CD28-CD57+ T cells from head and neck cancer patients produce high levels of cytotoxic granules and type II interferon but are not senescent.

T lymphocytes expressing CD57 and lacking costimulatory receptors CD27/CD28 have been reported to accumulate with aging, chronic infection, and cancer. These cells are described as senescent, with inability to proliferate but enhanced cytolytic and cytokine-producing capacity. However, robust functional studies on these cells taken directly from cancer patients are lacking. We isolated these T cells and their CD27/28+ counterparts from blood and tumor samples of 50 patients with previously untreated head and neck cancer. Functional studies confirmed that these cells have enhanced ability to degranulate and produce IFN-γ. They also retain the ability to proliferate, thus are not senescent. These data suggest that CD27/28-CD57+ CD8+ T cells are a subset of highly differentiated, CD45RA+ effector memory (TEMRA) cells with retained proliferative capacity. Patients with > 34% of these cells among CD8+ T cells in the blood had a higher rate of locoregional disease relapse, suggesting these cells may have prognostic significance.

Multifactorial determinants of NK cell repertoire organization: insights into age, sex, KIR genotype, HLA typing, and CMV influence.

Introduction: Polymorphisms in the KIR and HLA genes contribute to the diversity of the NK cell repertoire. Extrinsic factors also play a role in modifying this repertoire. The best example is cytomegalovirus, which promotes the expansion of memory-like NK cells. However, the mechanisms governing this phenotypic structure are poorly understood. Furthermore, the influence of age and sex has been understudied. Methods: In this study, we examined these parameters in a cohort of 200 healthy volunteer blood donors, focusing on the major inhibitory KIR receptors and CD94/NKG2A, as well as the differentiation marker CD57 and the memory-like population marker NKG2C. Flow cytometry and two joint analyses, unsupervised and semi-supervised, helped define the impact of various intrinsic and extrinsic markers on the phenotypic structure of the NK cell repertoire. Results: In the KIR NK cell compartment, the KIR3DL1 gene is crucial, as unexpressed alleles lead to a repertoire dominated by KIR2D interacting only with HLA-C ligands, whereas an expressed KIR3DL1 gene allows for a greater diversity of NK cell subpopulations interacting with all HLA class I ligands. KIR2DL2 subsequently favors the KIR2D NK cell repertoire specific to C1/C2 ligands, whereas its absence promotes the expression of KIR2DL1 specific to the C2 ligand. The C2C2Bw4+ environment, marked by strong -21T motifs, favors the expansion of the NK cell population expressing only CD57, whereas the absence of HLA-A3/A11 ligands favors the population expressing only NKG2A, a population highly represented within the repertoire. The AA KIR genotype favors NK cell populations without KIR and NKG2A receptors, whereas the KIR B+ genotypes favor populations expressing KIR and NKG2A. Interestingly, we showed that women have a repertoire enriched in CD57- NK cell populations, while men have more CD57+ NK cell subpopulations. Discussion: Overall, our data demonstrate that the phenotypic structure of the NK cell repertoire follows well-defined genetic rules and that immunological history, sex, and age contribute to shaping this NK cell diversity. These elements can contribute to the better selection of hematopoietic stem cell donors and the definition of allogeneic NK cells for cell engineering in NK cell-based immunotherapy approaches.cters are displayed correctly.

Research and Analysis of Molecules such as CD28, CD45RA, CD45RO, CD38, HLA-DR, and CD57 on T Cells in Multiple Myeloma.

