Natural Killer Cell Markers Research Articles

Decline in Club Cell Secretory Proteins, Exosomes Induction and Immune Responses to Lung Self-antigens, Kα1 Tubulin and Collagen V, Leading to Chronic Rejection After Human Lung Transplantation.

Chronic lung allograft dysfunction (CLAD), is a major hurdle for long-term lung allograft survival after lung transplant and roughly 50% of lung transplant recipients (LTxRs) develop CLAD within 5 years. The mechanisms of CLAD development remain unknown. Donor-specific immune responses to HLA and lung self-antigens (SAgs) are vital to the pathogenesis of CLAD. Reduction in Club cell secretory protein (CCSP) has been reported in bronchoalveolar lavage (BAL) fluid samples from LTxRs with bronchiolitis obliterans syndrome (BOS). CCSP levels in BAL fluid and development of antibodies to lung SAgs in plasma were determined by ELISA. Cytokines in BAL fluid were analyzed by 30-plex Luminex panel. Exosomes from BAL fluid or plasma were analyzed for SAgs, natural killer (NK) cells markers, and cytotoxic molecules. We demonstrate that LTxRs with BOS have lower CCSP levels up to 9 months before BOS diagnosis. LTxRs with antibodies to SAgs 1-year posttransplant also developed DSA (43%) and had lower CCSP. BOS with lower CCSP also induced Interleukin-8 and reduced vascular endothelial growth factor. Exosomes from BOS contained increased SAgs, NK cells markers, and cytotoxic molecules. We conclude lower CCSP leads to inflammation, pro-inflammatory cytokine production, immune responses to HLA and SAgs, and induction of exosomes. For the first time, we demonstrate that CCSP loss results in exosome release from NK cells capable of stimulating innate and adaptive immunity posttransplant. This increases the risk of BOS, suggesting a role of NK cell exosomes in CLAD development.

Expansion With IL-15 Increases Cytotoxicity of Vγ9Vδ2 T Cells and Is Associated With Higher Levels of Cytotoxic Molecules and T-bet.

Cancer immunotherapy has shown great advances during recent years, but it has yet to reach its full potential in all cancer types. Adoptive cell therapy (ACT) is now an approved treatment option for certain hematological cancers and has also shown success for some solid cancers. Still, benefit and eligibility do not extend to all patients. ACT with Vγ9Vδ2 T cells is a promising approach to overcome this hurdle. In this study, we aimed to explore the effect of different cytokine conditions on the expansion of Vγ9Vδ2 T cells in vitro. We could show that Vγ9Vδ2 T cell expansion is feasible with two different cytokine conditions: (a) 1,000 U/ml interleukin (IL)-2 and (b) 100 U/ml IL-2 + 100 U/ml IL-15. We did not observe differences in expansion rate or Vγ9Vδ2 T cell purity between the conditions; however, IL-2/IL-15-expanded Vγ9Vδ2 T cells displayed enhanced cytotoxicity against tumor cells, also in hypoxia. While this increase in killing capacity was not reflected in natural killer (NK) cell marker or activation marker expression, we demonstrated that IL-2/IL-15-expanded Vγ9Vδ2 T cells were characterized by an increased expression of perforin, granzyme B, and granulysin compared to IL-2-expanded cells. These cytotoxic molecules were not only increased in a resting state, but also released to a greater extent upon target recognition. In contrast, CD107a and cytokine expression did not differ between expansion conditions. However, IL-2/IL-15-expanded Vγ9Vδ2 T cells showed higher levels of transcription factor T-bet expression, which could indicate that T-bet and cytotoxic molecule levels confer the increased cytotoxicity. These results advocate the inclusion of IL-15 into ex vivo Vγ9Vδ2 T cell expansion protocols in future clinical studies.

Prognostic Significance of Tumor-Infiltrating Natural Killer Cells in Solid Tumors: A Systematic Review and Meta-Analysis.

