NK Cell Subsets Research Articles

Single-cell RNA sequencing reveals the heterogeneity of liver-resident immune cells in human

The liver plays a critical role in both immune defense and tolerance in the body. The liver-resident immune cells (LrICs) determine the immune properties, but the unique composition and heterogeneity of these cells are incompletely understood. Here, we dissect the diversity of LrICs by a comprehensive transcriptomic profiling using the unbiased single-cell RNA-sequencing (scRNA-seq). A total of 70, 706 of CD45+ immune cells from the paired liver perfusion, spleen and peripheral blood as references were profiled. We identified more than 30 discrete cell populations comprising 13 of T and NK cell, 7 of B cell, 4 of plasma cell, and 8 of myeloid cell subsets in human liver and donor-paired spleen and blood, and characterized their tissue distribution, gene expression and functional modules. Especially, four of CXCR6+ T and NK cell subsets were found to be present preferentially in the liver, where they manifested heterogeneity, distinct function and prominent homeostatic proliferation. We propose a universal category system of T and NK cells based on distinct chemokine receptors, confirmed subsequently by phenotype, transcriptional factors and functionality. We also identified adaptive changes by the spleen and liver-derived monocyte and macrophage populations. Finally, we give a global glimpse on B cell and plasma cell subsets in human spleen and liver. We, therefore, reveal the heterogeneity and functional diversity of LrICs in human. This study presents comprehensively the landscape of LrICs and will enable further study on their roles in various human diseases.

Characterization of the Impact of Daclizumab Beta on Circulating Natural Killer Cells by Mass Cytometry.

Daclizumab beta is a humanized monoclonal antibody that binds to CD25 and selectively inhibits high-affinity IL-2 receptor signaling. As a former treatment for relapsing forms of multiple sclerosis (RMS), daclizumab beta induces robust expansion of the CD56bright subpopulation of NK cells that is correlated with the drug’s therapeutic effects. As NK cells represent a heterogeneous population of lymphocytes with a range of phenotypes and functions, the goal of this study was to better understand how daclizumab beta altered the NK cell repertoire to provide further insight into the possible mechanism(s) of action in RMS. We used mass cytometry to evaluate expression patterns of NK cell markers and provide a comprehensive assessment of the NK cell repertoire in individuals with RMS treated with daclizumab beta or placebo over the course of 1 year. Treatment with daclizumab beta significantly altered the NK cell repertoire compared to placebo treatment. As previously reported, daclizumab beta significantly increased expression of CD56 on total NK cells. Within the CD56bright NK cells, treatment was associated with multiple phenotypic changes, including increased expression of NKG2A and NKp44, and diminished expression of CD244, CD57, and NKp46. These alterations occurred broadly across the CD56bright population, and were not associated with a specific subset of CD56bright NK cells. While the changes were less dramatic, CD56dim NK cells responded distinctly to daclizumab beta treatment, with higher expression of CD2 and NKG2A, and lower expression of FAS-L, HLA-DR, NTB-A, NKp30, and Perforin. Together, these data indicate that the expanded CD56bright NK cells share features of both immature and mature NK cells. These findings show that daclizumab beta treatment is associated with unique changes in NK cells that may enhance their ability to kill autoreactive T cells or to exert immunomodulatory functions.

Concordance between peripheral and decidual NK cell subsets and killer immunoglobulin-like receptors in women with recurrent spontaneous miscarriages

BackgroundThe role of decidual natural killer (dNK) cells in normal and complicated pregnancy and their relation with peripheral NK (pNK) cells remains unclear. The study aim was phenotypic analysis of pNK and dNK cells at time of miscarriage in recurrent spontaneous miscarriage (RSM) patients to assess whether measuring levels of pNK cell populations can reflect changes in dNK cells or not. MethodsThis study included 40 middle aged pregnant women in the 1st trimester subjected to evacuation because of a current miscarriage. They had a history of previous ≥ two unexplained miscarriages. Frequencies of pNK and dNK cells, based on the expression of CD56, CD16, inhibitory (CD158b) and activating (CD161) Killer immunoglobulin-like receptors (KIRs), were detected by flow cytometry. ResultsPercentages of CD56+ NK cells in peripheral blood and decidua were 17.5 % and 17.3 %, respectively. In both blood and decidua, CD56dim NK cells were exceeding CD56bright NK cells. The CD56dim CD16− NK cells were the predominating subset of NK cells, followed by CD56dim CD16dim. No substantial differences were detected in the levels of KIRs expression by the different NK subsets between blood and decidua. Abnormal up-regulation of both CD161 and CD158b on NK cells was observed in blood and decidua. ConclusionAt the time of miscarriage, patients with RSM have an extremely active immune system and an increased number of toxic NK cells both in blood and decidua. The pNK cells reflect dNK cell changes during miscarriage and may be a useful non-invasive predicting tool in reproductive failure setting.

