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.

An NK-like CAR T cell transition in CAR T cell dysfunction.

Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable success in hematological malignancies but remains ineffective in solid tumors, due in part to CAR T cell exhaustion in the solid tumor microenvironment. To study dysfunction of mesothelin-redirected CAR T cells in pancreatic cancer, we establish a robust model of continuous antigen exposure that recapitulates hallmark features of T cell exhaustion and discover, both in vitro and in CAR T cell patients, that CAR dysregulation is associated with a CD8+ T-to-NK-like T cell transition. Furthermore, we identify a gene signature defining CAR and TCR dysregulation and transcription factors, including SOX4 and ID3 as key regulators of CAR T cell exhaustion. Our findings shed light on the plasticity of human CAR T cells and demonstrate that genetic downmodulation of ID3 and SOX4 expression can improve the efficacy of CAR T cell therapy in solid tumors by preventing or delaying CAR T cell dysfunction.

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.

Tissue Determinants of Human NK Cell Development, Function, and Residence.

Immune responses in diverse tissue sites are critical for protective immunity and homeostasis. Here, we investigate how tissue localization regulates the development and function of human natural killer (NK) cells, innate lymphocytes important for anti-viral and tumor immunity. Integrating high-dimensional analysis of NK cells from blood, lymphoid organs, and mucosal tissue sites from 60 individuals, we identify tissue-specific patterns of NK cell subset distribution, maturation, and function maintained across age and between individuals. Mature and terminally differentiated NK cells with enhanced effector function predominate in blood, bone marrow, spleen, and lungs and exhibit shared transcriptional programs across sites. By contrast, precursor and immature NK cells with reduced effector capacity populate lymph nodes and intestines and exhibit tissue-resident signatures and site-specific adaptations. Together, our results reveal anatomic control of NK cell development and maintenance as tissue-resident populations, whereas mature, terminally differentiated subsets mediate immunosurveillance through diverse peripheral sites. VIDEO ABSTRACT.

Multi-Omics Resolves a Sharp Disease-State Shift between Mild and Moderate COVID-19.

We present an integrated analysis of the clinical measurements, immune cells, and plasma multi-omics of 139 COVID-19 patients representing all levels of disease severity, from serial blood draws collected during the first week of infection following diagnosis. We identify a major shift between mild and moderate disease, at which point elevated inflammatory signaling is accompanied by the loss of specific classes of metabolites and metabolic processes. Within this stressed plasma environment at moderate disease, multiple unusual immune cell phenotypes emerge and amplify with increasing disease severity. We condensed over 120,000 immune features into a single axis to capture how different immune cell classes coordinate in response to SARS-CoV-2. This immune-response axis independently aligns with the major plasma composition changes, with clinical metrics of blood clotting, and with the sharp transition between mild and moderate disease. This study suggests that moderate disease may provide the most effective setting for therapeutic intervention.

Denisovan, modern human and mouse TNFAIP3 alleles tune A20 phosphorylation and immunity.

Resisting and tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here genomic analyses of anatomically modern humans, extinct Denisovan hominins and mice revealed a TNFAIP3 allelic series with alterations in the encoded immune response inhibitor A20. Each TNFAIP3 allele encoded substitutions at non-catalytic residues of the ubiquitin protease OTU domain that diminished IκB kinase-dependent phosphorylation and activation of A20. Two TNFAIP3 alleles encoding A20 proteins with partial phosphorylation deficits seemed to be beneficial by increasing immunity without causing spontaneous inflammatory disease: A20 T108A;I207L, originating in Denisovans and introgressed in modern humans throughout Oceania, and A20 I325N, from an N-ethyl-N-nitrosourea (ENU)-mutagenized mouse strain. By contrast, a rare human TNFAIP3 allele encoding an A20 protein with 95% loss of phosphorylation, C243Y, caused spontaneous inflammatory disease in humans and mice. Analysis of the partial-phosphorylation A20 I325N allele in mice revealed diminished tolerance of bacterial lipopolysaccharide and poxvirus inoculation as tradeoffs for enhanced immunity.

