CD56bright NK Cells Research Articles

Dissecting the effect of a novel hypomorphic IL2RB mutation on immune dysregulation.

Abstract Human inborn errors of immunity (IEIs) are a class of genetic disorders caused by monogenic germline mutations that impair the function of the encoded protein. We recently described a novel IEI due to an IL2RB homozygous mutation (p.Pro222_Gln225Del) in two siblings who suffered from multi-organ autoimmunity and CMV susceptibility. This mutation resulted in decreased cell surface protein expression and increased serum IL-2/15 levels, with concomitant increased baseline STAT5 phosphorylation (pSTAT5) but poor pSTAT5 response to IL-2 and IL-15 stimulation. Counterintuitively, this IL-2Rβ hypomorphic mutation led to an expansion of memory CD8+ T and immature CD56bright NK cells, which express the highest levels of IL-2Rβ and require IL-2/15 signaling for survival and differentiation. Yet, their maturation and function appeared impaired, suggesting that both intrinsic (hypomorphic receptor) and extrinsic (serum cytokine milieu) mechanisms were at play. To further interrogate these receptor instrinsic/extrinsic mechanisms we developed a mouse model that harbors the homolog mutation (Il2rbMut/Mut). When mutant bone marrow cells reconstituted a WT host, serum IL-2/15 levels and IL-2Rβ protein surface expression partially normalized, though IL-2/15-induced signaling remained hypofunctional. Additionally, the dysregulated mutant IL-2Rβ-driven CD8+ T cell immunophenotype normalized but the NK phenotype did not. These data highlight the asymmetrical effect that a partial IL-2Rβ defect has on CD8 T and NK cell IL-2/15-dependent signaling and downstream cellular processes. These results imply that strategies to modify serum IL-2/15 levels may serve as a therapeutic approach to treating IL-2/15 signaling defects. Supported by grants from NIH (T32 AI074491, K23 AR070897)

Lack of DOCK8 impairs the primary biologic functions of human NK cells and abrogates CCR7 surface expression in a WASP-independent manner

Dedicator of Cytokinesis 8 (DOCK8) deficiency is a rare form of autosomal recessive combined immunodeficiency. The effect of DOCK8 deficiency on Natural Killer cell biology has not been fully elucidated yet. Thus, we undertook a detailed phenotypic and functional evaluation of NK cells from seven patients with DOCK8 deficiency.Patients' immature CD56bright NK cells were defective in IFN-γ secretion, while their mature CD56dim NK cells showed impaired cytotoxicity, partially rescued upon rIL-2 addition. Cross-linking of NK cell receptors revealed a specific defect in the CD3 zeta chain-dependent activation pathway in DOCK8 deficiency. Lack of DOCK8, but not of WASP, impaired CCR7 expression on human CD56bright NK cells, a critical receptor for their migration to secondary lymph nodes. Evaluation of a patient's lymph node showed a severe reduction in NK cells that showed increased intracellular expression of CCR7.Our data suggest that DOCK8 deficiency variably affects NK cell homeostasis in humans.

Single-cell RNA-seq reveals altered NK cell subsets and reduced levels of cytotoxic molecules in patients with ankylosing spondylitis.

Ankylosing spondylitis (AS) is an autoimmune disease with unknown aetiology. To unravel the mechanisms mediating AS pathogenesis, we profiled peripheral blood mononuclear cells (PBMCs) from AS patients and healthy subjects using 10X single‐cell RNA sequencing. The frequencies of immune cell subsets were evaluated by flow cytometry. NK cells were purified from PBMCs using isolation kit and were examined for gene expression by RT‐qPCR. Plasma levels of cytolytic molecules were examined by enzyme‐linked immunosorbent assay. Compared to healthy controls, AS patients showed a significant decrease in total NK cells as well as CD56dim NK subset, whereas CD56bright NK cells were increased. Additionally, impaired expression of cytotoxic genes in NK cells of AS patients was observed by bioinformatics algorithm and verified by RT‐qPCR and flow cytometry. Consistent with changes in transcriptomics, we found decreased plasma levels of granzymes, but not granulysin, in AS patients. Furthermore, Pearson correlation analysis revealed a negative correlation between plasma GZMB levels and disease activity (r = −0.5275, p = 0.0358). No correlation was observed between plasma cytolytic molecules and biochemical indexes (ESR and CRP). Our findings uncover altered NK cell subsets and cytotoxic profiles in peripheral circulation of AS patients at single‐cell resolution.