BACKGROUND: For many years it has been postulated that the immune system controls the progress of multiple myeloma (MM). However, the phenotypes of T cells in MM remain to be elucidated. In this study, we compared the phenotypes of T cells, which were obtained from the peripheral blood, in MM patients with those in healthy donors (HD). The expression of CCR7, CD57, CD28, HLA-DR, CD38, CD45RA, and CD45RO were assessed on T cells from MM patients and HDs using multicolor flow cytometry (MFC). METHODS: For this study, 17 newly diagnosed MM patients were selected, and 20 healthy people were selected as a control group. MFC was used to detect the markers on T cells. RESULTS: We detected significant increases in the expression levels of HLA-DR, CD38, and CD57on CD8+ T cells, significant decreases in the expression levels of CD28 and CD45RA on CD8+ T cells, and a decrease of CD4+ effec-tor T cells in MM patients, compared to the HD group. CONCLUSIONS: Our study shows that the accumulation of peripheral CD8+CD57+T cells, CD8+CD38high T cells, and CD8+HLA-DR+CD38high T cells is reflective of an ongoing antitumor T cell response and a progressive immune dysfunction in MM. During chemotherapy, the recovery of immune function can be monitored by detecting the proportion of activated molecules of T lymphocytes.

Lingering Effects of Early Institutional Rearing and Cytomegalovirus Infection on the Natural Killer Cell Repertoire of Adopted Adolescents.

Adversity during infancy can affect neurobehavioral development and perturb the maturation of physiological systems. Dysregulated immune and inflammatory responses contribute to many of the later effects on health. Whether normalization can occur following a transition to more nurturing, benevolent conditions is unclear. To assess the potential for recovery, blood samples were obtained from 45 adolescents adopted by supportive families after impoverished infancies in institutional settings (post-institutionalized, PI). Their immune profiles were compared to 39 age-matched controls raised by their biological parents (non-adopted, NA). Leukocytes were immunophenotyped, and this analysis focuses on natural killer (NK) cell populations in circulation. Cytomegalovirus (CMV) seropositivity was evaluated to determine if early infection contributed to the impact of an atypical rearing. Associations with tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ), two cytokines released by activated NK cells, were examined. Compared to the NA controls, PI adolescents had a lower percent of CD56bright NK cells in circulation, higher TNF-α levels, and were more likely to be infected with CMV. PI adolescents who were latent carriers of CMV expressed NKG2C and CD57 surface markers on more NK cells, including CD56dim lineages. The NK cell repertoire revealed lingering immune effects of early rearing while still maintaining an overall integrity and resilience.

CXCL12-CXCR4 mediates CD57+ CD8+ T cell responses in the progression of type 1 diabetes.

CD57+ CD8+ T cells, also referred as effector memory cells, are implicated in various conditions including tumor immunity, virus immunity, and most recently with autoimmunity. However, their roles in the progression and remission of T1D are still unclear. Here, we noted an increase in peripheral CD57+ CD8+ T cells in a T1D patient harboring an activator of transcription 3 (STAT3) mutation. Our in-depth study on the role of CD57+ CD8+ T cells within a T1D patient cohort revealed that these cells undergo significant compositional shifts during the disease's progression. Longitudinal cohort data suggested that CD57+ CD8+ T cell prevalence may be a harbinger of ß-cell function decline in T1D patients. Characterized by robust cytotoxic activity, heightened production of pro-inflammatory cytokines, and increased intracellular glucose uptake, these cells may be key players in the pathophysiology of T1D. Moreover, in vitro assays showed that the CXCL12-CXCR4 axis promotes the expansion and function of CD57+ CD8+ T cells via Erk1/2 signaling. Notably, the changes of serum CXCL12 concentrations were also found in individuals during the peri-remission phase of T1D. Furthermore, treatment with the CXCR4 antagonist LY2510924 reduced the immunological infiltration of CD57+ CD8+ T cells and mitigated hyperglycemia in a STZ-induced T1D mouse model. Taken together, our work has uncovered a novel role of the CXCL12-CXCR4 axis in driving CD57+ CD8+ T cells responses in T1D, and presented a promising therapeutic strategy for delaying the onset and progression of diabetes.

The Frequency and Function of NKG2C+CD57+ Adaptive NK Cells in Cytomagalovirus Co-Infected People Living with HIV Decline with Duration of Antiretroviral Therapy.

