Natural Killer Cell Dysfunction In Human Bladder Cancer Is Caused By Tissue-Specific Suppression of SLAMF6 Signaling.

NK cells are innate lymphocytes critical for surveillance of viruses and tumors, however the mechanisms underlying NK cell dysfunction in cancer are incompletely understood. We assessed the effector function of NK cells from bladder cancer patients and found severe dysfunction in NK cells derived from tumors versus peripheral blood. While both peripheral and tumor-infiltrating NK cells exhibited conserved patterns of inhibitory receptor over-expression, this did not explain the observed defects in NK surveillance in bladder tumors. Rather, TME-specific TGF-ß and metabolic perturbations such as hypoxia directly suppressed NK cell function. Specifically, an oxygen-dependent reduction in signaling through SLAMF6 was mechanistically responsible for poor NK cell function, as tumor-infiltrating NK cells cultured ex vivo under normoxic conditions exhibited complete restoration of function, while deletion of SLAMF6 abrogated NK cell cytolytic function even under normoxic conditions. Collectively, this work highlights the role of tissue-specific factors in dictating NK cell function, and implicates SLAMF6 signaling as a rational target for immuno-modulation to improve NK cell function in bladder cancer.

Ultrahigh frequencies of peripherally matured LGI1- and CASPR2-reactive B cells characterize the cerebrospinal fluid in autoimmune encephalitis.

Intrathecal synthesis of central nervous system (CNS)-reactive autoantibodies is observed across patients with autoimmune encephalitis (AE), who show multiple residual neurobehavioral deficits and relapses despite immunotherapies. We leveraged two common forms of AE, mediated by leucine-rich glioma inactivated-1 (LGI1) and contactin-associated protein-like 2 (CASPR2) antibodies, as human models to comprehensively reconstruct and profile cerebrospinal fluid (CSF) B cell receptor (BCR) characteristics. We hypothesized that the resultant observations would both inform the observed therapeutic gap and determine the contribution of intrathecal maturation to pathogenic B cell lineages. From the CSF of three patients, 381 cognate-paired IgG BCRs were isolated by cell sorting and scRNA-seq, and 166 expressed as monoclonal antibodies (mAbs). Sixty-two percent of mAbs from singleton BCRs reacted with either LGI1 or CASPR2 and, strikingly, this rose to 100% of cells in clonal groups with ≥4 members. These autoantigen-reactivities were more concentrated within antibody-secreting cells (ASCs) versus B cells (P < 0.0001), and both these cell types were more differentiated than LGI1- and CASPR2-unreactive counterparts. Despite greater differentiation, autoantigen-reactive cells had acquired few mutations intrathecally and showed minimal variation in autoantigen affinities within clonal expansions. Also, limited CSF T cell receptor clonality was observed. In contrast, a comparison of germline-encoded BCRs versus the founder intrathecal clone revealed marked gains in both affinity and mutational distances (P = 0.004 and P < 0.0001, respectively). Taken together, in patients with LGI1 and CASPR2 antibody encephalitis, our results identify CSF as a compartment with a remarkably high frequency of clonally expanded autoantigen-reactive ASCs whose BCR maturity appears dominantly acquired outside the CNS.

Cervical lymph nodes and ovarian teratomas as germinal centres in NMDA receptor-antibody encephalitis.