Abstract B84: Decipher the role of IL-33 as an activator of NK cells’ antitumor activity

Abstract Interleukin (IL)-33 is an alarmin belonging to the IL-1 family. Through its receptor ST2, IL-33 promotes both type 1 and type 2 immune responses, depending on the type of responding cells and the microenvironment in damaged tissues. Consistently, paradoxical roles of IL-33 have been reported in the context of cancer. These observations prompted us to investigate i) the expression of IL-33/ST2 in breast tumors and ii) the role of IL-33/ST2 in NK cell activation in physiologic and tumor contexts. Using immunohistochemistry (IHC) and transcriptomic analyses, we observed that IL-33 is expressed at higher levels in ductal carcinoma in situ (DCIS) compared to invasive breast cancer (IBC), especially in the stroma and in luminal BC subtype. In situ, IL-33 was mainly detected in endothelial cells and at lower extent in scattered cells within the stroma of breast tumors. Furthermore, we unraveled a new pathway for NK cells’ activation where IL-12 upregulates ST2 on human NK cells, which in turn become responsive to IL-33 by secreting high levels of IFN-γ and increasing their cytotoxic activity. This effect was specific to a subset of human CD56dim NK cells (20%) and was dependent on STAT-4 phosphorylation. In mice, we also observed a strong activation of spleen NK cell by IL-33 in combination with IL-12 in vitro. Finally, using IL-33ko mice, preliminary results show a contribution of endogenous IL-33 in the prevention of experimental lung metastasis development following B16 iv injection in therapeutic settings using exogenous administration of IL-12. Following up on these results, our aim is now to i) better characterize IL-33-responsive NK cells subpopulation in blood, ii) understand why human CD56bright NK cells do not upregulate ST2 despite response to IL-12 by phosphorylating STAT-4, iii) characterize NK cells’ response to IL-33 in human breast tumors, iv) evaluate ST2 expression by immune cells infiltrating breast and ovarian tumors, and v) confirm the activating role of IL-33 on NK cells’ biology in tumor models in vivo. All together, our observations are in favor of an NK-mediated antitumor role of IL-33 that we are currently pursuing as a potential novel therapeutic strategy in cancer. Citation Format: Anais Eberhardt, Elena Blanc, Emilie Lardenois, Sarah Renaudineau, Jennifer Herbulot, Benoit Dumont, Isabelle Durand, Emilie Charrier, Pauline Schmitt, Jean-Philippe Girard, Antoine Marçais, Thierry Walzer, Christophe Caux, Nathalie Bendriss-Vermare. Decipher the role of IL-33 as an activator of NK cells’ antitumor activity [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B84.

Comprehensive Phenotyping of Human PB NK Cells by Flow Cytometry.

The NK cell compartment provides powerful innate defenses against virus-infected and tumor cells. Specific NK cell receptors control this process and maintain the immune system homeostasis and prevent autoimmunity. A wide variety of NK cell subsets with different functional capabilities exist and this reflects not only the different maturation stages of NK cells but also different microenvironments in which they can operate. In this review, we will give an overview on the various NK cell subsets present in peripheral blood of healthy donors in order to clearly and univocally identify them on the basis of their phenotypic traits using flow cytometry. © 2020 International Society for Advancement of Cytometry.

Understanding the Synergy of NKp46 and Co-Activating Signals in Various NK Cell Subpopulations: Paving the Way for More Successful NK-Cell-Based Immunotherapy.