Human cytomegalovirus (CMV) infection drives the expansion and differentiation of natural killer (NK) cells with adaptive-like features. We investigated whether age and time on antiretroviral therapy (ART) influenced adaptive NK cell frequency and functionality. Flow cytometry was used to evaluate the frequency of adaptive and conventional NK cells in 229 CMV+ individuals of whom 170 were people living with HIV (PLWH). The frequency of these NK cell populations producing CD107a, CCL4, IFN-γ or TNF-α was determined following a 6-h antibody dependent (AD) stimulation. Though ART duration and age were correlated, longer time on ART was associated with a reduced frequency of adaptive NK cells. In general, the frequency and functionality of NK cells following AD stimulation did not differ significantly between treated CMV+PLWH and CMV+HIV- persons, suggesting that HIV infection, per se, did not compromise AD NK cell function. AD activation of adaptive NK cells from CMV+PLWH induced lower frequencies of IFN-γ or TNF-α secreting cells in older persons, when compared with younger persons.

Single-cell analysis reveals clonally expanded tumor-associated CD57+ CD8 T cells are enriched in the periphery of patients with metastatic urothelial cancer responding to PD-L1 blockade.

BACKGROUND: A growing body of evidence suggests that T-cell responses against neoantigens are critical regulators of response to immune checkpoint blockade. We previously showed that circulating neoantigen-specific CD8 T cells in patients with lung cancer responding to anti-Programmed death-ligand 1 (PD-L1) (atezolizumab) exhibit a unique phenotype with high expression of CD57, CD244, and KLRG1. Here, we extended our analysis on neoantigen-specific CD8 T cells to patients with metastatic urothelial cancer (mUC) and further profiled total CD8 T cells to identify blood-based predictive biomarkers of response to atezolizumab. METHODS: We identified tumor neoantigens from 20 patients with mUC and profiled their peripheral CD8 T cells using highly multiplexed combinatorial tetramer staining. Another set of patients with mUC treated with atezolizumab (n=30) or chemotherapy (n=40) were selected to profile peripheral CD8 T cells by mass cytometry. Using single-cell transcriptional analysis (single-cell RNA sequencing (scRNA-seq)), together with CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing) and paired T-cell receptor (TCR) sequencing, we further characterized peripheral CD8 T cells in a subset of patients (n=16). RESULTS: High frequency of CD57 was observed in neoantigen-specific CD8 T cells in patients with mUC responding to atezolizumab. Extending these findings to bulk CD8 T cells, we found higher frequency of CD57 expressing CD8 T cells before treatment in patients responding to atezolizumab (n=20, p<0.01) but not to chemotherapy. These findings were corroborated in a validation cohort (n=30, p<0.01) and notably were independent of known biomarkers of response. scRNA-seq analysis identified a clonally expanded cluster enriched within CD57+ CD8 T cells in responding patients characterized by higher expression of genes associated with activation, cytotoxicity, and tissue-resident memory markers. Furthermore, compared with CD57- CD8 T cells, TCRs of CD57+ CD8 T cells showed increased overlap with the TCR repertoire of tumor-infiltrating T cells. CONCLUSIONS: Collectively, we show high frequencies of CD57 among neoantigen-specific and bulk CD8 T cells in patients responding to atezolizumab. The TCR repertoire overlap between peripheral CD57+ CD8 T cells and tumor-infiltrating lymphocytes suggest that accumulation of peripheral CD57+ CD8 T cells is reflective of an ongoing antitumor T-cell response. Our findings provide evidence and rationale for using circulating CD8 T cells expressing CD57 as a readily accessible blood-based biomarker for selecting patients with mUC for atezolizumab therapy.

Anti-MAG neuropathy: From biology to clinical management.

The acquired chronic demyelinating neuropathies include a growing number of disease entities that have characteristic, often overlapping, clinical presentations, mediated by distinct immune mechanisms, and responding to different therapies. After the discovery in the early 1980s, that the myelin associated glycoprotein (MAG) is a target antigen in an autoimmune demyelinating neuropathy, assays to measure the presence of anti-MAG antibodies were used as the basis to diagnose the anti-MAG neuropathy. The route was open for describing the clinical characteristics of this new entity as a chronic distal large fiber sensorimotor neuropathy, for studying its pathogenesis and devising specific treatment strategies. The initial use of chemotherapeutic agents was replaced by the introduction in the late 1990s of rituximab, a monoclonal antibody against CD20+ B-cells. Since then, other anti-B cells agents have been introduced. Recently a novel antigen-specific immunotherapy neutralizing the anti-MAG antibodies with a carbohydrate-based ligand mimicking the natural HNK-1 glycoepitope has been described.