Autoantibodies against the extracellular domain of the N-methyl-d-aspartate receptor (NMDAR) NR1 subunit cause a severe and common form of encephalitis. To better understand their generation, we aimed to characterize and identify human germinal centres actively participating in NMDAR-specific autoimmunization by sampling patient blood, CSF, ovarian teratoma tissue and, directly from the putative site of human CNS lymphatic drainage, cervical lymph nodes. From serum, both NR1-IgA and NR1-IgM were detected more frequently in NMDAR-antibody encephalitis patients versus controls (both P < 0.0001). Within patients, ovarian teratoma status was associated with a higher frequency of NR1-IgA positivity in serum (OR = 3.1; P < 0.0001) and CSF (OR = 3.8, P = 0.047), particularly early in disease and before ovarian teratoma resection. Consistent with this immunoglobulin class bias, ovarian teratoma samples showed intratumoral production of both NR1-IgG and NR1-IgA and, by single cell RNA sequencing, contained expanded highly-mutated IgA clones with an ovarian teratoma-restricted B cell population. Multiplex histology suggested tertiary lymphoid architectures in ovarian teratomas with dense B cell foci expressing the germinal centre marker BCL6, CD21+ follicular dendritic cells, and the NR1 subunit, alongside lymphatic vessels and high endothelial vasculature. Cultured teratoma explants and dissociated intratumoral B cells secreted NR1-IgGs in culture. Hence, ovarian teratomas showed structural and functional evidence of NR1-specific germinal centres. On exploring classical secondary lymphoid organs, B cells cultured from cervical lymph nodes of patients with NMDAR-antibody encephalitis produced NR1-IgG in 3/7 cultures, from patients with the highest serum NR1-IgG levels (P < 0.05). By contrast, NR1-IgG secretion was observed neither from cervical lymph nodes in disease controls nor in patients with adequately resected ovarian teratomas. Our multimodal evaluations provide convergent anatomical and functional evidence of NMDAR-autoantibody production from active germinal centres within both intratumoral tertiary lymphoid structures and traditional secondary lymphoid organs, the cervical lymph nodes. Furthermore, we develop a cervical lymph node sampling protocol that can be used to directly explore immune activity in health and disease at this emerging neuroimmune interface.

LIR-1 educates expanded human NK cells and defines a unique antitumor NK cell subset with potent antibody-dependent cellular cytotoxicity.

OBJECTIVE: KIR and NKG2A receptors educate human NK cells to stay responsive to cells with diminished HLA class I. Here, we addressed whether the HLA class I-binding receptor LIR-1 (LILRB1/ILT2/CD85j), which is widely expressed on human NK cells, can mediate education and contribute to antitumor functions of NK cells. METHODS: Healthy donor NK cells either unstimulated, overnight cytokine-activated or ex vivo-expanded were used to target human cell lines. Phenotype and function were analysed using flow cytometry and 51Cr-release assays. RESULTS: We found that the inhibitory receptor LIR-1 can mediate NK cell education under specific conditions. This novel finding was exclusive to expanded NK cells and further characterisation of the cells revealed high expression of granzyme B and DNAM-1, which both previously have been linked to NK cell education. Corroborating the rheostat education model, LIR-1 co-expression with an educating KIR further increased the responsiveness of expanded NK cells. Inversely, antibody masking of LIR-1 decreased the responsiveness. LIR-1+ expanded NK cells displayed high intrinsic ADCC that, in contrast to KIR and NKG2A, was not inhibited by HLA class I. CONCLUSION: These findings identify a unique NK cell subset attractive for adoptive cell therapy to treat cancer. Given that LIR-1 binds most HLA class I molecules, this subset may be explored in both autologous and allogeneic settings to innately reject HLA class I- tumor cells as well as HLA class I+ target cells when combined with antitumor antibodies. Further studies are warranted to address the potential of this subset in vivo.

Distinctive binding properties of human monoclonal LGI1 autoantibodies determine pathogenic mechanisms.

Autoantibodies against leucine-rich glioma inactivated 1 (LGI1) are found in patients with limbic encephalitis and focal seizures. Here, we generate patient-derived monoclonal antibodies (mAbs) against LGI1. We explore their sequences and binding characteristics, plus their pathogenic potential using transfected HEK293T cells, rodent neuronal preparations, and behavioural and electrophysiological assessments in vivo after mAb injections into the rodent hippocampus. In live cell-based assays, LGI1 epitope recognition was examined with patient sera (n = 31), CSFs (n = 11), longitudinal serum samples (n = 15), and using mAbs (n = 14) generated from peripheral B cells of two patients. All sera and 9/11 CSFs bound both the leucine-rich repeat (LRR) and the epitempin repeat (EPTP) domains of LGI1, with stable ratios of LRR:EPTP antibody levels over time. By contrast, the mAbs derived from both patients recognized either the LRR or EPTP domain. mAbs against both domain specificities showed varied binding strengths, and marked genetic heterogeneity, with high mutation frequencies. LRR-specific mAbs recognized LGI1 docked to its interaction partners, ADAM22 and ADAM23, bound to rodent brain sections, and induced internalization of the LGI1-ADAM22/23 complex in both HEK293T cells and live hippocampal neurons. By contrast, few EPTP-specific mAbs bound to rodent brain sections or ADAM22/23-docked LGI1, but all inhibited the docking of LGI1 to ADAM22/23. After intrahippocampal injection, and by contrast to the LRR-directed mAbs, the EPTP-directed mAbs showed far less avid binding to brain tissue and were consistently detected in the serum. Post-injection, both domain-specific mAbs abrogated long-term potentiation induction, and LRR-directed antibodies with higher binding strengths induced memory impairment. Taken together, two largely dichotomous populations of LGI1 mAbs with distinct domain binding characteristics exist in the affinity matured peripheral autoantigen-specific memory pools of individuals, both of which have pathogenic potential. In human autoantibody-mediated diseases, the detailed characterization of patient mAbs provides a valuable method to dissect the molecular mechanisms within polyclonal populations.

