High-dimensional single-cell analysis of human natural killer cell heterogeneity.

Natural killer (NK) cells are innate lymphoid cells (ILCs) contributing to immune responses to microbes and tumors. Historically, their classification hinged on a limited array of surface protein markers. Here, we used single-cell RNA sequencing (scRNA-seq) and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) to dissect the heterogeneity of NK cells. We identified three prominent NK cell subsets in healthy human blood: NK1, NK2 and NK3, further differentiated into six distinct subgroups. Our findings delineate the molecular characteristics, key transcription factors, biological functions, metabolic traits and cytokine responses of each subgroup. These data also suggest two separate ontogenetic origins for NK cells, leading to divergent transcriptional trajectories. Furthermore, we analyzed the distribution of NK cell subsets in the lung, tonsils and intraepithelial lymphocytes isolated from healthy individuals and in 22 tumor types. This standardized terminology aims at fostering clarity and consistency in future research, thereby improving cross-study comparisons.

Five decades of natural killer cell discovery.

The first descriptions of "non-specific" killing of tumor cells by lymphocytes were reported in 1973, and subsequently, the mediators of the activity were named "natural killer" (NK) cells by Rolf Kiessling and colleagues at the Karolinska Institute in 1975. The activity was detected in mice, rats, and humans that had no prior exposure to the tumors, major histocompatibility complex (MHC) antigen matching of the effectors and tumor cells was not required, and the cells responsible were distinct from MHC-restricted, antigen-specific T cells. In the ensuing five decades, research by many labs has extended knowledge of NK cells beyond an in vitro curiosity to demonstrate their in vivo relevance in host defense against tumors and microbial pathogens and their role in regulation of the immune system. This brief Perspective highlights a timeline of a few selected advancements in NK cell biology from a personal perspective of being involved in this quest.

Hepatocellular carcinoma after direct-acting antivirals for hepatitis C is associated with KIR-HLA types predicting weak NK cell-mediated immunity.

BACKGROUND AND AIMS: Second-generation direct-acting antivirals (2G DAA) to cure HCV have led to dramatic clinical improvements. HCV-associated hepatocellular carcinoma (HCC), however, remains common. Impaired immune tumor surveillance may play a role in HCC development. Our cohort evaluated the effects of innate immune types and clinical variables on outcomes including HCC. METHODS: Participants underwent full HLA class I/KIR typing and long-term HCV follow-up. RESULTS: A total of 353 HCV+ participants were followed for a mean of 7 years. Cirrhosis: 25% at baseline, developed in 12% during follow-up. 158 participants received 2G DAA therapy. HCC developed without HCV therapy in 20 subjects, 24 HCC after HCV therapy, and 10 of these after 2G DAA. Two predictors of HCC among 2G DAA-treated patients: cirrhosis (OR, 10.0, p = 0.002) and HLA/KIR profiles predicting weak natural killer (NK) cell-mediated immunity (NK cell complementation groups 6, 9, 11, 12, OR of 5.1, p = 0.02). Without 2G DAA therapy: cirrhosis was the main clinical predictor of HCC (OR, 30.8, p < 0.0001), and weak NK-cell-mediated immunity did not predict HCC. CONCLUSION: Cirrhosis is the main risk state predisposing to HCC, but weak NK-cell-mediated immunity may predispose to post-2G DAA HCC more than intermediate or strong NK-cell-mediated immunity.

Pretreatment with IL-15 and IL-18 rescues natural killer cells from granzyme B-mediated apoptosis after cryopreservation.

Human natural killer (NK) cell-based therapies are under assessment for treating various cancers, but cryopreservation reduces both the recovery and function of NK cells, thereby limiting their therapeutic feasibility. Using cryopreservation protocols optimized for T cells, here we find that ~75% of NK cells die within 24 h post-thaw, with the remaining cells displaying reduced cytotoxicity. Using CRISPR-Cas9 gene editing and confocal microscopy, we find that cryopreserved NK cells largely die via apoptosis initiated by leakage of granzyme B from cytotoxic vesicles. Pretreatment of NK cells with a combination of Interleukins-15 (IL-15) and IL-18 prior to cryopreservation improves NK cell recovery to ~90-100% and enables equal tumour control in a xenograft model of disseminated Raji cell lymphoma compared to non-cryopreserved NK cells. The mechanism of IL-15 and IL-18-induced protection incorporates two mechanisms: a transient reduction in intracellular granzyme B levels via degranulation, and the induction of antiapoptotic genes.