The Adaptive Immune Landscape of the Colorectal Adenoma-Carcinoma Sequence.

BACKGROUND: The tumor immune microenvironment exerts a pivotal influence in tumor initiation and progression. The aim of this study was to analyze the immune context of sporadic and familial adenomatous polyposis (FAP) lesions along the colorectal adenoma-carcinoma sequence (ACS). METHODS: We analyzed immune cell counts (CD3+, CD4+, CD8+, Foxp3+, and CD57+), tumor mutation burden (TMB), MHC-I expression and PD-L1 expression of 59 FAP and 74 sporadic colorectal lesions, encompassing adenomas with low-grade dysplasia (LGD) (30 FAP; 30 sporadic), adenomas with high-grade dysplasia (22 FAP; 30 sporadic), and invasive adenocarcinomas (7 FAP; 14 sporadic). RESULTS: The sporadic colorectal ACS was characterized by (1) a stepwise decrease in immune cell counts, (2) an increase in TMB and MHC-I expression, and (3) a lower PD-L1 expression. In FAP lesions, we observed the same patterns, except for an increase in TMB along the ACS. FAP LGD lesions harbored lower Foxp3+ T cell counts than sporadic LGD lesions. A decrease in PD-L1 expression occurred earlier in FAP lesions compared to sporadic ones. CONCLUSIONS: The colorectal ACS is characterized by a progressive loss of adaptive immune infiltrate and by the establishment of a progressively immune cold microenvironment. These changes do not appear to be related with the loss of immunogenicity of tumor cells, or to the onset of an immunosuppressive tumor microenvironment.

Immunological Profiling of COVID-19 Patients with Pulmonary Sequelae.

Cellular immunity may be involved in organ damage and rehabilitation in patients with coronavirus disease 2019 (COVID-19). We aimed to delineate immunological features of COVID-19 patients with pulmonary sequelae (PS) 1 year after discharge. Fifty COVID-19 survivors were recruited and classified according to radiological characteristics, including 24 patients with PS and 26 patients without PS. Phenotypic and functional characteristics of immune cells were evaluated by multiparametric flow cytometry. Patients with PS had an increased proportion of natural killer (NK) cells and a lower percentage of B cells than patients without PS. Phenotypic and functional features of T cells in patients with PS were predominated by the accumulation of CD4-positive (CD4+) T cells secreting interleukin 17A (IL-17A), short-lived effector-like CD8+ T cells (CD27-negative [CD27-] CD62L-), and senescent T cells with excessive secretion of granzyme B/perforin/interferon gamma (IFN-γ). NK cells were characterized by the excessive secretion of granzyme B and perforin and the downregulation of NKP30 and NKP46; highly activated NKT and γδ T cells exhibited NKP30 and TIM-3 upregulation and NKB1 downregulation in patients with PS. However, immunosuppressive cells were comparable between the two groups. The interrelationship of immune cells in COVID-19 was intrinsically identified, whereby T cells secreting IL-2, IL-4, and IL-17A were enriched among CD28+ and CD57- cells and cells secreting perforin/granzyme B/IFN-γ/tumor necrosis factor alpha (TNF-α)-expressed markers of terminal differentiation. CD57+ NK cells, CD4+Perforin+ T cells, and CD8+ CD27+ CD62L+ T cells were identified as the independent predictors for residual lesions. Overall, our findings unveil the profound imbalance of immune landscape that may correlate with organ damage and rehabilitation in COVID-19. IMPORTANCE A considerable proportion of COVID-19 survivors have residual lung lesions such as ground-glass opacity and fiber streak shadow. To determine the relationship between host immunity and residual lung lesions, we performed an extensive analysis of immune responses in convalescent patients with COVID-19 1 year after discharge. We found significant differences in immunological characteristics between patients with pulmonary sequelae and patients without pulmonary sequelae 1 year after discharge. Our study highlights the profound imbalance of immune landscape in the COVID-19 patients with pulmonary sequelae, characterized by the robust activation of cytotoxic T cells, NK cells, and γδ T cells, as well as the deficiencies of immunosuppressive cells. Importantly, CD57+ NK cells, CD4+Perforin+ T cells, and CD8+ CD27+ CD62L+ T cells were identified as the independent predictors for residual lesions.