The NK cell population is characterized by distinct NK cell subsets that respond differently to the various activating stimuli. For this reason, the determination of the optimal cytotoxic activation of the different NK cell subsets can be a crucial aspect to be exploited to counter cancer cells in oncologic patients. To evaluate how the triggering of different combination of activating receptors can affect the cytotoxic responses of different NK cell subsets, we developed a microbead-based degranulation assay. By using this new assay, we were able to detect CD107a+ degranulating NK cells even within the less cytotoxic subsets (i.e., resting CD56bright and unlicensed CD56dim NK cells), thus demonstrating its high sensitivity. Interestingly, signals delivered by the co-engagement of NKp46 with 2B4, but not with CD2 or DNAM-1, strongly cooperate to enhance degranulation on both licensed and unlicensed CD56dim NK cells. Of note, 2B4 is known to bind CD48 hematopoietic antigen, therefore this observation may provide the rationale why CD56dim subset expansion correlates with successful hematopoietic stem cell transplantation mediated by alloreactive NK cells against host T, DC and leukemic cells, while sparing host non-hematopoietic tissues and graft versus host disease. The assay further confirms that activation of LFA-1 on NK cells leads to their granule polarization, even if, in some cases, this also takes to an inhibition of NK cell degranulation, suggesting that LFA-1 engagement by ICAMs on target cells may differently affect NK cell response. Finally, we observed that NK cells undergo a time-dependent spontaneous (cytokine-independent) activation after blood withdrawal, an aspect that may strongly bias the evaluation of the resting NK cell response. Altogether our data may pave the way to develop new NK cell activation and expansion strategies that target the highly cytotoxic CD56dim NK cells and can be feasible and useful for cancer and viral infection treatment.

Perirenal Adipose Tissue Displays an Age-Dependent Inflammatory Signature Associated With Early Graft Dysfunction of Marginal Kidney Transplants.

Background: Better understanding of the contribution of donor aging and comorbidity factors of expanded criteria donors (ECD) to the clinical outcome of a transplant is a challenge in kidney transplantation. We investigated whether the features of donor-derived stromal vascular fraction of perirenal adipose tissue (PRAT-SVF) could be indicative of the deleterious impact of the ECD microenvironment on a renal transplant.Methods: A comparative analysis of cellular components, transcriptomic and vasculogenic profiles was performed in PRAT-SVF obtained from 22 optimal donors and 31 ECD deceased donors. We then investigated whether these parameters could be associated with donor aging and early allograft dysfunction.Results: When compared with the PRAT-SVF of non-ECD donors, ECD PRAT-SVF displayed a lower proportion of stromal cells, a higher proportion of inflammatory NK cells. The global RNA sequencing approach indicated a differential molecular signature in the PRAT-SVF of ECD donors characterized by the over-expression of CXCL1 and IL1-β inflammatory transcripts. The vasculogenic activity of PRAT-SVF was highly variable but was not significantly affected in marginal donors. Periorgan recruitment of monocytes/macrophages and NK cells in PRAT-SVF was associated with donor aging. The presence of NK cell infiltrates was associated with lower PRAT-SVF angiogenic activity and with early allograft dysfunction evaluated on day 7 and at 1 month post-transplant.Conclusions: Our results indicate that human NK cell subsets are differentially recruited in the periorgan environment of aging kidney transplants. We provide novel evidence that PRAT-SVF represents a non-invasive and timely source of donor material with potential value to assess inflammatory features that impact organ quality and function.

Pretreatment Innate Cell Populations and CD4 T Cells in Blood Are Associated With Response to Immune Checkpoint Blockade in Melanoma Patients

The development of changes in T cells, referred to as T cell exhaustion, has been suggested as a cause of primary or acquired resistance to immunotherapy by immune checkpoint blockade (ICB). A limited number of studies, largely performed on tumor infiltrating lymphocytes (TILs), has provided evidence in support of this hypothesis, but whether similar changes occur in circulating blood lymphocytes has received little attention. In the present study, a comprehensive analysis of peripheral blood leukocytes from 42 patients taken over the course of treatment with anti-PD-1 was undertaken. The patients included those grouped as responders (who did not progress), primary non-responders (primary resistance) and those with acquired resistance (who initially responded then subsequently progressed). Analysis included surface markers of exhaustion, production of cytokines following in vitro stimulation, and assessment of transcription factor levels associated with T cell exhaustion. There were differences in innate cell populations between responders and non-responders at baseline and maintained throughout therapy. Frequencies of total and classical CD14+CD16− monocytes were higher and the major subset of NK cells (CD16hiCD56+) was significantly smaller in the primary resistance group compared with responders. However, differences in peripheral blood expression of exhaustion markers were not evident between the treatment groups. T cell exhaustion markers were expressed in practically all patients and the major observation was an increase in CD39 on CD4 T cells during treatment. The results confirm the association of Eomes transcription factor with T cell exhaustion but levels of expression and the ratio with T-bet over Eomes did not differ between the patient groups. Thus, peripheral blood expression of T cell exhaustion markers does not distinguish between responders and non-responders to anti-PD-1 therapy. CD4 T cell expression of IFNγ also differed in pre-treatment samples, indicating that predictors of response unrelated to exhaustion may be present in peripheral blood. The association of response with innate cell populations and CD4 T cell responses requires further study.