Background: Tumor-infiltrating natural killer (NK) cells (TINKs) are crucial immune cells in tumor defense, and might be related to tumor prognosis. However, the results were discrepant among different studies. The present meta-analysis was performed to comprehensively assess the prognostic value of NK cell markers in solid tumor tissues.Methods: PubMed, Web of Science, and EMBASE were searched to identify original researches reporting the prognostic significance of TINKs in solid tumors. NK cell markers CD56, CD57, NKp30, and NKp46 were included in the analysis. The hazard ratios (HRs) and 95% confidence intervals (CIs) of pooled overall survival (OS), disease-free survival (DFS), metastasis-free survival (MFS), progression-free survival (PFS), and recurrence-free survival (RFS) were calculated by STATA software 14.0 to assess the prognostic significance.Results : Of the 56 included studies, there were 18 studies on CD56, 31 studies on CD57, 1 study on NKp30, and 7 studies on NKp46. High levels of CD56, CD57, NKp30, and NKp46 were significantly correlated with better OS of patients with solid malignancies (HR = 0.473, 95%CI: 0.315–0.710, p < 0.001; HR = 0.484, 95%CI: 0.380–0.616, p < 0.001; HR = 0.34, 95%CI: 0.14–0.80, p = 0.014; HR = 0.622, 95%CI: 0.470–0.821, p < 0.001, respectively). Our results also revealed that CD56, CD57, and NKp46 could act as independent prognostic predictors for favorable OS (HR = 0.372, 95%CI: 0.261–0.531, p < 0.001; HR = 0.525, 95%CI: 0.346–0.797, p = 0.003; HR = 0.559, 95%CI: 0.385–0.812, p = 0.002, respectively).Conclusions : Our results indicated that high levels of NK cell markers in solid tumor tissues could predict favorable prognosis for solid tumor patients.

ISSUE HIGHLIGHTS - July 2020.

ISSUE HIGHLIGHTS - July 2020.

Effect of rapamycin on repeated immobilization stress-induced immune alterations in the rat spleen

Chronic stress modulates immune system functions via neuroendocrine pathways. Rapamycin inhibits activity of immune cells through the mTOR signaling pathway. We investigated the effect of rapamycin (15 mg/kg, 3-times/week) on neuroimmune-endocrine system in the spleen of rats exposed to 42 cycles of 2-h immobilization. Rapamycin enhanced the activity of hypothalamic-pituitary-adrenocortical axis induced by stress exposure, prevented stress-induced expression of natural killer cell markers while reversed stress-evoked decline of Th2 immune response markers. Overall, our findings suggest that rapamycin may act on immune functions not only directly by inhibiting of mTOR in immune cells but also indirectly via modulation of neuroendocrine system.

Immunophenotypic and functional characterisation of Mesodermal Killer (MK) cells: a novel cell type and potential cellular therapy for cancer

Background & Aim We have identified a novel cell type, named mesodermal killer (MK) cells, derived from adult bone marrow under proprietary culture conditions. This study characterized the MK cells by immunophenotype, cytokine profile, immunomodulatory function and cytotoxicity. Methods, Results & Conclusion Methods: We investigated MK cells before and after activation with TNFα or IFNγ for immunophenotype using high throughput-FACS, for secretome and for inflammatory response in the murine air pouch assay. We evaluated MK cell cytotoxicity using the chromium release assay and immunomodulatory function by co-culture with natural killer (NK) cells in transwells. We used immunocytochemistry, flow cytometry and RT-PCR to detect expression of granzyme/perforin genes and proteins and investigated death pathways by siRNA silencing of FasL and TRAIL. Results MK cells displayed a novel immunophenotype (and different from BM-MSCs) including expression of CD16, CD96, CD112, CD137L, FasL, TRAIL and CD277 and some NK cell markers. In contrast to NK cells, MK cells lacked CD56 expression. TNFα and IFNγ activation increased FasL expression. MK cells secreted GROa, IL-12, IL-2Ra, IL-8, TRAIL and IL-6, and GROa and IL-8 secretion was increased by TNFα. MK cell cytotoxicity was observed against leukemia (K562), multiple myeloma (RPMI-8226) and breast cancer (MCF7) cell lines. MK cell conditioned media had no effect on MCF7 and antibody dependent cell mediated cytotoxicity (ADCC) experiments also proved negative. MK cells expressed granzymes and perforin and these were upregulated following exposure to RPMI-8226 (in contrast to BM-MSCs). Pre-treatment of MK cells with EGTA (a non-specific inhibitor of granule exocytosis) demonstrated >50% decrease in cytotoxicity against MCF7 suggesting a partial role of granule exocytosis in MK cell cytotoxicity. Inhibition of TRAIL expression by siRNA led to a significant reduction in MK cell cytotoxicity suggesting TRAIL may also be involved. In contrast to BM-MSCs, MK cells enhanced the cytotoxicity of primary NK cells against K562, RPMI-8226 and U226 (plasma cell myeloma) and IFNγ-activated MK cells enhanced migration of NK cells and monocytes into the murine air pouch suggesting a pro-inflammatory response. Conclusion MK cells exhibit cytotoxicity against a variety of cancer cell lines, are capable of priming NK cells in vitro and are pro-inflammatory in vivo. These results suggest the clinical translation of the MK cells is warranted and animal efficacy studies will begin shortly.