Polyfunctional KLRG-1+CD57+ Senescent CD4+ T Cells Infiltrate Tumors and Are Expanded in Peripheral Blood From Breast Cancer Patients.

Senescent T cells have been described during aging, chronic infections, and cancer; however, a comprehensive study of the phenotype, function, and transcriptional program of this T cell population in breast cancer (BC) patients is missing. Compared to healthy donors (HDs), BC patients exhibit an accumulation of KLRG-1+CD57+ CD4+ and CD8+ T cells in peripheral blood. These T cells infiltrate tumors and tumor-draining lymph nodes. KLRG-1+CD57+ CD4+ and CD8+ T cells from BC patients and HDs exhibit features of senescence, and despite their inhibitory receptor expression, they produce more effector cytokines and exhibit higher expression of Perforin, Granzyme B, and CD107a than non-senescent subsets. When compared to blood counterparts, tumor-infiltrating senescent CD4+ T cells show similar surface phenotype but reduced cytokine production. Transcriptional profiling of senescent CD4+ T cells from the peripheral blood of BC patients reveals enrichment in genes associated with NK or CD8+-mediated cytotoxicity, TCR-mediated stimulation, and cell exhaustion compared to non-senescent T cells. Comparison of the transcriptional profile of senescent CD4+ T cells from peripheral blood of BC patients with those of HDs highlighted marked similarities but also relevant differences. Senescent CD4+ T cells from BC patients show enrichment in T-cell signaling, processes involved in DNA replication, p53 pathways, oncogene-induced senescence, among others compared to their counterparts in HDs. High gene expression of CD4, KLRG-1, and B3GAT1 (CD57), which correlates with increased overall survival for BC patients, underscores the usefulness of the evaluation of the frequency of senescent CD4+ T cells as a biomarker in the follow-up of patients.

CMV exposure drives long-term CD57+ CD4 memory T-cell inflation following allogeneic stem cell transplant.

Donor and recipient cytomegalovirus (CMV) serostatus correlate with transplant-related mortality that is associated with reduced survival following allogeneic stem cell transplant (SCT). Prior epidemiologic studies have suggested that CMV seronegative recipients (R-) receiving a CMV-seropositive graft (D+) experience inferior outcomes compared with other serostatus combinations, an observation that appears independent of viral reactivation. We therefore investigated the hypothesis that prior donor CMV exposure irreversibly modifies immunologic function after SCT. We identified a CD4+/CD57+/CD27- T-cell subset that was differentially expressed between D+ and D- transplants and validated results with 120 patient samples. This T-cell subset represents an average of 2.9% (D-/R-), 18% (D-/R+), 12% (D+/R-), and 19.6% (D+/R+) (P < .0001) of the total CD4+ T-cell compartment and stably persists for at least several years post-SCT. Even in the absence of CMV reactivation post-SCT, D+/R- transplants displayed a significant enrichment of these cells compared with D-/R- transplants (P = .0078). These are effector memory cells (CCR7-/CD45RA+/-) that express T-bet, Eomesodermin, granzyme B, secrete Th1 cytokines, and are enriched in CMV-specific T cells. These cells are associated with decreased T-cell receptor diversity (P < .0001) and reduced proportions of major histocompatibility class (MHC) II expressing classical monocytes (P < .0001), myeloid (P = .024), and plasmacytoid dendritic cells (P = .0014). These data describe a highly expanded CD4+ T-cell population and putative mechanisms by which prior donor or recipient CMV exposure may create a lasting immunologic imprint following SCT, providing a rationale for using D- grafts for R- transplant recipients.