Adaptive NK cells in people exposed to Plasmodium falciparum correlate with protection from malaria.

How antibodies naturally acquired during Plasmodium falciparum infection provide clinical immunity to blood-stage malaria is unclear. We studied the function of natural killer (NK) cells in people living in a malaria-endemic region of Mali. Multi-parameter flow cytometry revealed a high proportion of adaptive NK cells, which are defined by the loss of transcription factor PLZF and Fc receptor γ-chain. Adaptive NK cells dominated antibody-dependent cellular cytotoxicity responses, and their frequency within total NK cells correlated with lower parasitemia and resistance to malaria. P. falciparum-infected RBCs induced NK cell degranulation after addition of plasma from malaria-resistant individuals. Malaria-susceptible subjects with the largest increase in PLZF-negative NK cells during the transmission season had improved odds of resistance during the subsequent season. Thus, antibody-dependent lysis of P. falciparum-infected RBCs by NK cells may be a mechanism of acquired immunity to malaria. Consideration of antibody-dependent NK cell responses to P. falciparum antigens is therefore warranted in the design of malaria vaccines.

Adaptive NK cells can persist in patients with GATA2 mutation depleted of stem and progenitor cells.

Heterozygous GATA2 mutation is associated with immunodeficiency, lymphedema, and myelodysplastic syndrome. Disease presentation is variable, often coinciding with loss of circulating dendritic cells, monocytes, B cells, and natural killer (NK) cells. Nonetheless, in a proportion of patients carrying GATA2 mutation, NK cells persist. We found that peripheral blood NK cells in symptomatic patients uniformly lacked expression of the transcription factor promyelocytic leukemia zinc finger (PLZF), as well as expression of intracellular signaling proteins FcεRγ, spleen tyrosine kinase (SYK), and EWS/FLI1-Activated Transcript 2 (EAT-2) in a variegated manner. Moreover, consistent with an adaptive identity, NK cells from patients with GATA2 mutation displayed altered expression of cytotoxic granule constituents and produced interferon-γ upon Fc-receptor engagement but not following combined interleukin-12 (IL-12) and IL-18 stimulation. Canonical, PLZF-expressing NK cells were retained in asymptomatic carriers of GATA2 mutation. Developmentally, GATA-binding protein-2 (GATA-2) was expressed in hematopoietic stem cells, but not in NK-cell progenitors, CD3-CD56bright, canonical, or adaptive CD3-CD56dim NK cells. Peripheral blood NK cells from individuals with GATA2 mutation proliferated normally in vitro, whereas lineage-negative progenitors displayed impaired NK-cell differentiation. In summary, adaptive NK cells can persist in patients with GATA2 mutation, even after NK-cell progenitors expire. Moreover, our data suggest that adaptive NK cells are more long-lived than canonical, immunoregulatory NK cells.

Long-term Protective Changes in Adipose Tissue After Gastric Bypass.