PVRL2 Suppresses Antitumor Immunity through PVRIG- and TIGIT-independent Pathways.

Poliovirus receptor-related 2 (PVRL2, also known as nectin-2 or CD112) is believed to act as an immune checkpoint protein in cancer; however, most insight into its role is inferred from studies on its known receptor, poliovirus receptor (PVR)-related immunoglobulin domain protein (PVRIG, also known as CD112R). Here, we study PVRL2 itself. PVRL2 levels were found to be high in tumor cells and tumor-derived exosomes. Deletion of PVRL2 in multiple syngeneic mouse models of cancer showed a dramatic reduction in tumor growth that was immune dependent. This effect was even greater than that seen with deletion of PD-L1. PVRL2 was shown to function by suppressing CD8+ T and natural killer cells in the tumor microenvironment. The loss of PVRL2 suppressed tumor growth even in the absence of PVRIG. In contrast, PVRIG loss showed no additive effect in the absence of PVRL2. T-cell immunoreceptor with Ig and ITIM domains (TIGIT) blockade combined with PVRL2 deletion resulted in a near complete block in tumor growth. This effect was not recapitulated by the combined deletion of PVRL2 with its paralog, PVR, which is the ligand for TIGIT. These data uncover PVRL2 as a distinct inhibitor of the antitumor immune response with functions beyond that of its known receptor PVRIG. Moreover, the data provide a strong rationale for combinatorial targeting of PVRL2 and TIGIT for cancer immunotherapy.

Integrative systems biology reveals NKG2A-biased immune responses correlate with protection in infectious disease, autoimmune disease, and cancer.

Infection, autoimmunity, and cancer are principal human health challenges of the 21st century. Often regarded as distinct ends of the immunological spectrum, recent studies hint at potential overlap between these diseases. For example, inflammation can be pathogenic in infection and autoimmunity. T resident memory (TRM) cells can be beneficial in infection and cancer. However, these findings are limited by size and scope; exact immunological factors shared across diseases remain elusive. Here, we integrate large-scale deeply clinically and biologically phenotyped human cohorts of 526 patients with infection, 162 with lupus, and 11,180 with cancer. We identify an NKG2A+ immune bias as associative with protection against disease severity, mortality, and autoimmune/post-acute chronic disease. We reveal that NKG2A+ CD8+ T cells correlate with reduced inflammation and increased humoral immunity and that they resemble TRM cells. Our results suggest NKG2A+ biases as a cross-disease factor of protection, supporting suggestions of immunological overlap between infection, autoimmunity, and cancer.

ITAM-based receptors in natural killer cells.

The ability of cells of the immune system to acquire features such as increased longevity and enhanced secondary responses was long thought to be restricted to cells of the adaptive immune system. Natural killer (NK) cells have challenged this notion by demonstrating that they can also gain adaptive features. This has been observed in both humans and mice during infection with cytomegalovirus (CMV). The generation of adaptive NK cells requires antigen-specific recognition of virally infected cells through stimulatory NK receptors. These receptors lack the ability to signal on their own and rather rely on adaptor molecules that contain ITAMs for driving signals. Here, we highlight our understanding of how these receptors influence the production of adaptive NK cells and propose areas in the field that merit further investigation.

MICB Genomic Variant Is Associated with NKG2D-mediated Acute Lung Injury and Death.

