NK Cell Subsets Research Articles

Mass Cytometry Reveals the Broad Effect of CMV Reactivation on Adaptive and Innate Immune Recovery in HSCT Patients Undergoing Adoptive T Cell Therapy for Viral Infections

Introduction. Adoptive cell therapy (ACT) with virus specific T cells (VST) has been shown to be safe and effective in controlling viraemia in recipients of haemopoietic stem cell transplant (HSCT). Long term engraftment has been demonstrated in some studies but the fate of the transferred cells is not well understood. We hypothesised that the clinical effect of ACT is mediated by interaction of transferred cells with a wide range of innate and adaptive immune cells present in the recipient at the time of ACT treatment. In order to better understand the interaction of stem cell donor-derived and adoptively transferred third-party cells, we sought to apply immune profiling by mass cytometry.Methods. Longitudinal samples from healthy individuals and collected pre- and post-donor derived and third party ACT in HSCT recipients were assessed with a 37 marker panel to identify canonical immune subsets. Cryopreserved peripheral blood mononuclear cells were thawed in batches and stained with metal-labelled antibodies and markers for DNA content and viability. Samples were acquired on the Helios upgraded CyTOF2 mass cytometer. Following sample QC and normalisation of files, viable cells were manually gated to 75 canonical populations with cell subsets enumerated and represented graphically for each sample using visualised t-stochastic neighbour embedding (ViSNE). Cell subsets were expressed as percent of total cells as well as absolute cell number (x109/L) using lymphocyte and monocyte counts from an automated whole blood analyser. This approach was taken to incorporate the large numerical changes that occur in the early months post-HSCT. Mass cytometry data was analysed with unsupervised and supervised clustering algorithms to identify immune signatures. Significance of microarray (SAM) was used to identify the cell subset changes that contributed to the differences in clusters. t-stochastic neighbour embedding (tSNE) and principal component analysis (PCA) were used to correlate immune signatures with clinical variables including patient, transplant and donor baseline characteristics and post-transplant outcomes (including CMV reactivation and disease, acute GVHD, relapse and death).Results. A total of 104 samples from 9 healthy donors and 27 HSCT recipients who received ACT on phase I and II clinical trials were available for analysis. ViSNE revealed highly detailed relationships of the 75 cell subsets within individuals longitudinally and between patients. Unsupervised clustering on all patients and time points and the healthy individuals revealed three distinct immunological signatures (Figure 1). Healthy individuals (signature A) were clearly distinguished from all patients and no patient developed a normal immune profile over the timeframe of the study (up to 6 months post HSCT). In a subset of samples from HSCT recipients a distinct immune phenotype was seen (signature C), characterised by increased CD8+ subsets (CD8em, CD8emra (both of these with and without PD1), CD8+CD57+, CD8+CD31+), NK cell subsets (CD16-, NKp46loCD56lo), NKT cells and plasmacytoid dendritic cells. Of the clinical parameters assessed, signature C was most strongly correlated with CMV reactivation. Signature B showed lower cell numbers overall and included patients with acute GVHD treated with corticosteroids and patients in whom CMV reactivation progressed to CMV disease.Conclusion. Mass cytometry and sophisticated analysis reveal an unprecedented level of detail about the relationships of immune subsets to one another at single time points and their changes over time. This approach identifies distinct global immune signatures involving changes to multiple immune subsets in patients undergoing HSCT and ACT. We have identified an immune signature associated with CMV reactivation that indicates a broader effect of CMV reactivation on immune recovery than previously recognised. As this study was not randomised and did not include a control group who were not treated with ACT, further investigation is required to define the contribution of ACT to the observed immunological changes. This global approach to immunological assessment has the potential to improve understanding of the dynamics of immunological recovery post HSCT. [Display omitted] DisclosuresGottlieb:Indee: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees.

NK-Cell Alloreactivity Is Associated with Acute Gvhd and Decreased Relapse Incidence after T-Replete Haplo-Identical Allotransplant with High-Dose Post-Transplant Cyclophosphamide