OBJECTIVE: Although long-term weight regain may occur after bariatric surgery, many patients are protected against relapse or development of type 2 diabetes. The study objective was to investigate whether this involves beneficial changes in adipose function. RESEARCH DESIGN AND METHODS: Forty-nine obese women were investigated before and 2 and 5 years after Roux-en-Y gastric bypass (RYGB). At the 5-year follow-up, 30 subjects were pairwise matched for BMI and age to 30 control women. Clinical parameters and fine-needle biopsies from subcutaneous abdominal adipose tissue were obtained; fat cell size and number, lipolysis, adiponectin, and proinflammatory protein secretion were determined. RESULTS: After 2 years, BMI decreased from 43 to 29 kg/m2, which was accompanied by improvements in insulin sensitivity (HOMA of insulin resistance [HOMA-IR]), increased circulating and adipose secreted adiponectin, and decreased adipose lipolysis and fat cell size but no change in adipocyte number. Between 2 and 5 years after surgery, BMI had increased to 31 kg/m2. This was associated with slightly increased HOMA-IR and unaltered circulating or adipose secreted adiponectin but higher secretion of tumor necrosis factor-α and increased lipolysis and number of fat cells but no change in adipocyte size. All these parameters, except lipolysis, were significantly more favorable compared with those in matched control subjects. Furthermore, the relationship between HOMA-IR and circulating adiponectin was less steep than in control subjects. CONCLUSIONS: RYGB improves long-term insulin sensitivity and adipose phenotypes beyond the control state despite weight regain. Postoperative beneficial alterations in adipose function may be involved in the diabetes-protective effect of bariatric surgery.

Acquired somatic mutations in PNH reveal long-term maintenance of adaptive NK cells independent of HSPCs.

Natural killer (NK) cells have long been considered short-lived effectors of innate immunity. However, recent animal models and human studies suggest that subsets of NK cells have adaptive features. We investigate clonal relationships of various NK-cell subsets, including the adaptive population, by taking advantage of naturally occurring X-linked somatic PIGA mutations in hematopoietic stem and progenitor cells (HSPCs) from patients with paroxysmal nocturnal hemoglobinuria (PNH). The affected HSPCs and their progeny lack expression of glycosylphosphatidylinositol (GPI) anchors on their cell surface, allowing quantification of PIGA-mutant (GPI-negative) HSPC-derived peripheral blood cell populations. The fraction of GPI-negative cells within the CD56dim NK cells was markedly lower than that of neutrophils and the CD56bright NK-cell compartments. This discrepancy was most prominent within the adaptive CD56dim NK-cell population lacking PLZF expression. The functional properties of these adaptive NK cells were similar in PNH patients and healthy individuals. Our findings support the existence of a long-lived, adaptive NK-cell population maintained independently from GPIposCD56dim.

Reduced potency of cytotoxic T lymphocytes from patients with high-risk myelodysplastic syndromes.

INTRODUCTION: Myelodysplastic syndromes (MDS) are a group of clonal bone marrow disorders, with dysplasia, cytopenias and increased risk of progression to acute myeloid leukemia. A dysregulated immune system precipitates MDS, and to gain insights into the relevance of cytotoxic T lymphocyte (CTL) in this process, we examined the frequency and function of CX3CR1- and CD57-positive T lymphocytes from MDS patients. MATERIALS AND METHODS: Peripheral blood and/or bone marrow samples from 31 MDS patients and 12 healthy controls were examined by flow cytometry. Expression of cytotoxic granule constituents, immunological co-receptors, adhesion molecules and markers of activation were quantified on unstimulated lymphocytes. Degranulation, cytotoxicity and conjugate formation with target cells following co-culture of CTL with target cell lines or autologous bone marrow-derived CD34(+) cells were quantified by flow cytometry. RESULTS: CX3CR1 expression was increased in bone marrow from high-risk MDS patients compared to healthy controls. Expression of CD57 and CX3CR1 was closely correlated, identifying a CTL subset with high cytotoxic capacity. In vitro, TCR-induced redirected cytotoxicity was markedly decreased for high-risk MDS patients compared to controls. CTL from MDS patients with the lowest target cell cytotoxicity had reduced expression of adhesion molecules and formed fewer conjugates with target cells. DISCUSSION: Although phenotypically defined CTL numbers were increased in the bone marrow of MDS patients, we found that CTL from high-risk MDS patients exhibited a lower TCR-induced redirected cytotoxic capacity. Thus, decreased T cell cytotoxicity seems related to reduced adhesion to target cells and may contribute to impaired anti-leukemic immune surveillance in MDS.

Cytomegalovirus infection drives adaptive epigenetic diversification of NK cells with altered signaling and effector function.