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.

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.

The transcriptional landscape of αß T cell differentiation.

The differentiation of αßT cells from thymic precursors is a complex process essential for adaptive immunity. Here we exploited the breadth of expression data sets from the Immunological Genome Project to analyze how the differentiation of thymic precursors gives rise to mature T cell transcriptomes. We found that early T cell commitment was driven by unexpectedly gradual changes. In contrast, transit through the CD4(+)CD8(+) stage involved a global shutdown of housekeeping genes that is rare among cells of the immune system and correlated tightly with expression of the transcription factor c-Myc. Selection driven by major histocompatibility complex (MHC) molecules promoted a large-scale transcriptional reactivation. We identified distinct signatures that marked cells destined for positive selection versus apoptotic deletion. Differences in the expression of unexpectedly few genes accompanied commitment to the CD4(+) or CD8(+) lineage, a similarity that carried through to peripheral T cells and their activation, demonstrated by mass cytometry phosphoproteomics. The transcripts newly identified as encoding candidate mediators of key transitions help define the 'known unknowns' of thymocyte differentiation.

Identification of transcriptional regulators in the mouse immune system.

The differentiation of hematopoietic stem cells into cells of the immune system has been studied extensively in mammals, but the transcriptional circuitry that controls it is still only partially understood. Here, the Immunological Genome Project gene-expression profiles across mouse immune lineages allowed us to systematically analyze these circuits. To analyze this data set we developed Ontogenet, an algorithm for reconstructing lineage-specific regulation from gene-expression profiles across lineages. Using Ontogenet, we found differentiation stage-specific regulators of mouse hematopoiesis and identified many known hematopoietic regulators and 175 previously unknown candidate regulators, as well as their target genes and the cell types in which they act. Among the previously unknown regulators, we emphasize the role of ETV5 in the differentiation of γδ T cells. As the transcriptional programs of human and mouse cells are highly conserved, it is likely that many lessons learned from the mouse model apply to humans.

Activating Receptors for Self-MHC Class I Enhance Effector Functions and Memory Differentiation of NK Cells during Mouse Cytomegalovirus Infection.

Natural killer (NK) cells are important in host defense against pathogens, and they can subsequently differentiate into memory NK cells. The Ly49 and KIR gene families in rodents and humans encode both inhibitory and activating receptors for MHC class I. The physiological role of activating KIR or Ly49 receptors that recognize self-MHC class I during immune response to viral infections is unknown. Here, we address how the activating Ly49D receptor impacts the NK cell response to mouse cytomegalovirus (MCMV) infection by comparing the activation and differentiation of Ly49D-bearing NK cells in mice lacking or expressing H-2D(d), the cognate MHC class I ligand of Ly49D. After MCMV infection, Ly49D augmented IFN-γ production by MCMV-specific Ly49H(+) NK cells and preferentially promoted the generation of memory Ly49H(+) NK cells. Thus, activating receptors for self-MHC class I modulate the differentiation of MCMV-specific NK cells and are beneficial for host defense against MCMV infection.

Natural killer cell education and tolerance.

Natural killer (NK) cells play a key role in the immune response to certain infections and malignancies by direct cytolysis of infected or transformed cells and by secretion of potent immune mediators. NK cells express an array of activating receptors that recognize self-molecules. If not restrained by inhibitory receptors recognizing major histocompatibility complex (MHC) class I proteins on the surface of self cells, NK cells are able to kill normal, healthy cells. Not all NK cells express inhibitory receptors for self-MHC class I; thus, other tolerance mechanisms are necessary to prevent NK cell-mediated autoimmunity. Here we review the major mechanisms of NK cell education and tolerance.

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.

Mass cytometry reveals single-cell kinetics of cytotoxic lymphocyte evolution in CMV-infected renal transplant patients.