Background: NK cells express activating (act) and inhibitory (inh) receptors, which recognize MHC class-I alleles, termed “Killer cell Immunoglobulin-like Receptors” (KIRs). Previous clinical data from haplo-identical T-cell-depleted allotransplant have demonstrated that donor vs recipient NK cell alloreactivity (or KIR/HLA incompatibility, KIR/HLA inc) plays a major role as effectors against acute myeloid leukemia (AML). Recently, T-replete haplo-transplant using high-dose post-transplant cyclophosphamide (PTCY) has been a revolution in the field as it is associated with good rate of engraftment and very low incidence of severe acute and chronic GVHD.Methods: The main objective of this prospective study was to study the impact of NK alloreactivity after T-replete haplo-transplant using PTCY. The Baltimore conditioning regimen (Luznik, 2008) was used for lymphoid malignancies while fludarabine as part of this regimen was replaced by clofarabine (Clo-Baltimore) for patients with myeloid malignancies.KIR/HLA inc were studied between donor and recipient considering various act and inh KIR receptors. We then studied the correlation between the presence of KIR/HLA inc and inh KIR/HLA inc and the occurrence of GVHD and relapse post-transplant. Finally, we compared reconstitution of various NK cell subsets (KIR2DL3, KIR2DL1, NKp46+2B4-, NKp46-2B4+, NKG2A+) between cases with or without acute GVHD, KIR/HLA inc, and between Baltimore vs Clo-Baltimore patients. For this purpose, peripheral blood samples were collected at days+10, +20, +30, +60 and +90/100 post-transplant. All patients gave informed consent.Results: Between August 2014 and March 2017, 34 patients (males n=23, median age: 62.5 years) were included in the study. Twelve cases received a Baltimore regimen vs 22 a Clo-Baltimore regimen. All patients except one received peripheral blood stem cell as source of graft. 56% and 14.7% of patients developed grade 2-4 and 3-4 acute GVHD, respectively, at a median of 35 days post-transplant. In July 2017, 9 patients had relapsed and 10 are dead. No differences were observed in terms of acute GVHD incidence, relapse or deaths between Baltimore and Clo-Baltimore sub-groups.KIR/HLA inc was documented in 17 cases of whom 13 presented with acute grade 2-4 while it was only 6/17 cases among patients with no KIR/HLA inc (p=0.001). The same was observed when considering only inhKIR/HLA inc (n=11/13 vs n=8/21, p=0.006). Conversely, relapse was less frequent in patients with KIR/HLA inc (n=2/17 vs n=7/17, p=0.05). However, incidence of relapse was similar between patients developing GvHD (n 5/19) vs no (n=4/15), suggesting that decreased incidence of relapse was not due to GVHD but was related to KIR/HLA inc.The proportions of NK cells at each kinetic point considered after transplant were similar regardless of GVHD, KIR/HLA inc, or conditioning. Phenotypic analyses of NK cell subsets showed that patients developing GVHD had higher mature/activated NKp46+2B4- and NKG2A+NK cells at day +20 post-transplant. The proportion of KIR2DL3+ NK cells was also significantly higher in absence of KIR/HLA inc at day+30, in contrast to KIR2DL1+ NK cells that were similar between each sub-groups at any time considered.Conclusion: KIR/HLA inc are associated with acute GVHD and decreased relapse incidence after T-repleted haplo-identical allotransplantation using PTCY. NKp46+2B4- and NKG2A+NK cells were significantly higher in cases developing GVHD at day+20, suggesting a role of these activated NK cells in the occurrence of GVHD. Also, KIR/HLA inc impact the phenotypic NK cell reconstitution and may favor NK cell expansion with better leukemic effect. These results may be of crucial importance regarding the selection of haplo-donor. DisclosuresNo relevant conflicts of interest to declare.

Genetic Background of Chronic Active Epstein-Barr Virus Disease

IntroductionEpstein-Barr virus (EBV) is associated with various benign and malignant diseases. Chronic active EBV disease (CAEBV) is a rare EBV-associated disease characterized by persistent EBV infection in natural killer (NK) and/or T cells, which is mainly prevalent in East Asia. While patients with CAEBV initially present with persistent or recurrent symptoms similar to ones present in infectious mononucleosis (IM), they often develop peripheral NK/T-cell lymphoma or multiple organ failure in the long run, and prognosis is dismal.Numerous aspects of the molecular pathogenesis of CAEBV are unresolved; it is unclear whether this disease is a clonal disorder. Although several case reports indicate the presence of primary immunodeficiency (PID) in patients with persistent EBV disease, the prevalence of PID or other inherited diseases in patients with CAEBV remains unclear. To reveal the genetic background of CAEBV, we performed whole-exome and targeted sequencing.Patients and MethodsWe studied 83 patients with CAEBV. The diagnosis of CAEBV was based on the fulfillment of the following criteria: (1) persistent or recurrent symptoms similar to those present in IM, (2) increased quantities of EBV viral load in either affected tissues or peripheral blood, and (3) exclusion of other possible diagnoses. The Institutional Review Board of Nagoya University Graduate School of Medicine approved this study.We performed whole-exome sequencing (WES) of T-, B-, and NK-cell subsets. Subsequently, we performed targeted gene sequencing of 605 genes associated with PID, inherited blood diseases, and hematological malignancies.ResultsRegarding germline mutations, we identified two patients who carried nonsense mutations in EVC2, which led to the diagnosis of Weyers acrofacial dysostosis (WAD). We also identified a patient who carried POLH mutations, which led to the diagnosis of the variant type of xeroderma pigmentosum, and a patient who carried an APC mutation, which led to the diagnosis of familial adenomatous polyposis. Mutations leading to the diagnosis of PID were not identified in any patient.WES led to the identification of at least one somatic mutation in 58% of patients. We identified DDX3X (n= 14; 16%), KMT2D (n= 4; 4.8%), BCOR / BCORL1 (n= 3; 3.6%), KDM6A (n= 3; 3.6%), and TET2 (n= 2; 2.4%) as undergoing recurrent mutations.We analyzed multiple EBV-infected cell subsets within a patient. One patient (UPN125) developed CAEBV in which both T and NK cells were positive for EBV DNA. The patient's CD4+ and CD56+ cells carried 21 and 27 mutations, respectively, of which 8 were shared including a DDX3X p.Arg534Cys driver mutation. Similar to UPN125, CD3+ and CD56+ cells of UPN404 carried a shared DDX3X p.Thr198Pro mutation and private mutations. CD19+ cells of UPN404 also carried the identical DDX3X mutation.In the survival analysis, patients carrying DDX3X mutations at diagnosis demonstrated a significantly worse OS compared with others (5-year OS: 40.6% vs. 78.6%; P= 0.007). Patients carrying KMT2D mutations also demonstrated poor prognosis (5-year OS: 50.0% vs. 73.9%; P= 0.02).DiscussionIn the present study, we found that CAEBV was a malignant disease carrying somatic driver mutations, the spectrum of which showed similarity with that of extranodal NK/T-cell lymphoma (ENKTL) and Burkitt lymphoma. DDX3X mutations and other shared mutations, which were present in multiple cell subsets in a patient, strongly suggest the presence of common ancestry, possibly lymphoid progenitor cells, which acquired driver mutations.We identified the presence of somatic DDX3X mutations as a marker of poor prognosis. A prospective study can be designed using genetic testing for DDX3X mutations and subsequent interventions, possibly chemotherapies used for ENKTL and early hematopoietic stem cell transplantation, to improve patient outcome.Furthermore, we identified several germline mutations. WAD caused by EVC / EVC2 mutations is characterized by the malformation of mesoderm and ectoderm caused by decreased hedgehog signaling mediated via EVC/EVC2 . Together with POLH and APC mutations, EVC / EVC2 mutations may be a genetic predisposition to CAEBV.In conclusion, we revealed the genetic background of CAEBV, which will lead to further clarification of its molecular pathogenesis and the development of therapeutic strategy. Clinical trials utilizing genetic diagnosis and stratified therapy are warranted. DisclosuresNo relevant conflicts of interest to declare.