GPR56 inhibits chemotaxis of human Natural Killer and TEMRA lymphocytes

Abstract Within the human immune system, the expression of GPR56, an adhesion G-protein couple receptor, is restricted to cytotoxic Natural Killer (NK) and T lymphocytes, including T effector memory re-expressing CD45RA (TEMRA) cells. In primary NK cells, GPR56 acts as an inhibitory receptor, as it diminishes their ability to eliminate target cells. Overexpression of GPR56 in immortal NK-92 cells also leads to robust inhibition of cell chemotaxis. GPR56 is speculated to promote retention of cytotoxic lymphocytes in inflamed peripheral tissues. Thus, GPR56 has the potential to regulate immune responses that may lead to excessive inflammation and autoimmunity. The aim of the present study was, therefore, to examine whether GPR56 mediates cytotoxic immune cell functions and whether GPR56 expression is altered in autoimmune diseases. To tackle these questions, we first compared expression of GPR56 in healthy and autoimmune disease patient tissues using scRNA-seq datasets. Next, we tested whether stimulation of GPR56 with a monoclonal antibody modulated the migratory capacity of NK and CD8+ TEMRA cells. We found that chemotaxis toward CXCL12, IL-8, and CX3CL1 was strikingly inhibited by GPR56. Using bulk RNA-seq, we found GPR56-stimulation impacted a multitude of signaling cascades that regulate cell chemotaxis. Taken together, our results confirm that GPR56 is a marker for NK and TEMRA cells in healthy and autoimmune patient tissues and establish GPR56 as a key regulator of lymphocyte chemotaxis.

Isolation and characterization of canine natural killer cell-derived exosomes

Background & Aim Extracellular vesicles (EVs) are lipid bilayer-delimited particles that are naturally released from a cell and cannot replicate. Exosomes are membrane bound EVs that are produced in the endosomal compartment of most eukaryotic cells. Compared with EVs in general, it is unclear whether exosomes have unique characteristics or functions or can be separated or distinguished effectively from other EVs. Exosomes have been the focus of great interest, as they appear to be involved in numerous important cellular processes. They deliver bioactive macromolecules such as proteins, lipids, and nucleic acids, allowing intercellular communication in multicellular organisms. Exosomes are secreted by all cell types, including immune cells such as natural killer (NK) cells, and they are innate immune effector cells that contain lytic granules that are released to induce cellular cytotoxicity upon stimulation. NK cells are major effect or cells that are critical to the innate immune system and are typically considered to play a fundamental role, particularly in anti-viral and anti-tumor host responses. Methods, Results & Conclusion We peripheral blood mononuclear cells (PBMCs) were isolated from beagle dog. Subsequently, CD5 negative cells were isolated by immunomagnetic separation and cultured in specific culture condition supplemented with IL-2, IL-15 and IL-21. After 21 days, the culture medium with exosome-depleted fetal bovine serum was replaced, and the culture supernatant was harvested. The supernatant was centrifuged to remove cells and debris and then filtered using a 0.22 μm filter. The filtrate centrifuged at 100,000 g for 90 min at 4°C using ultracentrifugation. We also observed that the exosomes were isolated from culture medium of canine NK cells. As results, canine NK cells contained amounts of protein determined bicinochoninic acid assay with absorbance at 562 nm. Using western blotting, exosome markers (Flotillin-1, CD63, CD81, Alix, HSP70, and TSG101) and activated NK cell markers (perforin and granzyme B) were identified in the samples. The isolated exosomes typically possess a diameter of 40-200 nm, and are characterized by nanoparticle tracking analysis and electron microscope.