Dysregulated Antibody, Natural Killer Cell and Immune Mediator Profiles in Autoimmune Thyroid Diseases.

The pathogenesis of autoimmune thyroid diseases (AITD) is poorly understood and the association between different immune features and the germline variants involved in AITD are yet unclear. We previously observed systemic depletion of IgG core fucosylation and antennary α1,2 fucosylation in peripheral blood mononuclear cells in AITD, correlated with anti-thyroid peroxidase antibody (TPOAb) levels. Fucose depletion is known to potentiate strong antibody-mediated NK cell activation and enhanced target antigen-expressing cell killing. In autoimmunity, this may translate to autoantibody-mediated immune cell recruitment and attack of self-antigen expressing normal tissues. Hence, we investigated the crosstalk between immune cell traits, secreted proteins, genetic variants and the glycosylation patterns of serum IgG, in a multi-omic and cross-sectional study of 622 individuals from the TwinsUK cohort, 172 of whom were diagnosed with AITD. We observed associations between two genetic variants (rs505922 and rs687621), AITD status, the secretion of Desmoglein-2 protein, and the profile of two IgG N-glycan traits in AITD, but further studies need to be performed to better understand their crosstalk in AITD. On the other side, enhanced afucosylated IgG was positively associated with activatory CD335- CD314+ CD158b+ NK cell subsets. Increased levels of the apoptosis and inflammation markers Caspase-2 and Interleukin-1α positively associated with AITD. Two genetic variants associated with AITD, rs1521 and rs3094228, were also associated with altered expression of the thyrocyte-expressed ligands known to recognize the NK cell immunoreceptors CD314 and CD158b. Our analyses reveal a combination of heightened Fc-active IgG antibodies, effector cells, cytokines and apoptotic signals in AITD, and AITD genetic variants associated with altered expression of thyrocyte-expressed ligands to NK cell immunoreceptors. Together, TPOAb responses, dysregulated immune features, germline variants associated with immunoactivity profiles, are consistent with a positive autoreactive antibody-dependent NK cell-mediated immune response likely drawn to the thyroid gland in AITD.

The Toll-Like Receptor 3 Agonist Polyriboinosinic Polyribocytidylic Acid Increases the Numbers of NK Cells with Distinct Phenotype in the Liver of B6 Mice.

One of the activating factors of the cells of the innate immune system is the agonists of toll-like receptors (TLRs). Our earlier publications detailed how poly(I:C), a TLR3 agonist, elevates the NK cell population and the associated antigen-specific CD8+ T cell responses. This study involved a single treatment of the B6 mice with poly(I:C) intraperitoneally. To perform a detailed phenotypic analysis, mononuclear cells were prepared from each of the liver, peripheral blood, and spleen. These cells were then examined for their NK cell population by flow cytometric analysis following cell staining with indicated antibodies. The findings of the study showed that the NK cell population of the liver with an NK1.1highCD11bhighCD11chigh B220+Ly6G− phenotype was elevated following the treatment with poly(I:C). In the absence of CD11b molecule (CR3−/− mice), poly(I:C) can still increase the remained numbers of NK cells with NK1.1+CD11b− and NK1.1+Ly6G− phenotypes in the liver while their numbers in the blood decrease. After the treatment with anti-AGM1 Ab, which induced depletion of NK1.1+CD11b+ cells and partial depletion of CD3+NK1.1+ and NK1.1+CD11b− cell populations, poly(I:C) normalized the partial decreases in the numbers of NK cells concomitant with increased numbers of NK1.1−CD11b+ cell population in both liver and blood. Regarding mice with a TLR3−/− phenotype, their injection with poly(I:C) resulted in the partial elevation in the NK cell population as compared to wild-type B6 mice. To summarise, the TLR3 agonist poly(I:C) results in the elevation of a subset of liver NK cells expressing the two myeloid markers CD11c and CD11b. The effect of poly(I:C) on NK cells is partially dependent on TLR3 and independent of the presence of CD11b.

Tim-3 Hampers Tumor Surveillance of Liver-Resident and Conventional NK Cells by Disrupting PI3K Signaling.