The Impact of Single Dose Plerixaforas an Adjunct to G-CSF Based Peripheral Blood Stem Cell Mobilisation on the Graft Composition and Outcome for T Replete Haploidentical PBSC Transplantation with Post Transplantation Cyclophosphamide: A Pilot Study

▪Introduction : We conducted a prospective study on T cell and NK cell subset composition of the graftand the transplant outcome with single dose of subcutaneous Plerixafor(Px) to GCSF based mobilisation in T-replete Haploidentical Peripheral blood Stem cell transplantation with post-transplantation cyclophosphamide (PTCy) in 25 donors (G+Px group). This was compared with 26 donors who received G-CSF alone for mobilisation (G group).Material and Methods : With the aim to collect 10 x 106/kg CD34 cells in a single apheresis, donors with peripheral CD 34 count less than 50 cells /µl in the peripheral blood on day 4 of mobilization were administered Injection Plerixafor at the dose of 0.12mcg/kg at 12 midnight in addition to routine GCSF. This was followed by PBSC harvest starting 11 hours later at 11 A.M on the next day. The rest were continued G-CSF only as per protocol.Results: CD 34 cell count[G+Px-136(range, 52-351)vs G-139 (range,72-240) cells/µl ] in the peripheral blood on day 5 as well as that in the graft [G+Px- 2.7(range 1.1-6.0) VsG-2.3(range 0.5-5.3) X106 /ml, p=0.05] comparable between the two groups, despite a significantly lower peripheral CD34 cell count on day 4 in the pleraxifor group (G+Px-33(range, 6-47) vs G-81(58-168) cells/µl, p=0.0001). The total nucleated cell count was higher in the G- group[ 3.4(range 1.7-5.0) vs 3.1(range 1.15-4.73) X108/ml, p=0.05 ]. Despite the lower trend of CD4+ T cells[ 2.3(range 0.3-6.8) Vs 2.7(range 0.53-6.8) X107/ml, p=0.09 ]in the G-CSF group, mobilisation of Tregs was similar(7.1% vs 6.9% in G+Px group,p=0.1). There were no differences noted in the absolute number of NK cells [G+Px-5.3(range 0.8-11.6) vs 5.7.(range 1.7-12.9) X106/ml, p=0.9 ], or the CD56+16+/16- subsets. The time to engraftment was similar in both groups. The incidences of acute and chronic GVHD, non-relapse mortality and relapse were also similar.Conclusion: Addition of single dose pleraxifor to G-CSF mobilisation improves CD34 recovery and does not significantly alter the T and NK cell composition of the graft, including Tregs. No adverse impact was noted on engraftment, GVHD, NRM and relapse. Our study demonstrates that pleraxifor can be safely used as an adjunct to G-CSF to boost CD34 mobilisation without impacting conventional or regulatory T cells and NK cells in the graft. The preliminary data suggests no adverse impact on transplant outcomes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Tumor infiltrating NK cell (TINK) subsets and functional molecules in patients with breast cancer