Molecular Signatures of Natural Killer Cells in CMV-Associated Anterior Uveitis, A New Type of CMV-Induced Disease in Immunocompetent Individuals.

Cytomegalovirus (CMV) causes clinical issues primarily in immune-suppressed conditions. CMV-associated anterior uveitis (CMV-AU) is a notable new disease entity manifesting recurrent ocular inflammation in immunocompetent individuals. As patient demographics indicated contributions from genetic background and immunosenescence as possible underlying pathological mechanisms, we analyzed the immunogenetics of the cohort in conjunction with cell phenotypes to identify molecular signatures of CMV-AU. Among the immune cell types, natural killer (NK) cells are main responders against CMV. Therefore, we first characterized variants of polymorphic genes that encode differences in CMV-related human NK cell responses (Killer cell Immunoglobulin-like Receptors (KIR) and HLA class I) in 122 CMV-AU patients. The cases were then stratified according to their genetic features and NK cells were analyzed for human CMV-related markers (CD57, KLRG1, NKG2C) by flow cytometry. KIR3DL1 and HLA class I combinations encoding strong receptor-ligand interactions were present at substantially higher frequencies in CMV-AU. In these cases, NK cell profiling revealed expansion of the subset co-expressing CD57 and KLRG1, and together with KIR3DL1 and the CMV-recognizing NKG2C receptor. The findings imply that a mechanism of CMV-AU pathogenesis likely involves CMV-responding NK cells co-expressing CD57/KLRG1/NKG2C that develop on a genetic background of KIR3DL1/HLA-B allotypes encoding strong receptor-ligand interactions.

Markers of T Cell Exhaustion and Senescence and Their Relationship to Plasma TGF-ß Levels in Treated HIV+ Immune Non-responders.

Background: Immune non-responders (INR) are HIV+, ART-controlled (>2 yrs) people who fail to reconstitute their CD4 T cell numbers. Systemic inflammation and markers of T cell senescence and exhaustion are observed in INR. This study aims to investigate T cell senescence and exhaustion and their possible association with soluble immune mediators and to understand the immune profile of HIV-infected INR. Selected participants were <50 years old to control for the confounder of older age. Methods: Plasma levels of IL-6, IP10, sCD14, sCD163, and TGF-ß and markers of T cell exhaustion (PD-1, TIGIT) and senescence (CD57, KLRG-1) were measured in ART-treated, HIV+ participants grouped by CD4 T cell counts (n = 63). Immune parameters were also measured in HIV-uninfected, age distribution-matched controls (HC; n = 30). Associations between T cell markers of exhaustion and senescence and plasma levels of immune mediators were examined by Spearman rank order statistics. Results: Proportions of CD4 T cell subsets expressing markers of exhaustion (PD-1, TIGIT) and senescence (CD57, KLRG-1) were elevated in HIV+ participants. When comparing proportions between INR and IR, INR had higher proportions of CD4 memory PD-1+, EM CD57+, TEM TIGIT+ and CD8 EM and TEM TIGIT+ cells. Plasma levels of IL-6, IP10, and sCD14 were elevated during HIV infection. IP10 was higher in INR. Plasma TGF-ß levels and CD4 cycling proportions of T regulatory cells were lower in INR. Proportions of CD4 T cells expressing TIGIT, PD-1, and CD57 positively correlated with plasma levels of IL-6. Plasma levels of TGF-ß negatively correlated with proportions of TIGIT+ and PD-1+ T cell subsets. Conclusions: INR have lower levels of TGF-ß and decreased proportions of cycling CD4 T regulatory cells and may have difficulty controlling inflammation. IP10 is elevated in INR and is linked to higher proportions of T cell exhaustion and senescence seen in INR.