CD33 Delineates Two Functionally Distinct NK Cell Populations Divergent in Cytokine Production and Antibody-Mediated Cellular Cytotoxicity.

The generation and expansion of functionally competent NK cells in vitro is of great interest for their application in immunotherapy of cancer. Since CD33 constitutes a promising target for immunotherapy of myeloid malignancies, NK cells expressing a CD33-specific chimeric antigen receptor (CAR) were generated. Unexpectedly, we noted that CD33-CAR NK cells could not be efficiently expanded in vitro due to a fratricide-like process in which CD33-CAR NK cells killed other CD33-CAR NK cells that had upregulated CD33 in culture. This upregulation was dependent on the stimulation protocol and encompassed up to 50% of NK cells including CD56dim NK cells that do generally not express CD33 in vivo. RNAseq analysis revealed that upregulation of CD33+ NK cells was accompanied by a unique transcriptional signature combining features of canonical CD56bright (CD117high, CD16low) and CD56dim NK cells (high expression of granzyme B and perforin). CD33+ NK cells exhibited significantly higher mobilization of cytotoxic granula and comparable levels of cytotoxicity against different leukemic target cells compared to the CD33− subset. Moreover, CD33+ NK cells showed superior production of IFNγ and TNFα, whereas CD33− NK cells exerted increased antibody-dependent cellular cytotoxicity (ADCC). In summary, the study delineates a novel functional divergence between NK cell subsets upon in vitro stimulation that is marked by CD33 expression. By choosing suitable stimulation protocols, it is possible to preferentially generate CD33+ NK cells combining efficient target cell killing and cytokine production, or alternatively CD33− NK cells, which produce less cytokines but are more efficient in antibody-dependent applications.

The role of NK and NKT cells in the pathogenesis and improvement of multiple sclerosis following disease-modifying therapies.

BackgroundMultiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system (CNS) that T cells become autoreactive by recognizing CNS antigens. Both innate and adaptive immune systems are involved in the pathogenesis of MS. In recent years, the impact of innate immune cells on MS pathogenesis has received more attention. CD56bright NK cells, as an immunoregulatory subset of NK cells, can increase the production of cytokines that modulate adaptive immune responses, whereas CD56dim NK cells are more active in cytolysis functions. These two main subsets of NK cells may have different effects on the onset or progression of MS. Invariant NKT (iNKT) cells are other immune cells involved in the control of autoimmune diseases; however, variant NKT (vNKT) cells, despite limited information, could play a role in MS remission via an immunoregulatory pathway.AimWe aimed to evaluate the influence of MS therapeutic agents on NK and NKT cells and NK cell subtypes.Materials and MethodsThe possible mechanism of each MS therapeutic agent has been presented here, focusing on the effects of different disease‐modifying therapies on the number of NK and NKT subtypes.ResultsExpansion of CD56bright NK cells, reduction in the CD56dim cells, and enhancement in NKT cells are the more important innate immune cells alterations following the disease‐modifying therapies.ConclusionExpansion of CD56bright NK cells or reduction in the CD56dim cells has been associated with a successful response to different treatments in MS. iNKT and vNKT cells could have beneficial effects on MS improving. It seems that they are enhanced due to some of MS drugs, leading to disease improvement. However, a reduction in the number of NKT cells could be due to the adverse effects of some of MS drugs on the bone marrow.

Expansion of CD56dimCD16neg NK Cell Subset and Increased Inhibitory KIRs in Hospitalized COVID-19 Patients.

Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) infection induces elevated levels of inflammatory cytokines, which are mainly produced by the innate response to the virus. The role of NK cells, which are potent producers of IFN-γ and cytotoxicity, has not been sufficiently studied in the setting of SARS-CoV-2 infection. We confirmed a different distribution of NK cell subsets in hospitalized COVID-19 patients despite their NK cell deficiency. The impairment of this innate defense is mainly focused on the cytotoxic capacity of the CD56dim NK cells. On the one hand, we found an expansion of the CD56dimCD16neg NK subset, lower cytotoxic capacities, and high frequencies of inhibitory 2DL1 and 2DL1/S1 KIR receptors in COVID-19 patients. On the other hand, the depletion of CD56dimCD16dim/bright NK cell subsets, high cytotoxic capacities, and high frequencies of inhibitory 2DL1 KIR receptors were found in COVID-19 patients. In contrast, no differences in the distribution of CD56bright NK cell subsets were found in this study. These alterations in the distribution and phenotype of NK cells might enhance the impairment of this crucial innate line of defense during COVID-19 infection.