IntroductionNK cells have been introduced as the main innate arm of immunity against malignancies. Recent advances introduced new subsets of, and new effector molecules on NK cells suggesting new paradigms for NK cell functions in tumor immunity. Considering these new paradigms, in the current research we investigated the frequency of tumor infiltrating NK cell (TINK) subsets and their functional molecules in breast tumor tissues by flowcytometry method. MethodsBreast tumor tissues were obtained from 32 untreated patients with breast cancer. The tissues were then minced mechanically to acquire a single cell suspension and surface-stained with monoclonal antibodies against CD3, CD56, CD11b, CD27, NKG2A, NKG2D and CXCR3. For intracellular staining (ICS), the surface-stained cells were then fixed, permeabilized and stained with anti-Perforin and anti-Granzyme B antibodies. The samples were run and the data were acquired on a four-color flowcytometer. ResultsThe cell suspension derived from tumor tissue encompassed 3.10 ± 0.52 % CD3−CD56+(bright/dim) total NK cells. Based on the conventional classification the percentages of cytotoxic (CD3− CD56dim) and regulatory (CD3− CD56bright) NK cells were respectively 1.74 ± 0.24 % and 1.36 ± 0.48 %. According to the new classification the percentages of cytotoxic (CD3- CD56+ CD11b+ CD27−), regulatory (CD3−CD56+ CD11b+/- CD27+) and tolerant (CD3−CD56+ CD27- CD11b−) NK cells were respectively 0.48 ± 0.07, 1.55 ± 0.34 and 1.15 ± 0.51. A significant higher frequency of total NK cells (CD3−CD56+ (bright/dim)) in the breast tumor tissues of the patients whose tumor draining lymph nodes (TDLNs) has not been yet involved by tumor cells (LN− patients) compared with the ones with lymph nodes involvement (LN+) (5.91 ± 1.79 % Vs. 2.20 ± 0.20 %, P < 0.004). Furthermore, NK cells with overexpressed activating receptor; NKGD2 (CD3- CD56+(bright/dim) NKG2D+ NK cells) was observed to be elevated in LN− patients compared with the LN+ ones (70.01 ± 7.96 Vs. 42.5 ± 4.81, P < 0.011). Correlation analysis revealed the percentages of conventional regulatory NK cells (CD3- CD56bright) in breast tumor tissue to be in positive correlation with the tumor size (R = 0.380, P < 0.04). The mean percentage of this cell subset was also observed to be higher in patients with T3 tumor size compared with smaller T1 tumor size (1.61 ± 0.20 % vs. 0.75 ± 0.15 %, P < 0.023. ConclusionOur observations suggest that accumulation of NK cells as well as the expression of activating NKG2D receptor by TINKs may play roles in breast tumor regression especially in the LN− patients. As the tumor growths and the size of tumor increases the accumulation of regulatory NK cells may facilitate the tumor improvement. These observations may have implications in cancer NK cell-based immunotherapy.

Glucose Oligosaccharide and Long-Chain Glucomannan Feed Additives Induce Enhanced Activation of Intraepithelial NK Cells and Relative Abundance of Commensal Lactic Acid Bacteria in Broiler Chickens.

Restrictions on the use of antibiotics in the poultry industry stimulate the development of alternative nutritional solutions to maintain or improve poultry health. This requires more insight in the modulatory effects of feed additives on the immune system and microbiota composition. Compounds known to influence the innate immune system and microbiota composition were selected and screened in vitro, in ovo, and in vivo. Among all compounds, 57 enhanced NK cell activation, 56 increased phagocytosis, and 22 increased NO production of the macrophage cell line HD11 in vitro. Based on these results, availability and regulatory status, six compounds were selected for further analysis. None of these compounds showed negative effects on growth, hatchability, and feed conversion in in ovo and in vivo studies. Based on the most interesting numerical results and highest future potential feasibility, two compounds were analyzed further. Administration of glucose oligosaccharide and long-chain glucomannan in vivo both enhanced activation of intraepithelial NK cells and led to increased relative abundance of lactic acid bacteria (LAB) amongst ileum and ceca microbiota after seven days of supplementation. Positive correlations between NK cell subsets and activation, and relative abundance of LAB suggest the involvement of microbiota in the modulation of the function of intraepithelial NK cells. This study identifies glucose oligosaccharide and long-chain glucomannan supplementation as effective nutritional strategies to modulate the intestinal microbiota composition and strengthen the intraepithelial innate immune system.

The abnormal distribution of NK cell subsets before HAART treatment may be related to the level of immune reconstitution in HIV patient

BackgroundHIV infection leads to the damage of NK cells, which is closely associated with the disease's progression, but whether it affects immune reconstitution after Highly Active Antiretroviral Therapy (HAART) is not clear. MethodsFrom March 9 to October 31, 2017, a total of 75 confirmed cases with HIV in Wenzhou were collected and analyzed. NK cell subsets were measured and compared among three groups: the control group, the group whose CD4+ T cell counts <200/μL (named as low-CD4 group) and the group whose CD4+ T cell counts ≥200/μL (named as high-CD4 group). The lymphocytic subsets were dynamically monitored in patients with HIV after HAART. ResultsPatients in low-CD4 group have lower proportion of CD3−CD56dimCD16+ NK cell subset, but have higher proportion of CD3−CD56−CD16+ NK cell subsets, which were compared with patients in high-CD4 group (All P values <0.01). There is a positive correlation between the proportion of CD3−CD56−CD16+ NK cell subset and CD4+ T cell counts (r = 0.628, p < 0.001). Patients in the low-CD4 group have higher expression of PD-1 and PS on NK cells than patients in the high-CD4 group (All P values < 0.05). The odds ratio of the proportion of the CD3−CD56−CD16+ NK cell subset before treatment for HAART efficacy was 0.826 (p < 0.001). ConclusionsThere are abnormalities in the proportion and function of NK cell subsets in HIV patients, which are associated with disease progression. The proportion of the CD3−CD56−CD16+ NK cell subset before treatment is related with HAART efficacy.

Phenotypic and Functional Characteristics of a Novel Influenza Virus Hemagglutinin-Specific Memory NK Cell.