The mechanisms underlying human natural killer (NK) cell phenotypic and functional heterogeneity are unknown. Here, we describe the emergence of diverse subsets of human NK cells selectively lacking expression of signaling proteins after human cytomegalovirus (HCMV) infection. The absence of B and myeloid cell-related signaling protein expression in these NK cell subsets correlated with promoter DNA hypermethylation. Genome-wide DNA methylation patterns were strikingly similar between HCMV-associated adaptive NK cells and cytotoxic effector T cells but differed from those of canonical NK cells. Functional interrogation demonstrated altered cytokine responsiveness in adaptive NK cells that was linked to reduced expression of the transcription factor PLZF. Furthermore, subsets of adaptive NK cells demonstrated significantly reduced functional responses to activated autologous T cells. The present results uncover a spectrum of epigenetically unique adaptive NK cell subsets that diversify in response to viral infection and have distinct functional capabilities compared to canonical NK cell subsets.

Immunomodulatory activity of commonly used drugs on Fc-receptor-mediated human natural killer cell activation.

Natural killer (NK) cells mediate defense against neoplastic as well as infected cells. Yet, how their effector functions are affected by the large variety of pharmacological compounds commonly in use has not been investigated systematically. Here, we screened 1,200 in-use or previously approved drugs for their biological effect on freshly isolated human peripheral blood-derived NK cells. Mimicking antibody-dependent cellular cytotoxicity (ADCC), known to be important in antibody-based immunotherapies against, e.g., human malignancies, the cells were stimulated by Fc-receptor (CD16) engagement. Cellular responses were assessed by flow cytometry. Fifty-six compounds that significantly inhibited and twelve that enhanced one or more of the readouts of adhesion, exocytosis, and chemokine production were identified and confirmed as hits. Among the confirmed inhibitors, 80 % could be assigned to one of seven major pharmacological classes. These classes were ß2-adrenergic agonists, prostaglandins, phosphodiesterase-4 inhibitors, Ca(2+)-channel blockers, histamine H1-receptor antagonists, serotonin/dopamine receptor antagonists, and topoisomerase inhibitors that displayed distinct inhibitory patterns on NK cell responses. Among observed enhancers, interestingly, two ergosterol synthesis inhibitors were identified that specifically promoted exocytosis. In summary, these results provide a comprehensive knowledge base of the effect known drugs have on NK cells. More specifically, they provide an overview of drugs that may modulate NK cell-mediated ADCC in the context of clinical immunotherapies.

Epstein-Barr virus coinfection in children boosts cytomegalovirus-induced differentiation of natural killer cells.

During childhood, infections with cytomegalovirus (CMV) and Epstein-Barr virus (EBV) can occur in close temporal proximity. Active, as well as latent, CMV infection is associated with enlarged subsets of differentiated natural killer (NK) and cytotoxic T cells. How EBV infection may influence CMV-driven immune differentiation is not known. We found that EBV coinfection selectively influenced the NK cell compartment of CMV-seropositive (CMV(+)) children. Coinfected children had significantly higher proportions of peripheral-blood NKG2C(+) NK cells than CMV(+) EBV(-) children. Ex vivo NK cell degranulation after target cell stimulation and plasma IL-15 levels were significantly higher in CMV(+) children. EBV coinfection was related to the highest levels of plasma interleukin-15 (IL-15) and IL-12p70. Remarkably, in vitro EBV infection of peripheral blood mononuclear cells (PBMC) from EBV(-) CMV(+) children increased NKG2C(+) NK cell proportions. A similar tendency was seen in cocultures of PBMC with EBV(+) lymphoblastoid B-cell lines (LCL) and IL-15. After K562 challenge, NKG2C(+) NK cells excelled in regard to degranulation and production of gamma interferon, regardless of whether there was previous coculture with LCL. Taken together, our data suggest that dual latency with these herpesviruses during childhood could contribute to an in vivo environment supporting differentiation and maintenance of distinct NK cell populations. This viral imprint may affect subsequent immune responses through altered distributions of effector cells.

Systemic lupus erythematosus immune complexes increase the expression of SLAM family members CD319 (CRACC) and CD229 (LY-9) on plasmacytoid dendritic cells and CD319 on CD56(dim) NK cells.