NK Cells and Innate-Like T Cells After Autologous Hematopoietic Stem Cell Transplantation in Multiple Sclerosis.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system, in which autoreactive T and B cells play important roles. Other lymphocytes such as NK cells and innate-like T cells appear to be involved as well. To name a few examples, CD56bright NK cells were described as an immunoregulatory NK cell subset in MS while innate-like T cells in MS were described in brain lesions and with proinflammatory signatures. Autologous hematopoietic stem cell transplantation (aHSCT) is a procedure used to treat MS. This procedure includes hematopoietic stem/progenitor cell (HSPC) mobilization, then high-dose chemotherapy combined with anti-thymocyte globulin (ATG) and subsequent infusion of the patients own HSPCs to reconstitute a functional immune system. aHSCT inhibits MS disease activity very effectively and for long time, presumably due to elimination of autoreactive T cells. Here, we performed multidimensional flow cytometry experiments in peripheral blood lymphocytes of 27 MS patients before and after aHSCT to address its potential influence on NK and innate-like T cells. After aHSCT, the relative frequency and absolute numbers of CD56bright NK cells rise above pre-aHSCT levels while all studied innate-like T cell populations decrease. Hence, our data support an enhanced immune regulation by CD56bright NK cells and the efficient reduction of proinflammatory innate-like T cells by aHSCT in MS. These observations contribute to our current understanding of the immunological effects of aHSCT in MS.

Impact of NK Cell Activating Receptor Gene Variants on Receptor Expression and Outcome of Immunotherapy in Acute Myeloid Leukemia.

Natural killer cells are important effector cells in the immune response against myeloid malignancies. Previous studies show that the expression of activating NK cell receptors is pivotal for efficient recognition of blasts from patients with acute myeloid leukemia (AML) and that high expression levels impact favorably on patient survival. This study investigated the potential impact of activating receptor gene variants on NK cell receptor expression and survival in a cohort of AML patients receiving relapse-preventive immunotherapy with histamine dihydrochloride and low-dose IL-2 (HDC/IL-2). Patients harboring the G allele of rs1049174 in the KLRK1 gene encoding NKG2D showed high expression of NKG2D by CD56bright NK cells and a favorable clinical outcome in terms of overall survival. For DNAM-1, high therapy-induced receptor expression entailed improved survival, while patients with high DNAM-1 expression before immunotherapy associated with unfavorable clinical outcome. The previously reported SNPs in NCR3 encoding NKp30, which purportedly influence mRNA splicing into isoforms with discrete functions, did not affect outcome in this study. Our results imply that variations in genes encoding activating NK cell receptors determine receptor expression and clinical outcome in AML immunotherapy.

NK and CD8+ T cell phenotypes predict onset and control of CMV viremia after kidney transplant.

CMV causes mostly asymptomatic but lifelong infection. Primary infection or reactivation in immunocompromised individuals can be life-threatening. CMV viremia often occurs in solid organ transplant recipients and associates with decreased graft survival and higher mortality. Furthering understanding of impaired immunity that allows CMV reactivation is critical to guiding antiviral therapy and examining the effect of CMV on solid organ transplant outcomes. This study characterized longitudinal immune responses to CMV in 31 kidney transplant recipients with CMV viremia and matched, nonviremic recipients. Recipients were sampled 3 and 12 months after transplant, with additional samples 1 week and 1 month after viremia. PBMCs were stained for NK and T cell markers. PBMC transcriptomes were characterized by RNA-Seq. Plasma proteins were quantified by Luminex. CD8+ T cell transcriptomes were characterized by single-cell RNA-Seq. Before viremia, patients had high levels of IL-15 with concurrent expansion of immature CD56bright NK cells. After viremia, mature CD56dim NK cells and CD28–CD8+ T cells upregulating inhibitory and NK-associated receptors were expanded. Memory NK cells and NK-like CD28–CD8+ T cells were associated with control of viremia. These findings suggest that signatures of innate activation may be prognostic for CMV reactivation after transplant, while CD8+ T cell functionality is critical for effective control of CMV.

Identification of a prognostic immune-related signature for small cell lung cancer.