Natural killer (NK) cells are enriched within the liver. Apart from conventional NK (cNK) cells, recent studies identified a liver-resident NK (LrNK) subset, which constitutes about half of hepatic NK cells and exhibits distinct developmental, phenotypic, and functional features. However, it remains unclear whether and how LrNK cells, as well as cNK cells, participate in the development of hepatocellular carcinoma (HCC) individually. Here, we report that both LrNK and cNK cells are significantly decreased in HCC. The T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) was significantly upregulated in both tumor-infiltrating LrNK and cNK cells and suppressed their cytokine secretion and cytotoxic activity. Mechanistically, phosphatidylserine (PtdSer) engagement promoted phosphorylation of Tim-3, which then competed with PI3K p110 to bind p85, inhibiting downstream Akt/mTORC1 signaling and resulting in malfunctioning of both NK-cell subsets. Tim-3 blockade retarded HCC growth in a NK-cell-dependent manner. These studies for the first time report the presence and dysfunction of LrNK cells in HCC and show that Tim-3-mediated PI3K/mTORC1 interference is responsible for the dysfunction of both tumor-infiltrating cNK and LrNK cells, providing a new strategy for immune checkpoint-based targeting. SIGNIFICANCE: Tim-3 enhances hepatocellular carcinoma growth by blocking natural killer cell function.

Cytometry-based analysis of HLA-G functions according to ILT2 expression

HLA-G was described as a molecule inhibiting NK and T cells functions through its receptor, ILT2. However, most functional studies of HLA-G were so far performed on heterogeneous immune populations and regardless of ILT2 expression. This may lead to an underestimation of the effect of HLA-G. Thus, considering the immune subpopulations sensitive to HLA-G remained an important issue in the field. Here we present a new cytometry assay to evaluate HLA-G effects on both NK and CD8+ T cell cytotoxic functions.Using flow cytometry allows for the comparison of HLA-G function on multiple subsets and multiple functions in the same time. In particular, we sharpen the analysis by specifically studying the immune subpopulations expressing HLA-G receptor ILT2. We focused our work on: IFN-gamma production and cytotoxicity (CD107a expression) by CD8+ T cells and NK cells expressing or not ILT2. We compared the expression of these markers in presence of target cells, expressing or not HLA-G1, and added a blocking antibody to reverse HLA-G inhibition.This new method allows for the discrimination of cell subsets responding and non-responding to HLA-G1 in one tube. We confirm that HLA-G-specifically inhibits the ILT2+ CD8+ T cell and ILT2+ NK cell subsets but not ILT2-negative ones. By blocking HLA-G/ILT2 interaction using an anti-ILT2 antibody we restored the cytotoxicity level, corroborating the specific inhibition of HLA-G1. We believe that our methodology enables to investigate HLA-G immune functions easily and finely towards other immune cell lineages or expressing other receptors, and might be applied in several pathological contexts, such as cancer and transplantation.

Mesenchymal stem cells alter the frequency and cytokine profile of natural killer cells in abortion-prone mice.

Natural killer cells, which play a pivotal role in the establishment and maintenance of normal pregnancy, are the most abundant leukocytes at the fetomaternal interface that their subsets frequencies and cytokine profile are influential factors in the preservation of the decidual tolerogenic microenvironment. Any imbalance in NK cells' frequency and functions could be associated with pregnancy failure. Mesenchymal stem cells (MSCs) are shown to have immunomodulatory effects on NK cells and their cytokine profile. The purpose of this study is to evaluate the impact of MSCs therapy on the cytokine profiles and subpopulations of NK cells in a murine model of recurrent pregnancy loss. Adipose-derived MSCs were injected intraperitoneally to the abortion-prone mice on Day 4.5 of gestation. The abortion rate was determined after MSCs administration and the frequency and cytokine profiles of the different subsets of NK cells were determined using the flow cytometry. Our results showed that, in abortion-prone mice, the frequency of CD49b+ NK cells was significantly higher than normal pregnant mice that decreased after therapy. We also demonstrated that MSCs downregulated the production of IFN-γ and upregulated IL-4 and IL-10 production by uNK cells. These findings indicate that MSCs can decrease the infiltration of CD49b+ NK cells to the fetomaternal interface and modulate the cytokine profile of NK cells from inflammatory to tolerogenic profile and thereby improve the tolerogenic microenvironment at the fetomaternal interface in benefit of pregnancy maintenance.