Patients with systemic lupus erythematosus (SLE) display an activated type I IFN system due to unceasing IFN-α release from plasmacytoid dendritic cells (pDCs) stimulated by nucleic acid-containing immune complexes (ICs). NK cells strongly promote the IFN-α production by pDCs; therefore, we investigated surface molecules that could be involved in the pDC-NK cell cross-talk. In human PBMCs stimulated with RNA-containing ICs (RNA-ICs), the expression of the signaling lymphocyte activation molecule (SLAM) family receptors CD319 and CD229 on pDCs and CD319 on CD56(dim) NK cells was selectively increased. Upregulation of CD319 and CD229 on RNA-IC-stimulated pDCs was induced by NK cells or cytokines (e.g., GM-CSF, IL-3). IFN-α-producing pDCs displayed a higher expression of SLAM molecules compared with IFN-α⁻ pDCs. With regard to signaling downstream of SLAM receptors, pDCs expressed SHIP-1, SHP-1, SHP-2, and CSK but lacked SLAM-associated protein (SAP) and Ewing's sarcoma-activated transcript 2 (EAT2), indicating that these receptors may act as inhibitory receptors on pDCs. Furthermore, pDCs from patients with SLE had decreased expression of CD319 on pDCs and CD229 on CD56(dim) NK cells, but RNA-IC stimulation increased CD319 and CD229 expression. In conclusion, this study reveals that the expression of the SLAM receptors CD319 and CD229 is regulated on pDCs and NK cells by lupus ICs and that the expression of these receptors is specifically altered in SLE. These results, together with the observed genetic association between the SLAM locus and SLE, suggest a role for CD319 and CD229 in the SLE disease process.

Comparison of primary human cytotoxic T-cell and natural killer cell responses reveal similar molecular requirements for lytic granule exocytosis but differences in cytokine production.

Cytotoxic lymphocytes, encompassing cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, kill pathogen-infected, neoplastic, or certain hematopoietic cells through the release of perforin-containing lytic granules. In the present study, we first performed probability-state modeling of differentiation and lytic granule markers on CD8(+) T cells to enable the comparison of bona fide CTLs with NK cells. Analysis identified CD57(bright) expression as a reliable phenotype of granule marker-containing CTLs. We then compared CD3(+)CD8(+)CD57(bright) CTLs with NK cells. Healthy adult peripheral blood CD3(+)CD8(+)CD57(bright) CTLs expressed more granzyme B but less perforin than CD3(-)CD56(dim) NK cells. On stimulation, such CTLs degranulated more readily than other T-cell subsets, but had a propensity to degranulate that was similar to NK cells. Remarkably, the CTLs produced cytokines more rapidly and with greater frequency than NK cells. In patients with biallelic mutations in UNC13D, STX11, or STXBP2 associated with familial hemophagocytic lymphohistiocytosis, CTL and NK cell degranulation were similarly impaired. Therefore, cytotoxic lymphocyte subsets have similar requirements for Munc13-4, syntaxin-11, and Munc18-2 in lytic granule exocytosis. The present results provide a detailed comparison of human CD3(+)CD8(+)CD57(bright) CTLs and NK cells and suggest that analysis of CD57(bright) CTL function may prove useful in the diagnosis of primary immunodeficiencies including familial hemophagocytic lymphohistiocytosis.

Sensitive and viable quantification of inside-out signals for LFA-1 activation in human cytotoxic lymphocytes by flow cytometry.

An early step in immunosurveillance by cytotoxic lymphocytes is leukocyte functional antigen (LFA)-1-dependent adhesion to target cells, which is promoted by inside-out signals from receptors such as the T cell receptor and a variety of natural killer (NK) cell activating receptors. Inside-out signals induce a conformational change in LFA-1, resulting in an extension of the extracellular domain of the receptor. Here, we have evaluated several mAbs that specifically detect the extended conformation of LFA-1 and detail a protocol for flow cytometric quantification of ß2-integrin activation in human peripheral blood cytotoxic T cells and NK cells in response to target cell recognition. By comparison to the markers of degranulation and chemokine synthesis, e.g. surface CD107a expression and intracellular MIP-1ß expression, respectively, evaluation of LFA-1 conformational changes represent a sensitive and rapid parameter of primary cytotoxic lymphocyte activation that can facilitate isolation of viable cells. Potentially, combined with other read-outs of cytotoxic lymphocyte function, this technique is applicable to the assessment of various clinical conditions, including for the diagnosis of primary immunodeficiency syndromes and for evaluating the efficacy of tumor targeting by donor lymphocytes.