Differentially Expressed Genes of Natural Killer Cells Can Distinguish Rheumatoid Arthritis Patients from Healthy Controls.

Rheumatoid arthritis (RA) is one of the most prevalent autoimmune diseases, while its molecular triggers are not fully understood. A few studies have shown that natural killer (NK) cells may play either a pathogenic or a protective role in RA. In this study, we sought to explore NK cell markers that could be plausibly used in evaluating the differences among healthy controls and RA patients. Publicly available transcriptome datasets from RA patients and healthy volunteers were analyzed, in order to identify differentially expressed genes (DEGs) between 1. different immune cells as compared to NK cells, and 2. NK cells of RA patients and healthy controls. The identified DEGs were validated using 16 healthy controls and 17 RA patients. Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll density gradient method, while NK cells were isolated using RosetteSep technique. RNA was extracted and gene expression was assessed using RT-qPCR. All selected genes were differentially expressed in NK cells compared to PBMCs. CD56, CXCL16, PECAM-1, ITGB7, BTK, TLR10, and IL-1β were significantly upregulated, while CCL2, CCR4, RELA and IBTK were downregulated in the NK cells of RA patients when compared to healthy controls. Therefore, these NK specific genes might be used as promising biomarkers for RA diagnosis.

Multiplexed Proteomic Analysis for Diagnosis and Screening of Five Primary Immunodeficiency Disorders From Dried Blood Spots.

Early detection of Primary Immunodeficiencies Disorders (PIDDs) is of paramount importance for effective treatment and disease management. Many PIDDs would be strong candidates for newborn screening (NBS) if robust screening methods could identify patients from dried blood spots (DBS) during the neonatal period. As majority of congenital PIDDs result in the reduction or absence of specific proteins, direct quantification of these target proteins represents an attractive potential screening tool. Unfortunately, detection is often limited by the extremely low protein concentrations in blood cells and limited blood volume present in DBS. We have recently developed a robust novel method for quantification of low abundance proteins in DBS for PIDDs using peptide immunoaffinity enrichment coupled to selected reaction monitoring (immuno-SRM). Here, we further generated a multiplexed Immuno-SRM panel for simultaneous screening of eight signature peptides representing five PIDD-specific and two cell-type specific proteins from DBS. In samples from 28 PIDD patients including two carriers, representing X-Linked Agammaglobulinemia (XLA), Wiskott-Aldrich Syndrome (WAS), X-Linked Chronic Granulomatous Disease (XL-CGD), DOCK8 Deficiency and ADA deficiency, peptides representing each disease are significantly reduced relative to normal controls and patient identification had excellent agreement with clinical and molecular diagnosis. Also included in the multiplex panel are cell specific markers for platelets (CD42) and Natural Killer Cells (CD56). In patients with WAS, CD42 levels were found to be significantly reduced consistent with characteristic thrombocytopenia. A patient with WAS analyzed before and after bone marrow transplant showed normalized WAS protein and platelet CD42 after treatment highlighting the ability of immuno-SRM to monitor the effects of PIDD treatment. The assay was readily reproduced in two separate laboratories with similar analytical performance and complete agreement in patient diagnosis demonstrating the effective standardized methods. A high-throughput Immuno-SRM method screens PIDD-specific peptides in a 2.5-min runtime meeting high volume NBS workflow requirements was also demonstrated in this report. This high-throughput method returned identical results to the standard Immuno-SRM PIDD panel. Immuno-SRM peptide analysis represents a robust potential clinical diagnostic for identifying and studying PIDD patients from easily collected and shipped DBS and supports a significant potential for early PIDD diagnosis through newborn screening.

Natural-Killer-Derived Extracellular Vesicles: Immune Sensors and Interactors.