PurposeAs a subgroup of lung cancer, small cell lung cancer (SCLC) is characterized by a short tumor doubling time, high rates of early occurred distant cancer spread, and poor outcomes. Despite its exquisite sensitivity to chemotherapy and radiotherapy, acquired drug resistance and tumor progression are typical. This study aimed to develop a robust signature based on immune‐related genes to predict the outcome of patients with SCLC.MethodsThe expression data of 77 SCLC patients from George's cohort were divided into training set and testing set, and 1534 immune‐related genes from ImmPort database were used to generate and validate the signature. Cox proportional hazards and the Kaplan–Meier analysis were used for developing and testing the prognostic signature. Single‐sample gene set enrichment analysis was used to determine immune cell infiltration phenotypes.ResultsA 10‐gene model comprising NR3C1, NR1D2, TANK, ARAF, HDGF, INHBE, LRSAM1, PLXNA1, PML, and SP1 with the highest frequency after 1000 interactions, was chosen to construct immune‐related signature. This signature showed robust predictive value for SCLC patients’ survival in both training and testing sets. This signature was weakly associated with the clinic pathological values like TNM stage. Furthermore, patients with low risk presented with activation of immune signal pathways, and specific immune cell infiltration with high levels of CD56bright NK cells but low levels of CD8+ T cells, mast cells, and helper T cells.ConclusionThe present study developed immune‐related signature that may help predict the prognosis of SCLC patients, which reflects an unappreciated level of heterogeneity of immunophenotype associated with diverse prognosis for specific subsets in this highly lethal cancer type.

Perturbed NK-cell homeostasis associated with disease severity in chronic neutropenia

AbstractNeutrophils have been thought to play a critical role in terminal differentiation of NK cells. Whether this effect is direct or a consequence of global immune changes with effects on NK-cell homeostasis remains unknown. In this study, we used high-resolution flow and mass cytometry to examine NK-cell repertoires in 64 patients with neutropenia and 27 healthy age- and sex-matched donors. A subgroup of patients with chronic neutropenia showed severely disrupted NK-cell homeostasis manifesting as increased frequencies of CD56bright NK cells and a lack of mature CD56dim NK cells. These immature NK-cell repertoires were characterized by expression of the proliferation/exhaustion markers Ki-67, Tim-3, and TIGIT and displayed blunted tumor target cell responses. Systems-level immune mapping revealed that the changes in immunophenotypes were confined to NK cells, leaving T-cell differentiation intact. RNA sequencing of NK cells from these patients showed upregulation of a network of genes, including TNFSF9, CENPF, MKI67, and TOP2A, associated with apoptosis and the cell cycle, but different from the conventional CD56bright signatures. Profiling of 249 plasma proteins showed a coordinated enrichment of pathways related to apoptosis and cell turnover, which correlated with immature NK-cell repertoires. Notably, most of these patients exhibited severe-grade neutropenia, suggesting that the profoundly altered NK-cell homeostasis was connected to the severity of their underlying etiology. Hence, although our data suggest that neutrophils are dispensable for NK-cell development and differentiation, some patients displayed a specific gap in the NK repertoire, associated with poor cytotoxic function and more severe disease manifestations.

P-080: Single-cell transcriptomic analysis of bone marrow NK cells reveals loss of activated cytotoxic NK cells in Multiple Myeloma