Rationale: Acute lung injury (ALI) carries a high risk of mortality but has no established pharmacologic therapy. We previously found that experimental ALI occurs through natural killer (NK) cell NKG2D receptor activation and that the cognate human ligand, MICB, was associated with ALI after transplantation. Objectives: To investigate the association of a common missense variant, MICBG406A, with ALI. Methods: We assessed MICBG406A genotypes within two multicenter observational study cohorts at risk for ALI: primary graft dysfunction (N = 619) and acute respiratory distress syndrome (N = 1,376). Variant protein functional effects were determined in cultured and ex vivo human samples. Measurements and Main Results: Recipients of MICBG406A-homozygous allografts had an 11.1% absolute risk reduction (95% confidence interval [CI], 3.2-19.4%) for severe primary graft dysfunction after lung transplantation and reduced risk for allograft failure (hazard ratio, 0.36; 95% CI, 0.13-0.98). In participants with sepsis, we observed 39% reduced odds of moderately or severely impaired oxygenation among MICBG406A-homozygous individuals (95% CI, 0.43-0.86). BAL NK cells were less frequent and less mature in participants with MICBG406A. Expression of missense variant protein MICBD136N in cultured cells resulted in reduced surface MICB and reduced NKG2D ligation relative to wild-type MICB. Coculture of variant MICBD136N cells with NK cells resulted in less NKG2D activation and less susceptibility to NK cell killing relative to the wild-type cells. Conclusions: These data support a role for MICB signaling through the NKG2D receptor in mediating ALI, suggesting a novel therapeutic approach.

Hypoimmune induced pluripotent stem cells survive long term in fully immunocompetent, allogeneic rhesus macaques.

Genetic engineering of allogeneic cell therapeutics that fully prevents rejection by a recipient's immune system would abolish the requirement for immunosuppressive drugs or encapsulation and support large-scale manufacturing of off-the-shelf cell products. Previously, we generated mouse and human hypoimmune pluripotent (HIP) stem cells by depleting HLA class I and II molecules and overexpressing CD47 (B2M-/-CIITA-/-CD47+). To determine whether this strategy is successful in non-human primates, we engineered rhesus macaque HIP cells and transplanted them intramuscularly into four allogeneic rhesus macaques. The HIP cells survived unrestricted for 16 weeks in fully immunocompetent allogeneic recipients and differentiated into several lineages, whereas allogeneic wild-type cells were vigorously rejected. We also differentiated human HIP cells into endocrinologically active pancreatic islet cells and showed that they survived in immunocompetent, allogeneic diabetic humanized mice for 4 weeks and ameliorated diabetes. HIP-edited primary rhesus macaque islets survived for 40 weeks in an allogeneic rhesus macaque recipient without immunosuppression, whereas unedited islets were quickly rejected.

CCR5 drives NK cell-associated airway damage in pulmonary ischemia-reperfusion injury.

Primary graft dysfunction (PGD) limits clinical benefit after lung transplantation, a life-prolonging therapy for patients with end-stage disease. PGD is the clinical syndrome resulting from pulmonary ischemia-reperfusion injury (IRI), driven by innate immune inflammation. We recently demonstrated a key role for NK cells in the airways of mouse models and human tissue samples of IRI. Here, we used 2 mouse models paired with human lung transplant samples to investigate the mechanisms whereby NK cells migrate to the airways to mediate lung injury. We demonstrate that chemokine receptor ligand transcripts and proteins are increased in mouse and human disease. CCR5 ligand transcripts were correlated with NK cell gene signatures independently of NK cell CCR5 ligand secretion. NK cells expressing CCR5 were increased in the lung and airways during IRI and had increased markers of tissue residency and maturation. Allosteric CCR5 drug blockade reduced the migration of NK cells to the site of injury. CCR5 blockade also blunted quantitative measures of experimental IRI. Additionally, in human lung transplant bronchoalveolar lavage samples, we found that CCR5 ligand was associated with increased patient morbidity and that the CCR5 receptor was increased in expression on human NK cells following PGD. These data support a potential mechanism for NK cell migration during lung injury and identify a plausible preventative treatment for PGD.

An NKG2A biased immune response confers protection for infection, autoimmune disease, and cancer.