Cytomegalovirus (CMV) infection is associated with graft rejection in renal transplantation. Memory-like natural killer (NK) cells expressing NKG2C and lacking FcεRIγ are established during CMV infection. Additionally, CD8+ T cells expressing NKG2C have been observed in some CMV-seropositive patients. However, in vivo kinetics detailing the development and differentiation of these lymphocyte subsets during CMV infection remain limited. Here, we interrogated the in vivo kinetics of lymphocytes in CMV-infected renal transplant patients using longitudinal samples compared with those of nonviremic (NV) patients. Recipient CMV-seropositive (R+) patients had preexisting memory-like NK cells (NKG2C+CD57+FcεRIγ-) at baseline, which decreased in the periphery immediately after transplantation in both viremic and NV patients. We identified a subset of prememory-like NK cells (NKG2C+CD57+FcεRIγlow-dim) that increased during viremia in R+ viremic patients. These cells showed a higher cytotoxic profile than preexisting memory-like NK cells with transient up-regulation of FcεRIγ and Ki67 expression at the acute phase, with the subsequent accumulation of new memory-like NK cells at later phases of viremia. Furthermore, cytotoxic NKG2C+CD8+ T cells and γδ T cells significantly increased in viremic patients but not in NV patients. These three different cytotoxic cells combinatorially responded to viremia, showing a relatively early response in R+ viremic patients compared with recipient CMV-seronegative viremic patients. All viremic patients, except one, overcame viremia and did not experience graft rejection. These data provide insights into the in vivo dynamics and interplay of cytotoxic lymphocytes responding to CMV viremia, which are potentially linked with control of CMV viremia to prevent graft rejection.

Molecular definition of the identity and activation of natural killer cells.

Using whole-genome microarray data sets of the Immunological Genome Project, we demonstrate a closer transcriptional relationship between NK cells and T cells than between any other leukocytes, distinguished by their shared expression of genes encoding molecules with similar signaling functions. Whereas resting NK cells are known to share expression of a few genes with cytotoxic CD8(+) T cells, our transcriptome-wide analysis demonstrates that the commonalities extend to hundreds of genes, many encoding molecules with unknown functions. Resting NK cells demonstrate a 'preprimed' state compared with naive T cells, which allows NK cells to respond more rapidly to viral infection. Collectively, our data provide a global context for known and previously unknown molecular aspects of NK cell identity and function by delineating the genome-wide repertoire of gene expression of NK cells in various states.

SURE gel electrophoresis: A method for improved detection and purification of dilute nucleic acid samples.

Agarose gel electrophoresis is performed routinely by molecular biologists as both an analytical and a preparative method for characterization of nucleic acids. Gel analysis of highly dilute DNA solutions is challenging because of the limited sensitivity of detection available with conventional methods. In this study a new approach is described for concentrating samples directly within gels called SURE (successive reloading) electrophoresis. The approach involves loading of dilute samples multiple times into a single well, with each loading followed by a brief pulse of electrical current before the next sample is loaded. The procedure generates single bands created by molecular stacking that exhibit strongly enhanced signal intensities and minimal band broadening. Using optimized voltages and time intervals as many as 20 successive loadings could be performed and up to 800 µL could be loaded into a single well. Gel extraction and fluorescent quantitation demonstrated that approximately 97 % of the DNA from each loading was incorporated into the resultant band. Highly dilute DNA samples (<0.0007 ng per microliter) could be readily detected after six loadings. The method produced good results with either TAE or TBE as electrophoresis buffers, using loading dyes with or without SDS, and in both minigels and large gels.

Natural Killer Cell Memory.

Natural killer (NK) cells have historically been considered short-lived cytolytic cells that can rapidly respond against pathogens and tumors in an antigen-independent manner and then undergo cell death. Recently, however, NK cells have been shown to possess traits of adaptive immunity and can acquire immunological memory in a manner similar to that of T and B cells. In this review, we discuss evidence of NK cell memory and the mechanisms involved in the generation and survival of these innate lymphocytes.

Epigenetic modification and antibody-dependent expansion of memory-like NK cells in human cytomegalovirus-infected individuals.