<h3>Background</h3> Multiple Myeloma (MM) disease progression and therapy response are influenced by cues of the microenvironment including tumor control by a cytotoxic immune response. Natural killer (NK) cells are notable mediators of the cytotoxic immune response to MM, and important effector cells in recent immune-mediated therapies. NK cells are drivers of antibody-dependent-cellular cytotoxicity in therapies based on anti-CD38 monoclonal antibodies such as Daratumumab. Classically, NK cells are divided based upon CD56 expression into a cytokine-producing CD56bright subset, releasing cytokines such as IFN-γ, TNF-a and GM-CSF, and a cytotoxic CD56dim subset. However, accumulating evidence suggests much larger heterogeneity in the NK cell compartment and modulation of these NK cell subsets could impact response to NK cell-driven immunotherapies. Here, we used single-cell RNA sequencing to investigate the heterogeneity and MM-driven alterations of the NK cell compartment in the bone marrow of newly diagnosed MM patients undergoing first-line Daratumumab-containing therapy. <h3>Methods</h3> We performed single-cell RNA sequencing of the CD38+ and CD38- fractions of viably frozen bone marrow aspirates from 19 newly diagnosed MM patients and 5 non-cancer control patients. NK cells were identified in silico by transcription of KLRF1, KLRD1, GNLY and NKG7 resulting in a single-cell transcriptomic dataset of 30,373 NK cells from MM patients and 8,865 NK cells from control patients. <h3>Results</h3> After integration of the datasets, bone marrow NK cells formed eight distinct transcriptomic clusters. Conventional CD56bright and CD56dim NK-cells were identified by increased transcription of GZMK or GZMB, respectively. The GZMK+CD56bright NK cells contained both a cluster of naive and a cluster of activated NK cells. The GZMB+CD56dim NK cells consisted of 5 subclusters. To identify MM-induced alterations in NK cell subsets we compared GZMK+CD56bright vs GZMB+CD56dim cluster composition and distribution between control and MM patients. Control bone marrow was dominated by GZMB-transcribing cytotoxic CD56dim NK cells, represented by a low cytokine-producing GZMK+CD56bright vs cytotoxic GZMB+CD56dim ratio. In contrast, MM bone marrow was characterized by heterogeneity in the ratio of cytokine-producing GZMK+CD56bright vs cytotoxic GZMB+CD56dim NK cells. A subset of patients presented with a complete reversal of this ratio. This altered composition was due to a loss of cytotoxic GZMB+CD56dim NK cells, and more specifically a loss of NK cells with a transcriptome suggesting recent activation. <h3>Conclusion</h3> Here we present a transcriptomic overview of NK cells in MM bone marrow at the single-cell level. A subset of MM patients has a loss of activated cytotoxic GZMB+CD56dim NK cells, suggestive of reduced cytotoxic anti-tumor responses. Current analyses are focused on clinical implications of NK cell alterations in response to Daratumumab-based therapies.

Upregulated Expression of CYBRD1 Predicts Poor Prognosis of Patients with Ovarian Cancer.

Cytochrome b reductase 1 (CYBRD1) promotes the development of ovarian serous cystadenocarcinoma (OV). We assessed the function of CYBRD1 in OV underlying The Cancer Genome Atlas (TCGA) database. The correlation between clinicopathological characteristics and CYBRD1 expression was estimated. The Cox proportional hazards regression model and the Kaplan–Meier method were applied to identify clinical features related to overall survival and disease-specific survival. Gene set enrichment analysis (GSEA) was applied to identify the relationship between CYBRD1 expression and immune infiltration. CYBRD1 expression in OV was significantly associated with poor outcomes of primary therapy and FIGO stage. Patients with high levels of CYBRD1 expression were prone to the development of a poorly differentiated tumor and experience of an unfavorable outcome. CYBRD1 expression had significant association with shorter OS and acts as an independent predictor of poor outcome. Moreover, enhanced CYBRD1 expression was positively associated with Tem, NK cells, and mast cells but negatively associated with CD56 bright NK cells and Th2 cells. CYBRD1 expression may serve as a diagnostic and prognostic indicator of OV patients. The mechanisms of poor prognosis of CYBRD1-mediated OV may include increased iron uptake, regulation of immune microenvironment, ferroptosis related pathway, and ERK signaling pathway, among which ferroptosis and ERK signaling pathway may be important pathways of CYBRD1-mediated OV. Furthermore, we verified that CYBRD1 was upregulated in OV and significant correlated with lymph nodes metastasis, advanced stage, poor-differentiated tumor, and poor clinical prognosis in East Hospital cohort. The results of this study may provide guidance for the development of optimal treatment strategies for OV.

P-033. Changes of natural cytotoxicity receptors of CD56dim and CD56bright NK cells in peripheral blood and decidua in preeclampsia

P-033. Changes of natural cytotoxicity receptors of CD56dim and CD56bright NK cells in peripheral blood and decidua in preeclampsia

CLL-156: Novel CCL22 Mutations Drive Chronic Lymphoproliferative Disorder of NK Cells Through Biased GPCR Signaling