Natural killer (NK) cells contribute to immunosurveillance and first-line defense in the control of tumor growth and metastasis diffusion. NK-cell-derived extracellular vesicles (NKEVs) are constitutively secreted and biologically active. They reflect the protein and genetic repertoire of originating cells, and exert antitumor activity in vitro and in vivo. Cancer can compromise NK cell functions, a status potentially reflected by their extracellular vesicles. Hence, NKEVs could, on the one hand, contribute to improve cancer therapy by interacting with tumor and/or immune cells and on the other hand, sense the actual NK cell status in cancer patients. Here, we investigated the composition of healthy donors' NKEVs, including NK microvesicles and exosomes, and their interaction with uncompromised cells of the immune system. To sense the systemic NK cell status in cancer patients, we developed an immune enzymatic test (NKExoELISA) that measures plasma NK-cell-derived exosomes, captured as tsg101+CD56+ nanovesicles. NKEV mass spectrometry and cytokine analysis showed the expression of NK cell markers, i.e., NKG2D and CD94, perforin, granzymes, CD40L, and other molecules involved in cytotoxicity, homing, cell adhesion, and immune activation, together with EV markers tsg101, CD81, CD63, and CD9 in both NK-derived exosomes and microvesicles. Data are available via Proteome Xchange with identifier PXD014894. Immunomodulation studies revealed that NKEVs displayed main stimulatory functions in peripheral blood mononuclear cells (PBMCs), inducing the expression of human leukocyte antigen DR isotype (HLA-DR) and costimulatory molecules on monocytes and CD25 expression on T cells, which was maintained in the presence of lipopolysaccharide (LPS) and interleukin (IL)-10/transforming growth factor beta (TGFβ), respectively. Furthermore, NKEVs increased the CD56+ NK cell fraction, suggesting that effects mediated by NKEVs might be potentially exploited in support of cancer therapy. The measurement of circulating NK exosomes in the plasma of melanoma patients and healthy donors evidenced lower levels of tsg101+CD56+ exosomes in patients with respect to donors. Likewise, we detected lower frequencies of NK cells in PBMCs of these patients. These data highlight the potential of NKExoELISA to sense alterations of the NK cell immune status.

Dissecting the NK Cell Population in Hematological Cancers Confirms the Presence of Tumor Cells and Their Impact on NK Population Homeostasis

The innate immune lymphocyte lineage natural killer (NK) cell protects against multiple pathogens and tumor cells and contains multiple subsets that could have specific activity against different attacks. The presence of viral-sculpted NK populations has already been described, but the presence of cancer-sculpted NK is unknown. Cancer induces a broad NK cell dysfunction, but this is not related to a specific population. Here we investigate NK cell population by Uniform Manifold Approximation and Projection (UMAP) embed maps in Hodgkin lymphoma (HL) and acute myeloid leukemia (AML) patients at diagnosis and, at least 30 days, after treatment, which correlates with tumor cell clearance. Whereas we identified that the NK lineage largely responded to tumor presence by generating antitumor NK cells and renewing NK cell population with immature subsets, we failed to identify a specific “memory-like” subset with the NK cell markers used. Moreover, in patients in relapse we found basically the same NK populations that those found at diagnostic, suggesting that NK cells equally respond to the first or second tumor rise. Finally, previous CMV infection largely affects the tumor-associated changes in NK population, but the CMV-associated CD57+NKG2C+ NK population does not look like to play any role. Funding Statement: The imaging facility MRI, member of the national infrastructure France BioImaging is supported by the French National Research Agency (ANR-10-INBS-04, «Investments for the future»). The “Region Languedoc Roussillon” supports the clinical data and samples (HEMODIAG_2020). This work was supported by the PRT-K program 2018 (MV and GC; 2018-021) and Canceropole GSO Emergence (MV; 2018/2019). This work was also supported by La Ligue Regionale contre le Cancer (GC), the “Investissements d’avenir” Grant LabEx MAbImprove: ANR-10-LABX-53 (GC/MV) and the NK 001 projet financed by the “Fonds Europeen de Developpement Regional “ (FEDER-FSE-IEJ 2014/2020) and by “Region Occitanie Pyrenees Mediterranee”. Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: The use of human specimens for scientific purposes was approved by the French National Ethics Committee. All methods were carried out in accordance with the approved guidelines and regulations of this committee. Written informed consent was obtained from each patient or donor prior to surgery.