Increased Frequency of Dysfunctional Siglec-7-CD57+PD-1+ Natural Killer Cells in Patients With Non-alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is a progressive disorder that can develop into liver fibrosis and hepatocellular carcinoma. Natural killer (NK) cells have been shown to protect against liver fibrosis and tumorigenesis, suggesting that they may also play a role in the pathogenesis of NAFLD. Sialic acid-binding immunoglobulin-like lectins (Siglecs) are a family of inhibitory and activating receptors expressed by many cell types, including NK cells. Here, we investigated the phenotypic profiles of peripheral blood and intrahepatic NK cells, including expression of Siglecs and immune checkpoint molecules, and their association with NK cell function in patients with NAFLD. Immune cells in the peripheral blood of 42 patients with biopsy-proven NAFLD and 13 healthy volunteers (HVs) were identified by mass cytometry. The function of various NK cell subpopulations was assessed by flow cytometric detection of intracellular IFN-γ and CD107a/LAMP-1, a degranulation marker, after in vitro stimulation. We found that peripheral blood from NAFLD patients, regardless of fibrosis stage, contained significantly fewer total CD56+ NK cell and CD56dim NK cell populations compared with HVs, and the CD56dim cells from NAFLD patients were functionally impaired. Among the Siglecs examined, NK cells predominantly expressed Siglec-7 and Siglec-9, and both the expression levels of Siglec-7 and Siglec-9 on NK cells and the frequencies of Siglec-7+CD56dim NK cells were reduced in NAFLD patients. Notably, Siglec-7 levels on CD56dim NK cells were inversely correlated with PD-1, CD57, and ILT2 levels and positively correlated with NKp30 and NKp46 levels. Further subtyping of NK cells identified a highly dysfunctional Siglec-7-CD57+PD-1+CD56dim NK cell subset that was increased in patients with NAFLD, even those with mild liver fibrosis. Intrahepatic NK cells from NAFLD patients expressed elevated levels of NKG2D and CD69, suggesting a more activated phenotype than normal liver NK cells. These data identify a close association between NK cell function and expression of Siglec-7, CD57, and PD-1 that could potentially be therapeutically targeted in NAFLD.

Comparative Study of Senescent Th Biomarkers in Healthy Donors and Early Arthritis Patients. Analysis of VPAC Receptors and Their Influence.

Pro-inflammatory CD4+CD28- T cells are characteristic of immunosenescence, but also of several autoimmune/inflammatory diseases. Vasoactive intestinal peptide (VIP) acts as an anti-inflammatory and immunomodulatory mediator on these cells. Our objective was to study the mutual influence between senescent Th cells and VIP axis in early arthritis (EA), comparing with non-EA donors. We characterized the correlation between senescent Th cells and clinic parameters of EA as well as the behavior of senescent Th biomarkers by real-time PCR. Clinical data were systematically recorded at baseline and after 6 months of follow-up. The number of CD4+CD28- T cells measured by sorting is higher in patients who initially meet ACR classification criteria for rheumatoid arthritis (RA) compared to those who were classified as undifferentiated arthritis (UA). A slight positive correlation between EA CD4+CD28- T cells and CRP or ESR and a negative correlation with bone mineral density were found. Th senescent biomarkers in EA CD4+CD28- T cells were similar to donors, however some of them increased after 6 months of follow-up. VPAC receptors were analyzed by real-time PCR and immunofluorescence, and CD4+CD28- T cells showed higher expression of VPAC2 and lower of VPAC1, VPAC2 showing a significant increased expression in EA cells. Sorted CD4+CD28- T cells were in vitro expanded in presence of VIP, wherein VIP increased senescent biomarker CD27, while it diminished CD57 or NKG2 senescent biomarkers. Our study demonstrates for the first time the existence of a link between senescent Th cells and the VIP axis.

Mass Cytometry Analysis of the NK Cell Receptor-Ligand Repertoire Reveals Unique Differences between Dengue-Infected Children and Adults.