Immune memory represents the most efficient defense against invasion and transmission of infectious pathogens. In contrast to memory T and B cells, the roles of innate immunity in recall responses remain inconclusive. In this study, we identified a novel mouse spleen NK cell subset expressing NKp46 and NKG2A induced by intranasal influenza virus infection. These memory NK cells specifically recognize N-linked glycosylation sites on influenza hemagglutinin (HA) protein. Different from memory-like NK cells reported previously, these NKp46+ NKG2A+ memory NK cells exhibited HA-specific silence of cytotoxicity but increase of gamma interferon (IFN-γ) response against influenza virus-infected cells, which could be reversed by pifithrin-μ, a p53-heat shock protein 70 (HSP70) signaling inhibitor. During recall responses, splenic NKp46+ NKG2A+ NK cells were recruited to infected lung and modulated viral clearance of virus and CD8+ T cell distribution, resulting in improved clinical outcomes. This long-lived NK memory bridges innate and adaptive immune memory response and promotes the homeostasis of local environment during recall response.IMPORTANCE In this study, we demonstrate a novel hemagglutinin (HA)-specific NKp46+ NKG2A+ NK cell subset induced by influenza A virus infection. These memory NK cells show virus-specific decreased cytotoxicity and increased gamma interferon (IFN-γ) on reencountering the same influenza virus antigen. In addition, they modulate host recall responses and CD8 T cell distribution, thus bridging the innate immune and adaptive immune responses during influenza virus infection.

High Dose Intravenous IgG Therapy Modulates Multiple NK Cell and T Cell Functions in Patients With Immune Dysregulation.

Intravenous immunoglobulin (IVIG) is an effective immunomodulatory treatment for immune dysregulation diseases. However, the mechanisms by which it reduces systemic inflammation are not well understood. NK cell cytotoxicity is decreased by IVIG in women with reduced fertility, but IVIG effects on NK cells in immune dysregulation are less clear. We hypothesized that IVIG modulation of lymphocyte function, especially in NK cells, is important for resolution of inflammation. Our aim was to identify IVIG-induced changes in a cohort of patients with Kawasaki disease (KD) and those that occur broadly in pediatric patients with various immune dysregulatory diseases. Peripheral blood mononuclear cells (PBMCs) of patients with KD or autoimmune/inflammatory diseases were phenotyped pre and post high dose IVIG treatment by flow cytometry. In KD patients, after IVIG infusion Treg cell frequency and the proportion of activated CD25+ immunoregulatory CD56bright NK cells was increased, and multiple lymphocyte subsets showed increased expression of the lymphoid tissue homing receptor CD62L. Importantly, IVIG treatment decreased the frequency of cells expressing the degranulation marker CD107a among cytotoxic CD56dim NK cells, which was reflected in a significant reduction in target cell killing and in decreased production of multiple pro-inflammatory mediators. Interestingly, the activating receptor CD336 was expressed on a higher proportion of CD56bright NK cells after IVIG in both KD and autoimmune/inflammatory patients while other NK receptors were increased differentially in each cohort. In autoimmune/inflammatory patients IVIG induced the proliferation marker CD71 on a higher percentage of CD56dim NK cells, and in contrast to KD patients, CD107a+ cells were increased in this subset. Furthermore, when PBMCs were stimulated ex vivo with IL-2 or Candida antigen in autologous plasma, more of the CD4+ T cells of KD patients expressed CD25 after IVIG therapy but fewer cytotoxic T cells were degranulated based on CD107a expression. In summary, IVIG treatment in patients with immune dysregulation has multiple effects, especially on NK cell subsets and CD4+ T cells, which are compatible with promoting resolution of inflammation. These novel findings provide insight into the immunomodulatory actions of IVIG in autoimmune and inflammatory conditions.

The Role of ZEB2 in Human CD8 T Lymphocytes: Clinical and Cellular Immune Profiling in Mowat–Wilson Syndrome

The Zeb2 gene encodes a transcription factor (ZEB2) that acts as an important immune mediator in mice, where it is expressed in early-activated effector CD8 T cells, and limits effector differentiation. Zeb2 homozygous knockout mice have deficits in CD8 T cells and NK cells. Mowat–Wilson syndrome (MWS) is a rare genetic disease resulting from heterozygous mutations in ZEB2 causing disease by haploinsufficiency. Whether ZEB2 exhibits similar expression patterns in human CD8 T cells is unknown, and MWS patients have not been comprehensively studied to identify changes in CD8 lymphocytes and NK cells, or manifestations of immunodeficiency. By using transcriptomic assessment, we demonstrated that ZEB2 is expressed in early-activated effector CD8 T cells of healthy human volunteers following vaccinia inoculation and found evidence of a role for TGFß-1/SMAD signaling in these cells. A broad immunological assessment of six genetically diagnosed MWS patients identified two patients with a history of recurrent sinopulmonary infections, one of whom had recurrent oral candidiasis, one with lymphopenia, two with thrombocytopenia and three with detectable anti-nuclear antibodies. Immunoglobulin levels, including functional antibody responses to protein and polysaccharide vaccination, were normal. The MWS patients had a significantly lower CD8 T cell subset as % of lymphocytes, compared to healthy controls (median 16.4% vs. 25%, p = 0.0048), and resulting increased CD4:CD8 ratio (2.6 vs. 1.8; p = 0.038). CD8 T cells responded normally to mitogen stimulation in vitro and memory CD8 T cells exhibited normal proportions of subsets with important tissue-specific homing markers and cytotoxic effector molecules. There was a trend towards a decrease in the CD8 T effector memory subset (3.3% vs. 5.9%; p = 0.19). NK cell subsets were normal. This is the first evidence that ZEB2 is expressed in early-activated human effector CD8 T cells, and that haploinsufficiency of ZEB2 in MWS patients had a slight effect on immune function, skewing T cells away from CD8 differentiation. To date there is insufficient evidence to support an immunodeficiency occurring in MWS patients.