Innate and adaptive immunity associated with resolution of acute woodchuck hepatitis virus infection in adult woodchucks.

Viral and/or host factors that are directly responsible for the acute versus chronic outcome of hepatitis B virus (HBV) infection have not been identified yet. Information on immune response during the early stages of HBV infection in humans is mainly derived from blood samples of patients with acute hepatitis B (AHB), which are usually obtained after the onset of clinical symptoms. Features of intrahepatic immune response in these patients are less studied due to the difficulty of obtaining multiple liver biopsies. Woodchuck hepatitis virus (WHV) infection in woodchucks is a model for HBV infection in humans. In the present study, five adult woodchucks were experimentally infected with WHV and then followed for 18 weeks. Blood and liver tissues were frequently collected for assaying markers of WHV replication and innate and adaptive immune responses. Liver tissues were further analyzed for pathological changes and stained for important immune cell subsets and cytokines. The increase and subsequent decline of viral replication markers in serum and liver, the elicitation of antibodies against viral proteins, and the induction of virus-specific T-cell responses indicated eventual resolution of acute WHV infection in all animals. Intrahepatic innate immune makers stayed unchanged immediately after the infection, but increased markedly during resolution, as determined by changes in transcript levels. The presence of interferon-gamma and expression of natural killer (NK) cell markers suggested that a non-cytolytic response mechanism is involved in the initial viral control in liver. This was followed by the expression of T-cell markers and cytolytic effector molecules, indicating the induction of a cytolytic response mechanism. Parallel increases in regulatory T-cell markers suggested that this cell subset participates in the overall immune cell infiltration in liver and/or has a role in regulating AHB induced by the cytolytic response mechanism. Since the transcript levels of immune cell markers in blood, when detectable, were lower than in liver, and the kinetics, except for NK-cells and interferon-gamma, did not correlate well with their intrahepatic expression, this further indicated enrichment of immune cells within liver. Conclusion: The coordinated interplay of innate and adaptive immunity mediates viral clearance in the woodchuck animal model of HBV infection. The initial presence of NK-cell associated interferon-gamma response points to an important role of this cytokine in HBV resolution.

Combination Therapy of Allogeneic HSCT and In Vivo Activated iNKT Cells Prevents Acute Gvhd While Augmenting GVL Effect

Adult acute lymphoblastic leukemia (ALL) is a highly aggressive cancer with poor clinical prognosis and high relapse rate. Whereas allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective treatment for highly differentiated hematologic malignancies, allo-HSCT provides only limited benefits for treating ALL with relapses frequent. Additionally, the development of graft-versus-host-disease (GVHD) remains a major concern for allo-HSCT recipients. Invariant natural killer T (iNKT) cells are a subset of T lymphocytes that express both a semi-invariant T cell receptor and natural killer cell markers. They recognize lipid antigens presented by non-polymorphic CD1d and have been reported to directly target CD1d+ solid tumors. Many B-ALL, including the P210 B-ALL cell line, are also CD1d+. Clinically, increased iNKT cells have simultaneously been correlated with reduced risk of developing acute GVHD and lower rates of malignant relapse. Because iNKT cells comprise only a tiny fraction of total T cells, we established a protocol to insert an iNKT TCR into murine HSC, thereby generating retrogenic mice with 5-10x expansion of iNKT cells in spleen and liver. Although engineered T cell therapy, including CD19-CART therapy, has proven to be effective in inducing remission in many B-ALL patients, when used as a monotherapy, relapse remains a major obstacle. We therefore tested whether a combination of allo-HSCT and iNKT cell therapy could robustly prevent GVHD while preserving graft-versus-leukemia effects (GVL). To test whether the addition of iNKT cells into allo-graft was capable of preventing acute GVHD (aGVHD), lethally irradiated BALB/C were given B6 bone marrow (2.5 x 106) and whole splenocytes (2.5 x 106, 5 x 106, or 10 x 106) from either B6 or retrogenic B6-iNKT mice. Mice that received 5 x 106 B6 splenocytes developed lethal aGVHD and became moribund by Day 10. Half of the mice that received 2.5 x 106 B6 splenocytes develop lethal aGVHD and died by Day 20. In contrast, only half of the mice that received 5 x 106 or 10 x 106 B6-iNKT splenocytes developed lethal aGVHD by Day 20, and all recipients of 2.5 x 106 B6 splenocytes remained alive by Day 20, suggesting the addition of iNKT cells into allograft may reduce the severity of aGVHD. To test whether adding iNKT cells to allograft could enhance GVL without causing GVHD, BALB/C mice with pre-established P210 B-ALL leukemia were given allo-HSCT with or without iNKT cell addition. Initially, our data showed freshly isolated iNKT cells were rapidly cleared following transplantation. We therefore included lethally irradiated P210 loaded with α-Galactosylceramide (α-Galcer) as adjuvant, and were able to see robust expansion of iNKT cells in vitro and in vivo (&gt;20% vs &lt;1% in WT in BM and Liver). Additionally, we found that HSCT recipients that also received iNKT cells were able to fully clear bone marrow residing B-ALL cells by Day8, compared to less than half in control, suggesting an important role of iNKT cells for targeting bone marrow residing B-ALL cells to prevent relapse. Taken together, combination therapy of allogeneic HSCT with infusion of donor-type iNKT cells activated via irradiated α-Galcer-loaded host-type ALL represent a novel approach for preventing GVHD and augmenting GVL effect against ALL. Disclosures No relevant conflicts of interest to declare.