Infection, autoimmunity, and cancer are the principal human health challenges of the 21st century and major contributors to human death and disease. Often regarded as distinct ends of the immunological spectrum, recent studies have hinted there may be more overlap between these diseases than appears. For example, pathogenic inflammation has been demonstrated as conserved between infection and autoimmune settings. T resident memory (TRM) cells have been highlighted as beneficial for infection and cancer. However, these findings are limited by patient number and disease scope; exact immunological factors shared across disease remain elusive. Here, we integrate large-scale deeply clinically and biologically phenotyped human cohorts of 526 patients with infection, 162 with lupus, and 11,180 with cancer. We identify an NKG2A+ immune bias as associative with protection against disease severity, mortality, and autoimmune and post-acute chronic disease. We reveal that NKG2A+ CD8+ T cells correlate with reduced inflammation, increased humoral immunity, and resemble TRM cells. Our results suggest that an NKG2A+ bias is a pan-disease immunological factor of protection and thus supports recent suggestions that there is immunological overlap between infection, autoimmunity, and cancer. Our findings underscore the promotion of an NKG2A+ biased response as a putative therapeutic strategy.

Exhausted intratumoral Vδ2- γδ T cells in human kidney cancer retain effector function.

Gamma delta (γδ) T cells reside within human tissues including tumors, but their function in mediating antitumor responses to immune checkpoint inhibition is unknown. Here we show that kidney cancers are infiltrated by Vδ2- γδ T cells, with equivalent representation of Vδ1+ and Vδ1- cells, that are distinct from γδ T cells found in normal human tissues. These tumor-resident Vδ2- T cells can express the transcriptional program of exhausted αß CD8+ T cells as well as canonical markers of terminal T-cell exhaustion including PD-1, TIGIT and TIM-3. Although Vδ2- γδ T cells have reduced IL-2 production, they retain expression of cytolytic effector molecules and co-stimulatory receptors such as 4-1BB. Exhausted Vδ2- γδ T cells are composed of three distinct populations that lack TCF7, are clonally expanded and express cytotoxic molecules and multiple Vδ2- T-cell receptors. Human tumor-derived Vδ2- γδ T cells maintain cytotoxic function and pro-inflammatory cytokine secretion in vitro. The transcriptional program of Vδ2- T cells in pretreatment tumor biopsies was used to predict subsequent clinical responses to PD-1 blockade in patients with cancer. Thus, Vδ2- γδ T cells within the tumor microenvironment can contribute to antitumor efficacy.

The interweaved signatures of common-gamma-chain cytokines across immunologic lineages.

"γc" cytokines are a family whose receptors share a "common-gamma-chain" signaling moiety, and play central roles in differentiation, homeostasis, and communications of all immunocyte lineages. As a resource to better understand their range and specificity of action, we profiled by RNAseq the immediate-early responses to the main γc cytokines across all immunocyte lineages. The results reveal an unprecedented landscape: broader, with extensive overlap between cytokines (one cytokine doing in one cell what another does elsewhere) and essentially no effects unique to any one cytokine. Responses include a major downregulation component and a broad Myc-controlled resetting of biosynthetic and metabolic pathways. Various mechanisms appear involved: fast transcriptional activation, chromatin remodeling, and mRNA destabilization. Other surprises were uncovered: IL2 effects in mast cells, shifts between follicular and marginal zone B cells, paradoxical and cell-specific cross-talk between interferon and γc signatures, or an NKT-like program induced by IL21 in CD8+ T cells.

The CD16 and CD32b Fc-gamma receptors regulate antibody-mediated responses in mouse natural killer cells.

Natural killer (NK) cells are innate lymphocytes capable of mediating immune responses without prior sensitization. NK cells express Fc-gamma receptors (FcγRs) that engage the Fc region of IgG. Studies investigating the role of FcγRs on mouse NK cells have been limited due to lack specific reagents. In this study, we characterize the expression and biological consequences of activating mouse NK cells through their FcγRs. We demonstrate that most NK cells express the activating CD16 receptor, and a subset of NK cells also expresses the inhibitory CD32b receptor. Critically, these FcγRs are functional on mouse NK cells and can modulate antibody-mediated responses. We also characterized mice with conditional knockout alleles of Fcgr3 (CD16) or Fcgr2b (CD32b) in the NK and innate lymphoid cell (ILC) lineage. NK cells in these mice did not reveal any developmental defects and were responsive to cross-linking activating NK receptors, cytokine stimulation, and killing of YAC-1 targets. Importantly, CD16-deficient NK cells failed to induce antibody-directed cellular cytotoxicity of antibody-coated B-cell lymphomas in in vitro assays. In addition, we demonstrate the important role of CD16 on NK cells using an in vivo model of cancer immunotherapy using anti-CD20 antibody treatment of B-cell lymphomas.