HLA-DR-Positive NK Cells Expand in Response to MycobacteriumTuberculosis Antigens and Mediate Mycobacteria-Induced T Cell Activation.

NK cells play an important role in the control of tuberculosis infection: they are not only able to kill the infected cells, but also control the activity of macrophages and development of the adaptive immune response. Still, there is little information on the role of specific NK cell subsets in this network. In this study, we focused on the mycobacteria-driven responses of the NK cells expressing HLA-DR – a type of MHC class II. We have revealed that this subset is increased in the peripheral blood of patients with primary diagnosed tuberculosis, and expands in response to in vitro stimulation with ultrasonically destroyed Mycobacterium tuberculosis cells (sonicate). The expanded HLA-DR+ NK cells had less differentiated phenotype, higher proliferative activity and increased expression of NKp30 and NKp46 receptors. HLA-DR+CD56dim NK cells showed higher IFNγ production and degranulation level than the respective HLA-DR− NK cells in response to both 24 h and 7 day stimulation with sonicate, while HLA-DR+CD56bright NK cells mostly demonstarted similar high responsiveness to the same stimulating conditions as their HLA-DR−CD56bright counterparts. After preliminary incubation with destroyed mycobacteria, cytokine-activated HLA-DR-expressing NK cells were able to mediate mycobacteria-induced and HLA-DR-dependent cytokine production in autologous CD4+ T cells. Thus, functionally active HLA-DR+ cells seem to be one of the NK cell subsets providing an important link to the adaptive immunity.

Selective downregulation of natural killer activating receptors on NK cells and upregulation of PD-1 expression on T cells in children with severe and/or recurrent Herpes simplex virus infections

Severe, recurrent or atypical Herpes simplex virus (HSV) infections are still posing clinical and diagnostic problem in clinical immunology facilities. However, the molecular background of this disorder is still unclear. The aim of this study was to investigate the expression of activating receptors on NK cells (CD16, NKp46, NKG2D, NKp80, 2B4, CD48 and NTB-A) and checkpoint molecule PD-1 on T lymphocytes and NK cells, in patients with severe and/or recurrent infections with HSV and age-matched healthy control subjects. As a result, we noticed that patients with severe and/or recurrent infection with HSV had significantly lower percentage of CD16brightCD56dim and higher percentage of CD16dimCD56bright NK cell subsets, when compared to control subjects, which may be associated with abnormal NK cell maturation during chronic HSV infection. Patients had also significantly downregulated expression of CD16 receptor on CD16bright NK cells. The expression of activating receptors was significantly reduced on patients’ NK cells - either both the percentage of NK cells expressing the receptor and MFI of its expression (NKp46, NKp80 and 2B4 on CD16brightCD56dim cells and NKp46 on CD16dimCD56bright cells) or only MFI (NKG2D on both NK cell subsets). It should be noted that the reduction of receptor expression was limited to NK cells, since there was no differences in the percentage of receptor-positive cells or MFI on T cells. However, NTB-A receptor was the only one which expression was not only simultaneously changed in patients’ NK and T cells, but also significantly upregulated on CD16dimCD56bright NK cell and CD8+ cell subsets. Patients had also upregulated proportion of CD4+ T cells expressing PD-1. Thus, we suggest that an increased percentage of PD-1+ cells may represent an independent indirect mechanism of downregulation of antiviral response, separate from the reduction of NK cell activating receptors expression. Altogether, our studies indicate two possible mechanisms which may promote perpetuation of HSV infection: 1) selective inhibition of activating receptors on NK cells, but not on T cells, and 2) upregulation of checkpoint molecule PD-1 on CD4+ T cells.

Immune cell composition at the feto-maternal interface in health and disease

Abstract Objectives: We hypothesize that innate and adaptive leukocyte infiltration, function and localization within the placenta is altered in health and disease and that immune cell interactions play a crucial role in maintaining immune tolerance at the fetal-maternal interface. Methods: To evaluate the complexity and differences of the immune landscape in decidua basalis we examine tissue samples from healthy women and women who developed gestational diabetes (GDM). We use flow cytometry (18-plex), fluorescent multiplex immunohistochemistry and gene expression analysis. Results: Preliminary data suggest abundant innate immune cell infiltration (macrophages and neutrophils) as well as accumulation of T-cell and NK cell subsets at the fetal-maternal interface. In gestational diabetes mellitus pregnancies, we were able to observe lower T cell infiltration when compared to healthy, which was not seen in obese pregnant women. However, in both groups (obese and GDM) there was a trend for lower T regulatory cell infiltration. For monocyte and macrophage populations a trend towards lower immune cell infiltration was observed in both obese and GDM pregnancies when compared to healthy. 70–80% of macrophages show M2 polarization, which suggest that they have an important role in anti-inflammatory processes during pregnancies. We could observe also abundant neutrophil infiltration (up to 20% of all leukocytes), although there was no difference between heathy and diseased pregnancies. Conclusion: Our study comprehends a detailed picture of the immune landscape at the fetal-maternal interface in normal and GMD pregnancies, which will aid our understanding of possible dysfunctional immune mechanisms.