Human signal transducer and activator of transcription 5b (STAT5b) mutation causes dysregulated human natural killer cell maturation and impaired lytic function

Patients with signal transducer and activator of transcription 5b (STAT5b) deficiency have impairment in T-cell homeostasis and natural killer (NK) cells which leads to autoimmunity, recurrent infections, and combined immune deficiency. In this study we characterized the NK cell defect in STAT5b-deficient human NK cells, as well as Stat5b-/- mice. We used multiparametric flow cytometry, functional NK cell assays, microscopy, and a Stat5b-/- mouse model to elucidate the effect of impaired and/or absent STAT5b on NK cell development and function. This alteration generated a nonfunctional CD56bright NK cell subset characterized by low cytokine production. The CD56dim NK cell subset had decreased expression of perforin and CD16 and a greater frequency of cells expressing markers of immature NK cells. We observed low NK cell numbers and impaired NK cell maturation, suggesting that STAT5b is involved in terminal NK cell maturation in Stat5b-/- mice. Furthermore, human STAT5b-deficient NK cells had low cytolytic capacity, and fixed-cell microscopy showed poor convergence of lytic granules. This was accompanied by decreased expression of costimulatory and activating receptors. Interestingly, granule convergence and cytolytic function were restored after IL-2 stimulation. Our results show that in addition to the impaired terminal maturation of NK cells, human STAT5b mutation leads to impairments in early activation events in NK cell lytic synapse formation. Our data provide further insight into NK cell defects caused by STAT5b deficiency.

168 Coexpression of TCR/CD3 complex and natural killer cell markers, CD16 and/or CD56, by Epstein-Barr virus-infected T-cells in hydroa vacciniforme-like lymphoproliferative disorders

168 Coexpression of TCR/CD3 complex and natural killer cell markers, CD16 and/or CD56, by Epstein-Barr virus-infected T-cells in hydroa vacciniforme-like lymphoproliferative disorders

Defining a novel subset of CD1d-dependent type II natural killer T cells using natural killer cell-associated markers.