CD16+ natural killer cells in bronchoalveolar lavage are associated with antibody-mediated rejection and chronic lung allograft dysfunction.

Acute and chronic rejections limit the long-term survival after lung transplant. Pulmonary antibody-mediated rejection (AMR) is an incompletely understood driver of long-term outcomes characterized by donor-specific antibodies (DSAs), innate immune infiltration, and evidence of complement activation. Natural killer (NK) cells may recognize DSAs via the CD16 receptor, but this complement-independent mechanism of injury has not been explored in pulmonary AMR. CD16+ NK cells were quantified in 508 prospectively collected bronchoalveolar lavage fluid samples from 195 lung transplant recipients. Associations between CD16+ NK cells and human leukocyte antigen mismatches, DSAs, and AMR grade were assessed by linear models adjusted for participant characteristics and repeat measures. Cox proportional hazards models were used to assess CD16+ NK cell association with chronic lung allograft dysfunction and survival. Bronchoalveolar lavage fluid CD16+ NK cell frequency was associated with increasing human leukocyte antigens mismatches and increased AMR grade. Although NK frequencies were similar between DSA+ and DSA- recipients, CD16+ NK cell frequencies were greater in recipients with AMR and those with concomitant allograft dysfunction. CD16+ NK cells were associated with long-term graft dysfunction after AMR and decreased chronic lung allograft dysfunction-free survival. These data support the role of CD16+ NK cells in pulmonary AMR.

NKG2D receptor activation drives primary graft dysfunction severity and poor lung transplantation outcomes.

Clinical outcomes after lung transplantation, a life-saving therapy for patients with end-stage lung diseases, are limited by primary graft dysfunction (PGD). PGD is an early form of acute lung injury with no specific pharmacologic therapies. Here, we present a large multicenter study of plasma and bronchoalveolar lavage (BAL) samples collected on the first posttransplant day, a critical time for investigations of immune pathways related to PGD. We demonstrated that ligands for NKG2D receptors were increased in the BAL from participants who developed severe PGD and were associated with increased time to extubation, prolonged intensive care unit length of stay, and poor peak lung function. Neutrophil extracellular traps (NETs) were increased in PGD and correlated with BAL TNF-α and IFN-γ cytokines. Mechanistically, we found that airway epithelial cell NKG2D ligands were increased following hypoxic challenge. NK cell killing of hypoxic airway epithelial cells was abrogated with NKG2D receptor blockade, and TNF-α and IFN-γ provoked neutrophils to release NETs in culture. These data support an aberrant NK cell/neutrophil axis in human PGD pathogenesis. Early measurement of stress ligands and blockade of the NKG2D receptor hold promise for risk stratification and management of PGD.

Diminished cell proliferation promotes natural killer cell adaptive-like phenotype by limiting FcεRIγ expression.

Human adaptive-like natural killer (NK) cells express low levels of FcεRIγ (FcRγ-/low) and are reported to accumulate during COVID-19 infection; however, the mechanism underlying and regulating FcRγ expression in NK cells has yet to be fully defined. We observed lower FcRγ protein expression in NK cell subsets from lung transplant patients during rapamycin treatment, suggesting a link with reduced mTOR activity. Further, FcRγ-/low NK cell subsets from healthy donors displayed reduced mTOR activity. We discovered that FcRγ upregulation is dependent on cell proliferation progression mediated by IL-2, IL-15, or IL-12, is sensitive to mTOR suppression, and is inhibited by TGFß or IFNα. Accordingly, the accumulation of adaptive-like FcRγ-/low NK cells in COVID-19 patients corresponded to increased TGFß and IFNα levels and disease severity. Our results show that an adaptive-like NK cell phenotype is induced by diminished cell proliferation and has an early prognostic value for increased TGFß and IFNα levels in COVID-19 infection associated with disease severity.