Defining cellular correlates of protection and vaccine failure to influenza across two human cohorts

Abstract Influenza viruses are endemic viral pathogens causing mild to severe respiratory illness in humans. Immunologic protection against influenza is determined by immune correlates of protection– factors associated with reduced infection or severe disease. While antibodies specific to viral surface proteins are known correlates, waning seasonal vaccine efficacy and reported infection of patients despite elevated antibody titers suggest that humoral responses alone do not provide complete protective immunity. Indeed, evidence points to a larger role for cell-mediated immunity (CMI; innate cells and antigen-specific T cells) in conferring protection. CMI correlates, which act independently from humoral responses, have yet to be identified. Here, we analyzed samples from adult human subjects across two influenza infection and vaccination cohorts to identify distinct CMI correlates of protection to influenza. We profiled CMI responses using high-dimension flow cytometry from PBMCs collected pre- and post-exposure to influenza infection or vaccination. Statistical comparison of cell frequency and infection status identified candidate CMI correlates, which were validated using logistic regression accounting for vaccination, demographic (age, sex, BMI), and serologic (antibody) covariates. We identified 9 individual and 6 cell clusters across myeloid and lymphoid compartments associated with protection. The strongest correlations were observed in Th17, cTfh, and subsets of NK and dendritic cells. Further, AUROC models identified 3 baseline CMI infection classifiers. Lastly, our study identified correlates of vaccine failure– pre-exposure CMI profiles correlated with positive infection status despite vaccination.

Meta-analysis of the CD56-negative NK cell subset indicates altered functional responses and unique KIR regulation

Abstract Chronic infections such as HIV-1, HCV, and some herpesviruses, expand a subset of NK cells that lack CD56 expression (CD56neg) and are less responsive to HLA-negative targets than CD56+CD16+ (CD56dim) cells. The 39 research papers that have reported on CD56neg NK cells have considered that this subset comprises anergic, dysfunctional or adaptive NK cells. Our objective was to assess these possibilities through meta-analysis of these papers, with inclusion of new data extracted from openly deposited CyTOF datasets (n=7). Data on CD56neg NK cells was extracted from papers. CyTOF datasets were downloaded and markers expressed by CD56neg cells identified. Meta-analysis was undertaken following the Cochrane guidelines using Random Effect Model assuming study variability. We found through analysis of “healthy” control groups (n=30) that the CD56neg subset represents 5.42% of peripheral blood NK cells, and confirmed that HIV-1 infection expands this population by 9.00%. Four studies presenting comparable functional data report decreased IFNγ and CD107a CD56neg cell responses to HLA-lacking targets compared to CD56dim cells (−5.99% and −4.24% respectively). Comparison to CD56dim marker expression showed no difference in Ki67 implying CD56neg cells are not anergic. The CD56neg subset also has a decreased proportion of CD57+NKG2C+ cells so are not adaptive NK cells. Lower KIR expression and range of KIR combinations were seen in CD56neg cells indicating their reduced responsiveness is linked to altered KIR:HLA signalling capacity. The expansion of this subset may be due to disrupted HLA expression induced by chronic viral infections, or may be an adaptation to long-term infection to mediate ongoing activation of the NK compartment.

Peptide: MHC-based DNA vaccination strategy to activate natural killer cells by targeting killer cell immunoglobulin-like receptors

BackgroundNatural killer (NK) cells are increasingly being recognized as agents for cancer immunotherapy. The killer cell immunoglobulin-like receptors (KIRs) are expressed by NK cells and are immunogenetic determinants of the...

Origin of human uterine natural killer cells

Abstract Uterine NK cells (uNK) are a distinct immune population which influence pregnancy outcome. While murine studies have suggested that tissue-resident uNKs predominate in virgin endometrium, the origin of uNKs in humans is unknown. To define subsets of uterine NKs in humans and determine their developmental origin, we performed single-cell RNA-sequencing on CD56+ uNKs sorted from endometrial biopsies of 4 healthy controls (HCs) (17,146 cells) and 5 uterus transplant (UTx) recipients (30,351 cells). SNP polymorphisms and HLA alleles were used to identify cells of donor (tissue-resident) or recipient (peripheral) origin in UTx recipients. Developmental trajectories were built with Monocle v.3.0. Ten major clusters were identified in HCs, with 48% of endometrial NK cells (eNKs) occupying 3 clusters (eNK1–3) that shared gene signatures with known human decidual NK cell subsets (dNK1–3). Pseudotime analysis revealed that the eNK1–3 clusters arose from proliferating subsets and were less mature than cytotoxic uNKs expressing ZEB2, TBX21, and FCGR3A (CD16). uNKs from UTx recipients were similarly distributed across the clusters, with a trend towards fewer cells in the FCGR3A-expressing mature subset. Notably, recipient-derived uNKs dominated all uNK subsets within four months post-transplant but became less frequent in UTx recipients &amp;gt;1 year after transplant (95% vs. 72%; early vs. late). The proportion of donor-derived to recipient-derived cells remained constant across subsets in all UTx recipients. In contrast to mouse models, our study suggests that human uNKs derive primarily from blood-borne peripheral immigrants and that environmental factors influence uNK differentiation more than genotype or developmental origin.

