump implantation) over a 9-year period and had preoperative laboratory values available were included. The authors determined abnormal values on the basis of a clinical protocol utilized at their practice, which combined the normal ranges of the laboratory tests and clinical relevance. This protocol had cutoff values of 12 seconds and 39 seconds for PT and PTT, respectively, and < 120,000 platelets/µl. The authors identified risk factors for these abnormalities and described interventions. RESULTS: Of the 1767 patients who met the inclusion criteria, 136 had abnormal preoperative laboratory values. Five of these 136 patients had values that were misclassified as abnormal because they were within the normal ranges at the outside facility where they were tested. Fifty-one patients had laboratory values outside the ranges of our protocol, but the surgeons reviewed and approved these patients without further intervention. Of the remaining 80 patients, 8 had known coagulopathies and 24 were receiving warfarin/heparin. The remaining 48 patients were receiving other anticoagulant/antiplatelet medications. These included apixaban/rivaroxaban/dabigatran anticoagulants (n = 22; mean ± SD PT 13.7 ± 2.5 seconds) and aspirin/clopidogrel/other antiplatelet medications (n = 26; mean ± SD PT 14.4 ± 5.8 seconds). Eight new coagulopathies were identified and further investigated with hematological analysis. CONCLUSIONS: New anticoagulants and antiplatelet medications are not monitored with PT/PTT, but they affect coagulation status and laboratory values. Although platelet function tests aid in a subset of medications, it is more difficult to assess the coagulation status of patients receiving novel anticoagulants. PT/PTT may provide value preoperatively.

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.

Identification of the predominant human NK cell effector subset mediating ADCC against HIV-infected targets coated with BNAbs or plasma from PLWH.

The potential of immunotherapy strategies utilizing broadly neutralizing antibodies (BNAbs), such as 3BNC117 and 10-1074, to limit viral replication while also facilitating clearance of HIV infected cells has heightened interest in identifying the predominant NK effector subset(s) capable of mediating antibody dependent cellular cytotoxicity (ADCC). Utilizing advanced polychromatic flow cytometry, we identified that CD57 positive NK cells from ART-suppressed in People Living With HIV (PLWH) expressed significantly higher levels of the CD16 FcγR receptor, 2B4 ADCC coreceptor, and HLA-DR activation marker while NKG2C positive NK cells expressed significantly higher levels of the CD2 ADCC coreceptor (p < 0.001, n = 32). Functionally, CD57 positive NK cells from ART-suppressed PLWH with either high or low NKG2C expansion exhibited significantly enhanced degranulation and IFN-γ production against heterologous gp120-coated ADCC targets coated with HIV reference plasma compared to CD57 negative NK cells (p = 0.0029, n = 11). CD57 positive NK cells from control donors lacking NKG2C expansion also exhibited significantly more degranulation and IFN-γ production at every timepoint tested against both heterologous ADCC targets (p = 0.019, n = 9) and HIV-1 infected autologous CD4+ primary T cells coated with BNAbs. Together, our data support CD57 positive and NKG2C positive NK cells as the predominant ADCC effector subsets capable of targeting HIV-infected CD4+ cells in the presence of 3BNC117 and 10-1074 immunotherapy.

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.

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

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