Context: Chronic lymphoproliferative disorder of natural killer cells (CLPD-NK) is a subset of large granular lymphocyte (LGL) leukemia included as a provisional entity in the 2016 WHO classification of lymphoid neoplasms, which is characterized by clonal expansion of NK cells with no standardized therapy. STAT3 and TET2 mutations were previously each reported in 15%–30% of cases; however, the genomic basis of the remaining cases is still unknown. Objective: We aimed to perform a comprehensive genomic and transcriptomic analysis of CLPD-NK to elucidate the genomic landscape, investigate mechanisms of pathogenesis, and improve diagnostic classification and treatment strategies. Methods: CLPD-NK samples (n=121) were examined by whole-genome, targeted, and/or whole-transcriptome sequencing. Structural modeling, G-protein signaling, β-arrestin signaling, G-protein-coupled receptor (GPCR) for CCL22, CCR4 internalization, and chemotaxis were examined. Cell line-derived xenografts were used to examine cell proliferation and phenotype. Results: Somatic mutations in the chemokine CCL22 were found in 21.5% of CLPD-NK patients but not in 2837 cases of other hematological malignancies, including T-LGL and other NK leukemias. Clinically, there was a trend to an association with cutaneous complications and/or neurological symptoms. CCL22 mutations were enriched at the highly conserved residues Leu45, Pro46, and Pro79 and were mutually exclusive of STAT3 mutations. CCL22 mutations were associated with dysregulated gene expression, similar to normal CD56bright NK cells, with enrichment for activation of cytokine signaling pathways, representing a distinct subgroup. Most CCL22 mutations were substitutions to Arg, a basic residue, and Leu45Arg was predicted to promote electrostatic interactions with CCR4-Tyr22/Glu23 acidic residues. The mutations resulted in ligand-biased CCR4 signaling, in which activation of G-protein signaling was unchanged, but recruitment of β-arrestin, which mediates receptor internalization, was impaired, which, in turn, resulted in enhanced cell chemotaxis. CCL22 mutations drove enhanced cell proliferation in vivo in hIL-15-transgenic mice and induced an expression profile resembling that of primary samples, indicating the importance of microenvironmental crosstalk in pathogenesis. Conclusions: Somatic CCL22 mutations defined a new subgroup of CLPD-NK similar to CD56bright normal NK cells and represented a novel mechanism of leukemogenesis in which gain-of-function chemokine mutations drive tumorigenesis by biased GPCR signaling and dysregulation of microenvironmental crosstalk.

NK cells that differ in expression of NKp46 might play different roles in endometrium

NKp46 is a natural cytotoxicity receptor expressed by NK cells and its expression is decreased in reproductive failure patients. NKp46 can be subdivided into NKp46dim and NKp46bright according to different fluorescence staining intensities. We investigated the role of the NKp46 receptor in determining the reproductive outcomes. Uterine endometrium was collected from 34 women with reproductive failure and divided into the pregnant and failed groups based on the results of a pregnancy reaction test during a 1-year follow-up period. NKp46 receptor and other activating or inhibitory receptors expressed on NK cells as well as intracellular cytokine production by NK cells were analyzed by multicolor flow cytometry. In the failed group, the percentage of NKp46dim NK cells (P < 0.05) was significantly higher and percentages of NKp46bright NK cells (P < 0.01) and CD16−/CD56bright NK cells (P < 0.05) were significantly lower than those in the pregnant group. NKp46dim NK cells were significantly and positively correlated with CD16+/NKp46dim NK cells; NKp46bright NK cells were significantly and positively correlated with CD16−/NKp46bright NK cells. CD16+/NKp46dim NK cells were significantly and positively correlated with IFN-γ- and/or TNF-α-producing NK cells; CD16−/NKp46bright NK cells were significantly and positively correlated with TGF-β1-producing NK cells. We suggest that the NKp46 receptor plays different roles in reproduction based on the different fluorescence intensities associated with NK cells, i.e. NKp46dim NK cells are involved in killing cells, whereas NKp46bright NK cells are involved in cytokine production, indicating that NKp46 could be a predictive marker to see a tolerate condition for embryos.

Third-party bone marrow–derived mesenchymal stromal cell infusion before liver transplantation: A randomized controlled trial

Mesenchymal stromal cells (MSC) have emerged as a promising therapy to minimize the immunosuppressive regimen or induce tolerance in solid organ transplantation. In this randomized open-label phase Ib/IIa clinical trial, 20 liver transplant patients were randomly allocated (1:1) to receive a single pretransplant intravenous infusion of third-party bone marrow–derived MSC or standard of care alone. The primary endpoint was the safety profile of MSC administration during the 1-year follow-up. In all, 19 patients completed the study, and none of those who received MSC experienced infusion-related complications. The incidence of serious and non-serious adverse events was similar in the two groups. Circulating Treg/memory Treg and tolerant NK subset of CD56bright NK cells increased slightly over baseline, albeit not to a statistically significant extent, in MSC-treated patients but not in the control group. Graft function and survival, as well as histologic parameters and intragraft expression of tolerance-associated transcripts in 1-year protocol biopsies were similar in the two groups. In conclusion, pretransplant MSC infusion in liver transplant recipients was safe and induced mild positive changes in immunoregulatory T and NK cells in the peripheral blood. This study opens the way for a trial on possible tolerogenic efficacy of MSC in liver transplantation. ClinicalTrials.gov identifier: NCT02260375.