Dengue virus (DENV) is a significant cause of morbidity in many regions of the world, with children at the greatest risk of developing severe dengue. NK cells, characterized by their ability to rapidly recognize and kill virally infected cells, are activated during acute DENV infection. However, their role in viral clearance versus pathogenesis has not been fully elucidated. Our goal was to profile the NK cell receptor-ligand repertoire to provide further insight into the function of NK cells during pediatric and adult DENV infection. We used mass cytometry to phenotype isolate NK cells and PBMCs from a cohort of DENV-infected children and adults. Using unsupervised clustering, we found that pediatric DENV infection leads to a decrease in total NK cell frequency with a reduction in the percentage of CD56dimCD38bright NK cells and an increase in the percentage of CD56dimperforinbright NK cells. No such changes were observed in adults. Next, we identified markers predictive of DENV infection using a differential state test. In adults, NK cell expression of activation markers, including CD69, perforin, and Fas-L, and myeloid cell expression of activating NK cell ligands, namely Fas, were predictive of infection. In contrast, increased NK cell expression of the maturation marker CD57 and myeloid cell expression of inhibitory ligands, such as HLA class I molecules, were predictive of pediatric DENV infection. These findings suggest that acute pediatric DENV infection may result in diminished NK cell activation, which could contribute to enhanced pathogenesis and disease severity.

Exposure to small molecule cocktails allows induction of neural crest lineage cells from human adipose-derived mesenchymal stem cells.

Neural crest cells (NCCs) are a promising source for cell therapy and regenerative medicine owing to their multipotency, self-renewability, and capability to secrete various trophic factors. However, isolating NCCs from adult organs is challenging, because NCCs are broadly distributed throughout the body. Hence, we attempted to directly induce NCCs from human adipose-derived mesenchymal stem cells (ADSCs), which can be isolated easily, using small molecule cocktails. We established a controlled induction protocol with two-step application of small molecule cocktails for 6 days. The induction efficiency was evaluated based on mRNA and protein expression of neural crest markers, such as nerve growth factor receptor (NGFR) and sex-determining region Y-box 10 (SOX10). We also found that various trophic factors were significantly upregulated following treatment with the small molecule cocktails. Therefore, we performed global profiling of cell surface makers and identified distinctly upregulated markers, including the neural crest-specific cell surface markers CD271 and CD57. These results indicate that our chemical treatment can direct human ADSCs to developing into the neural crest lineage. This offers a promising experimental platform to study human NCCs for applications in cell therapy and regenerative medicine.

The Oncometabolite 5'-Deoxy-5'-Methylthioadenosine Blocks Multiple Signaling Pathways of NK Cell Activation.

Tumor cells develop various mechanisms to escape immune surveillance. In this context, oncometabolites secreted by tumor cells due to deregulated metabolic pathways, have been in the spotlight of researchers during the last years. 5'-Deoxy-5'-methylthioadenosine (MTA) phosphorylase (MTAP) deficiency in tumors results in the accumulation of MTA within the tumor microenvironment and thereby negatively influencing immune functions of various immune cells, including T and NK cells. The influence of MTA on T cell activation has been recently described in more detail, while its impact on NK cells is still largely unknown. Therefore, we aimed to illuminate the molecular mechanism of MTA-induced NK cell dysfunction. NK cell cytotoxicity against target cells was reduced in the presence of MTA in a dose-dependent manner, while NK cell viability remained unaffected. Furthermore, we revealed that MTA blocks NK cell degranulation and cytokine production upon target cell engagement as well as upon antibody stimulation. Interestingly, the immune-suppressive effect of MTA was less pronounced in healthy donors harboring an expansion of NKG2C+ NK cells. Finally, we demonstrated that MTA interferes with various signaling pathways downstream of the CD16 receptor upon NK cell activation, including the PI3K/AKT/S6, MAPK/ERK, and NF-κB pathways. In summary, we revealed that MTA blocks NK cell functions like cytotoxicity and cytokine production by interfering with the signaling cascade of activating NK cell receptors. Specific targeting of MTA metabolism in MTAP-deficient tumors therefore could offer a promising new strategy to reverse immune dysfunction of NK cells within the tumor microenvironment.