Long-lived "memory-like" NK cells have been identified in individuals infected by human cytomegalovirus (HCMV), but little is known about how the memory-like NK cell pool is formed. Here, we have shown that HCMV-infected individuals have several distinct subsets of memory-like NK cells that are often deficient for multiple transcription factors and signaling proteins, including tyrosine kinase SYK, for which the reduced expression was stable over time and correlated with epigenetic modification of the gene promoter. Deficient expression of these proteins was largely confined to the recently discovered FcRγ-deficient NK cells that display enhanced antibody-dependent functional activity. Importantly, FcRγ-deficient NK cells exhibited robust preferential expansion in response to virus-infected cells (both HCMV and influenza) in an antibody-dependent manner. These findings suggest that the memory-like NK cell pool is shaped and maintained by a mechanism that involves both epigenetic modification of gene expression and antibody-dependent expansion.

Mortality in adults with sickle cell disease: Results from the sickle cell disease implementation consortium (SCDIC) registry.

The cause of death in people affected by sickle cell disease (SCD) is often challenging to define as prior studies have used retrospective or administrative data for analysis. We used a prospective longitudinal registry to assess mortality and clinical co-morbidities among subjects enrolled in the Sickle Cell Disease Implementation Consortium (SCDIC) registry. At enrollment, we collected the following data: patient-reported demographics, SCD phenotype, baseline laboratory values, comorbidities, and current medications. Subjects were followed for a median of 4.7 years before the present analysis. The relationship of clinical co-morbidities (at time of enrollment) to mortality was determined using survival analysis, adjusting for SCD phenotype and gender. There was a total of 2439 people with SCD enrolled in the SCDIC registry. One hundred and twenty-eight participants (5%) died during the observation period (2017-2022). Six people died from trauma and were excluded from further analysis. Proximate cause of death was unwitnessed in 17% of the deaths, but commonest causes of death include cardiac (18%), acute chest or respiratory failure (11%), sudden unexplained death (8%). Enrollment characteristics of the individuals who died (n = 122) were compared to those of survivors (n = 2317). Several co-morbidities at enrollment increased the odds of death on univariate analysis. All co-morbidities were included in a multivariable model. After backward elimination, iron overload, pulmonary hypertension, and depression, remained statistically significant predictors of the risk of death. SCD reduces life expectancy. Improved comprehensive and supportive care to prevent end-organ damage and address comorbidities is needed for this population.

Influence of Self-MHC Class I Recognition on the Dynamics of NK Cell Responses to Cytomegalovirus Infection.

Although interactions between inhibitory Ly49 receptors and their self-MHC class I ligands in C57BL/6 mice are known to limit NK cell proliferation during mouse CMV (MCMV) infection, we created a 36-marker mass cytometry (CyTOF) panel to investigate how these inhibitory receptors impact the NK cell response to MCMV in other phenotypically measurable ways. More than two thirds of licensed NK cells (i.e., those expressing Ly49C, Ly49I, or both) in uninfected mice had already differentiated into NK cells with phenotypes indicative of Ag encounter (KLRG1+Ly6C-) or memory-like status (KLRG1+Ly6C+). These pre-existing KLRG1+Ly6C+ NK cells resembled known Ag-specific memory NK cell populations in being less responsive to IL-18 and IFN-α stimulation in vitro and by selecting for NK cell clones with elevated expression of a Ly49 receptor. During MCMV infection, the significant differences between licensed and unlicensed (Ly49C-Ly49I-) NK cells disappeared within both CMV-specific (Ly49H+) and nonspecific (Ly49H-) responses. This lack of heterogeneity carried into the memory phase, with only a difference in CD16 expression manifesting between licensed and unlicensed MCMV-specific memory NK cell populations. Our results suggest that restricting proliferation is the predominant effect licensing has on the NK cell population during MCMV infection, but the inhibitory Ly49-MHC interactions that take place ahead of infection contribute to their limited expansion by shrinking the pool of licensed NK cells capable of robustly responding to new challenges.