Profiles of NK cell subsets are associated with successful tyrosine kinase inhibitor discontinuation in chronic myeloid leukemia and changes following interferon treatment.

Recent studies have shown that approximately 50% of patients with chronic myeloid leukemia (CML) receiving tyrosine kinase inhibitor (TKI) therapy with a sustained deep molecular response (DMR) (BCR-ABL1IS ≤ 0.01%) can achieve treatment-free remission (TFR, stopping TKI without relapse) and that prior interferon (IFN)-α therapy and higher NK cell counts at and after TKI discontinuation are associated with TFR. We recently reported that post-TKI discontinuation of IFN-α therapy could prevent molecular relapse (MR, BCR-ABL1IS > 0.1%). Here, we evaluated whether NK cells are associated with MR and investigated the effects of post-TKI discontinuation IFN-α therapy on lymphocyte subsets. A total of 34 patients measuring blood lymphocyte subclasses were included. In the 22 patients who did not receive IFN-α therapy, at 1month after TKI discontinuation, the nonrelapsed patients showed a significantly higher proportion and count of NK cells than the relapsed patients. In particular, the proportion and count of CD56dim NK cells were significantly higher in the nonrelapsed patients than in the relapsed patients. In the 12 patients who received IFN-α therapy, the level of CD56bright NK cells increased significantly after 3 and 6months of IFN-α therapy. In summary, NK cells, in particular CD56dim NK cells, were associated with MR after TKI discontinuation in patients with CML. Additionally, IFN-α therapy gradually increased the level of CD56bright NK cells in patients with CML.

Low Natural Killer (NK) Cell Counts and Functionality Are Associated With Molecular Relapse After Imatinib Discontinuation In Patients (pts) With Chronic Phase (CP)-Chronic Myeloid Leukemia (CML) With Undetectable BCR-ABL Transcripts For At Least 2 Years: Preliminary Results From Immunostim, On Behalf Of STIM Investigators