Natural killer T (NKT) cells are αβ T cell receptor (TCR) expressing innate‐like T cells that display natural killer (NK) cell markers. Based on TCR characteristics, they are divided into two groups restricted to the MHC class I‐like molecule CD1d. Type I NKT cells, most extensively studied, are identified by a semi‐invariant Vα14‐Jα18 (mouse, Vα24‐Jα18 in humans) TCR reactive to the prototypic ligand α‐galactosylceramide presented on CD1d. In contrast, type II NKT cells display diverse TCR reacting to different CD1d‐presented ligands. There are no reagents that identify all type II NKT cells, limiting their exploration. Here, we searched for novel type II NKT cells by comparing Jα18−/−MHCII−/− mice that harbour type II but not type I NKT cells, and CD1d−/−MHCII−/− mice, lacking all NKT cells. We identified significantly larger populations of CD4+ and CD4−CD8− (double negative, DN) TCRβ+ cells expressing NKG2D or NKG2A/C/E in Jα18−/−MHCII−/− mice compared with CD1d−/−MHCII−/− mice, suggesting that 30%‐50% of these cells were type II NKT cells. They expressed CD122, NK1.1, CXCR3 and intermediate/low levels of CD45RB. Further, the CD4+ subset was CD69+, while the DN cells were CD49b+ and CD62L+. Both subsets expressed the NKT cell‐associated promyelocytic leukaemia zinc finger (PLZF) transcription factor and Tbet, while fewer cells expressed RORγt. NKG2D+ CD4+ and DN populations were producers of IFN‐γ, but rarely IL‐4 and IL‐17. Taken together, we identify a novel subset of primary CD4+ and DN type II NKT cells that expresses NKG2 receptors have typical NKT cell phenotypes and a TH1‐like cytokine production.

Human mesenchymal stem cells promote tumor growth via MAPK pathway and metastasis by epithelial mesenchymal transition and integrin \u03b15 in hepatocellular carcinoma

Mesenchymal stem cells (MSCs) appear to be a potential vehicle for anticancer drugs due to their excellent tumor tropism ability. However, the interactions between MSCs and hepatocellular carcinoma (HCC) are quite controversial and the underlying mechanisms are ambiguous. In this study, an investigation was conducted into the effect of human MSCs (hMSCs) on tumor proliferation and metastasis both in xenograft and orthotopic models. It was discovered that hMSCs could promote tumor growth though activating mitogen-activated protein kinase (MAPK) signaling pathway and promote metastasis by epithelial mesenchymal transition (EMT) in vivo. To test whether hMSCs could induce immunosuppressive effects, the expression of the Natural killer (NK) cell marker CD56 was measured by immunohistochemical staining and the expression of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) were measured by qRT-PCR. It was found out that CD56 expression significantly decreased, while TNF-α and IL-6 expression increased in the hMSCs-treated tissues. Mechanistically, RNA sequencing was performed, which led to a discovery that integrin α5 (ITGA5) was over-expressed in hMSCs-treated HCC. ITGA5 siRNAs blocked the hMSCs-induced migration and invasion of HCC, while over-expression of ITGA5 promoted the migration and invasion ability in HCC-hMSCs, indicating that the expression of ITGA5 is associated with hMSCs-induced tumor metastasis. These findings suggest that hMSCs may play a vital role in HCC proliferation and metastasis and could be identified as a putative therapeutic target in HCC.

TGF-β in Mice Ameliorates Experimental Autoimmune Encephalomyelitis in Regulating NK Cell Activity

Multiple sclerosis is a disease characterized by inflammation and demyelination located in the central nervous system. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model for multiple sclerosis (MS). Although the roles of T cells in MS/EAE have been well investigated, little is known about the functions of other immune cells in the neuroinflammation model. Here we found that an essential cytokine transforming growth factor β (TGF-β) which could mediate the differentiation of Th17/regulatory T cells was implicated in the natural killer (NK) cells’ activity in EAE. In EAE mice, TGF-β expression was first increased at the onset and then decreased at the peak, but the expressions of TGF-β receptors and downstream molecules were not affected in EAE. When we immunized the mice with MOG antigen, it was revealed that TGF-β treatment reduced susceptibility to EAE with a lower clinical score than the control mice without TGF-β. Consistently, inflammatory cytokine production was reduced in the TGF-β treated group, especially with downregulated pathogenic interleukin-17 in the central nervous system tissue. Furthermore, TGF-β could increase the transcription level of NK cell marker NCR1 both in the spleen and in the CNS without changing other T cell markers. Meanwhile TGF-β promoted the proliferation of NK cell proliferation. Taken together, our data demonstrated that TGF-β could confer protection against EAE model in mice through NK cells, which would be useful for the clinical therapy of MS.

P62: Evaluation of Natural Killer cell markers CD16, CD56, and CD57 in the endometrium of women with recurrent pregnancy loss

P62: Evaluation of Natural Killer cell markers CD16, CD56, and CD57 in the endometrium of women with recurrent pregnancy loss