IFNγ-producing NK cells in adipose tissue are associated with hyperglycemia and insulin resistance in obese women.

Innate lymphoid cells (ILCs) play an important role in the maintenance of immune and metabolic homeostasis in adipose tissue (AT). The crosstalk between AT ILCs and adipocytes and other immune cells coordinates adipocyte differentiation, beiging, glucose metabolism and inflammation. Although the metabolic and homeostatic functions of mouse ILCs have been extensively investigated, little is known about human adipose ILCs and their roles in obesity and insulin resistance (IR). Here we characterized T and NK cell populations in omental AT (OAT) from women (n = 18) with morbid obesity and varying levels of IR and performed an integrated analysis of metabolic parameters and adipose tissue transcriptomics. In OAT, we found a distinct population of CD56-NKp46+EOMES+ NK cells characterized by expression of cytotoxic molecules, pro-inflammatory cytokines, and markers of cell activation. AT IFNγ+ NK cells, but not CD4, CD8 or γδ T cells, were positively associated with glucose levels, glycated hemoglobin (HbA1c) and IR. AT NK cells were linked to a pro-inflammatory gene expression profile in AT and developed an effector phenotype in response to IL-12 and IL-15. Moreover, integrated transcriptomic analysis revealed a potential implication of AT IFNγ+ NK cells in controlling adipose tissue inflammation, remodeling, and lipid metabolism. Our results suggest that a distinct IFNγ-producing NK cell subset is involved in metabolic homeostasis in visceral AT in humans with obesity and may be a potential target for therapy of IR.

The Longest Persistence of Viable SARS-CoV-2 With Recurrence of Viremia and Relapsing Symptomatic COVID-19 in an Immunocompromised Patient-A Case Study.

BackgroundImmunocompromised patients show prolonged shedding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal swabs. We report a case of prolonged persistence of viable SARS-CoV-2 associated with clinical relapses of coronavirus disease 2019 (COVID-19) in a patient with mantle cell lymphoma who underwent treatment with rituximab, bendamustine, cytarabine with consequent lymphopenia and hypogammaglobulinemia.MethodsNasopharyngeal swabs and blood samples were tested for SARS-CoV-2 by real-time polymerase chain reaction (RT-PCR). On 5 positive nasopharyngeal swabs, we performed viral culture and next-generation sequencing. We analyzed the patient’s adaptive and innate immunity to characterize T- and NK-cell subsets.ResultsSARS-CoV-2 RT-PCR on nasopharyngeal swabs samples remained positive for 268 days. All 5 performed viral cultures were positive, and genomic analysis confirmed a persistent infection with the same strain. Viremia resulted positive in 3 out of 4 COVID-19 clinical relapses and cleared each time after remdesivir treatment. The T- and NK-cell dynamic was different in aviremic and viremic samples, and no SARS-CoV-2-specific antibodies were detected throughout the disease course.ConclusionsIn our patient, SARS-CoV-2 persisted with proven infectivity for >8 months. Viremia was associated with COVID-19 relapses, and remdesivir treatment was effective in viremia clearance and symptom remission, although it was unable to clear the virus from the upper respiratory airways. During the viremic phase, we observed a low frequency of terminal effector CD8+ T lymphocytes in peripheral blood; these are probably recruited in inflammatory tissue for viral eradication. In addition, we found a high level of NK-cell repertoire perturbation with relevant involvement during SARS-CoV-2 viremia.

Clinical applications of monitoring immune status with 90 immune cell subsets in human whole blood by 10-color flow cytometry.

The immune system may involve and predict the different prognosis and therapy consequences. So, it's important to monitor and evaluate the immune status before and after treatments. Flow cytometry is the best technology to perform immune monitoring, because it can detect immune cells using small amount of sample in a short time. The whole blood is the ideal sample for immune status monitoring, since it includes almost all the immune cells and it's relatively easy to obtain and less invasive than bone marrow or lymph node. Here we developed and validated a 10-color panel with only four tubes containing 29 antibodies to monitor 90 immune cell subsets in 2ml whole blood samples. The major immune cell populations detected by our panel included T cell subsets (CD3+ total T, Th, Tc, Treg, CD8hi , CD8low , αβTCR, γδTCR, naïve, and memory T), T cell activation markers (CD25, CD69, and HLA-DR) and one immune checkpoint PD1, B cell subsets (B1, switched memory, non-switched, naïve B, and CD27- IgD- B cells), neutrophils, basophils, four monocytic cell subsets, dendritic cells (pDCs and mDCs), and four NK cell subsets. These panels of antibodies had been applied to monitor immune status (percentage and absolute number) in total 303 cases with various diseases, such as leukemia (AML, CML, MM, and ALL), lymphoma (B cells and NK/T cells), cancers (colon, lung, prostate, and breast), immune deficiencies, and autoimmune diseases. We provided proof of feasibility for clinical monitoring immune status and guiding immunotherapy by multicolor flow cytometry testing.