BackgroundIn pts with CP-CML, the notion that tyrosine kinase inhibitors (TKI) may never be stopped is currently challenged in the setting of clinical trials. In the pioneering French STIM trial, pts on imatinib for at least 3 years who had achieved and maintained undetectable BCR-ABL transcripts for at least 2 years had a 24-month molecular relapse-free survival of 39% with no relapses beyond 24 months. A low Sokal risk group and a long-lasting imatinib treatment were significantly associated with an increased freedom from molecular relapse but the role of other factors such as cellular immunity cannot be excluded. The aim of IMMUNOSTIM performed on a subset of 51 pts enrolled in the STIM trial was to analyze the potential implication of NK-cell and T-cell immunity in molecular relapse-free survival after imatinib discontinuation. MethodsPeripheral blood was drawn prior to and after imatinib cessation. All analyses were performed in a centralized fashion. Blood cell counts and differentials were determined using an automated blood cell count analyzer. Flow cytometry was performed on fresh cells to quantify CD56dim and CD56bright NK cells, CD3+ T cells, naïve and memory CD4+ and CD8+ T cells and CD4+ regulatory (CD25+CD127neg/low) T cells (TREGs). Expression of activating and inhibitory NK cell receptors was also analyzed. Functional analyses were performed using frozen mononuclear cells. The focus was put on NK cell function by means of a CD107a degranulation assay against the K562 cell line and an IFN-g secretion assay upon IL-12 and IL-18 stimulation. ResultsStudied pts' characteristics were comparable to that of the entire STIM study population. Median age was 63 years, 64.7% of pts were females and 52.9% has received prior IFN. Sokal risk group was low in 54.9%, intermediate in 35.3%, high in 7.8% and unknown in 2%. Median imatinib treatment duration was 57 months (36-94). Median follow-up after imatinib discontinuation was 42 months (24-54). A molecular relapse occurred in 28 pts after a median time off therapy of 2 months (1-20). Median BCR-ABL/ABL transcript ratio at relapse confirmation was 0.015% IS (0.006-1.546). Molecular relapse-free survival at 24 months was 47.1% (95% CI: 31.4-58.7). A significantly higher proportion of low low Sokal risk group and a trend toward longer imatinib treatment duration were found in pts who did not relapse compared to those who relapsed.At baseline, median leucocyte and lymphocyte counts were 4820/mm3 (2550-7860) and 1310/mm3 (720-2610), respectively and were comparable in pts who relapsed and those who did not. Median CD3+, CD4+, CD4+ TREGs, CD8+ T cells and CD4/CD8 ratio were 879/mm3 (315-1949), 534/mm3 (228-1248), 37/mm3 (4-94), 279/mm3 (72-998) and 2 (0.5-14.2), respectively, with no significant difference between pts who relapsed and those who did not. Analysis of CD4+ and CD8+ naïve, effector and central memory T cells did not reveal any differences between the 2 groups.In contrast, NK cell counts at imatinib cessation were significantly lower in pts who relapsed than in those who did not relapse (145/mm3 (67-450) versus 233/mm3 (70-727), p=0.0146). This decrease specifically affected the cytotoxic CD56dim population. NK cells numbers did not significantly increase in either group after imatinib discontinuation.NK receptor expression analysis did not show any significant defect in the expression of the activating NKG2D receptor and the CD94, NKG2A and KIR inhibitory receptors. However, the cytotoxic activity of CD56dim NK cells toward K562 and the IFN-g secretion potential of CD56bright NK cells upon IL-12 and IL-18 stimulation were significantly decreased when compared to those of healthy donors. Analyses are ongoing to further dissect NK cell function in pts who relapsed and those who did not. Updated results will be presented. ConclusionsOur stud shows a specific association between peripheral blood NK cells and clinical outcome after imatinib discontinuation in pts with long-lasting molecularly undetectable residual disease. These results suggest that NK cell-based immune surveillance may contribute to CML control after TKI cessation. Whether NK cells may be used as biomarkers among other factors to predict disease relapse after TKI cessation needs to be investigated. The understanding of NK cell defects may help in building strategies to restore an optimal NK cell-mediated anti-leukemic activity. Disclosures:Rea:BMS, Novartis, Pfizer, Ariad, Teva: Honoraria. Nicolini:Novartis, Ariad and Teva: Consultancy; Novartis & Bristol Myers Squibb: Research Funding; Novartis, BMS, Teva, Pfizer, Ariad: Honoraria; Novartis, BMS, Teva: Speakers Bureau; Novartis, Ariad, Teva, Pfizer: Membership on an entity's Board of Directors or advisory committees. Rousselot:BMS, Ariad, Pfizer: Honoraria from BMS, Ariad, Pfizer. Grants from BMS Other.

Ovarian Cancer-Associated Ascites Have High Proportions of Cytokine-Responsive CD56bright NK Cells.

Ovarian cancer is the most lethal gynecological malignancy, with serous histotype as the most prevalent epithelial ovarian cancer (EOC). Peritoneal ascites is a frequent comorbidity in advanced EOC. EOC-associated ascites provide a reliable sampling source for studying lymphocytes directly from tumor environment. Herein, we carried out flow cytometry-based analysis to readdress issues on NK and T lymphocyte subsets in women with advanced EOC, additionally evaluating phenotypic modulation of their intracellular pathways involved in interleukin (IL)-2 and IL-15 signaling. Results depicted ascites as an inflammatory and immunosuppressive environment, presenting significantly (p < 0.0001) higher amounts of IL-6 and IL-10 than in the patients’ blood, as well as significantly (p < 0.05) increased expression of checkpoint inhibitory receptors (programmed death protein-1, PD-1) and ectonucleotidase (CD39) on T lymphocytes. However, NK lymphocytes from EOC-associated ascites showed higher (p < 0.05) pS6 phosphorylation compared with NK from blood. Additionally, in vitro treatment of lymphocytes with IL-2 or IL-15 elicited significantly (p < 0.001) phosphorylation of the STAT5 protein in NK, CD3 and CD8 lymphocytes, both from blood and ascites. EOC-associated ascites had a significantly (p < 0.0001) higher proportion of NK CD56bright lymphocytes than blood, which, in addition, were more responsive (p < 0.05) to stimulation by IL-2 than CD56dim NK. EOC-associated ascites allow studies on lymphocyte phenotype modulation in the tumor environment, where inflammatory profile contrasts with the presence of immunosuppressive elements and development of cellular self-regulating mechanisms.