Treatment effects of fingolimod in multiple sclerosis: Selective changes in peripheral blood lymphocyte subsets.

BackgroundTreatment with fingolimod reduces inflammation in multiple sclerosis (MS) by inhibiting lymphocyte egress from lymph nodes. We aimed to map, in detail, the alterations in peripheral blood lymphocyte subpopulations in relation to clinical outcome in MS patients treated with fingolimod.MethodsPaired blood samples from relapsing-remitting MS patients (n = 19) were collected before and after one year of treatment with fingolimod (0.5 mg/day). Absolute counts and relative proportions of a broad set of T- B- and NK-cell subsets were analyzed by flow cytometry. Blood samples from 18 healthy controls were used for baseline comparisons.ResultsTreatment with fingolimod markedly decreased the absolute numbers of all major lymphocyte subsets, except for NK cells. The reduction was most pronounced within the T helper (Th) and B cell populations (p<0.001). By phenotyping differentiation status of T cells, dramatic reductions within the naïve and central memory (CM) cell populations were found (p<0.001), while a less pronounced reduction was observed among effector memory (EM) cells (p<0.001). The numbers of regulatory T cells (Tregs) were also decreased (p<0.001), but to a lesser extent than other T cell populations, resulting in a relative preservation of Tregs with a memory phenotype (p = 0.002).ConclusionsOur results confirm that fingolimod therapy markedly reduces lymphocyte counts in peripheral blood of MS patients. Subgroup analysis of T cells showed that naïve and CM Th cells were the most profoundly affected and that memory Tregs were relatively preserved.

NKG2C+NKG2A-CD56dim Subset of NK Cells Show Increased Anti-Leukemia Potential in Presence of CTLA4Ig in-Vitro and Is the Key Determinant of Early Relapse and Long-Term Disease-Free Survival without Gvhd Following CTLA4Ig-DLI Based Haploidentical HCT

<h3>Background & Methods</h3> CTLA4Ig-primed donor lymphocyte infusions (CTLA4Ig-DLI) following haploidentical HCT were earlier shown to be associated with early proliferation of CD56dim NK cells with lower incidence of disease relapse. We prospectively studied the reconstitution of NKG2C+ subset of NK cells in 60 patients with advanced leukemia (myeloid-28/lymphoid-32), along with functional assays. Conditioning was Flu-Bu-Mel followed by CTLA4Ig-DLI on days +7, +21 and +35. GVHD prophylaxis was PTCy followed by cyclosporine for 60 days [BBMT,2019]. All donors and recipients were CMV seropositive. <h3>Results</h3> All patients engrafted with incidences of grade 2-4 acute and chronic GVHD being 12% and 19.8% respectively. NRM was 5% with an overall survival (OS) of 86.5% at a median of 26 months. Disease progression (DP) was 17.6% between days+28 to +144 (median 90 days). This tended to be lower in patients with myeloid leukemia (7.7% vs 26.3% in lymphoid; p=0.06) and NK-KIR B haplotype (10.9% vs 36.6%, p=0.03). Higher CD56dim NK cells at days+30 (135 vs 17 cells/µl), +60 (150 vs 19 cells/µl) and +90 (121 vs 33 cells/µl) (p< 0.01) without any difference in T cell subsets correlated with lack of DP. NKG2C+/NKG2A- subset of CD56dim NK cells were significantly higher at days +30 (5.3% vs 1.0%), +60 (18.5% vs 1.4%) and + 90 (30.6% vs 2.1%) in those without DP (p <0.0001). The surge of NKG2C+ NK cell subset was independent of CMV reactivation and was sustained at a mean value of 27.2%, 32.8%, 34.5% and 28.1% at 6 months, 1, 2 at 3 years, with no DP in survivors beyond 6 months(n = 50). Functionally, NKG2C+ NK cells showed a higher production of IFN-gamma in patients without DP at 30 to 90 days. In normal donors, IFN-gamma production was increased several folds on incubation with CTLA4Ig for 24 hours, supporting the anti-leukemia potential of CTLA4Ig-DLI. On multivariate analysis, NKG2C+/ NKG2A- NK cells had the strongest impact on DP (OR-0.3[0.1-0.6]). Likewise, OS strongly correlated with NKG2C+NK cells (p <0.001). Low levels of NKG2C+NK cells at day+60 and +180 correlated with increased acute and chronic GVHD respectively. They reverted to higher and sustained levels after resolution of chronic GVHD. <h3>Conclusions</h3> In patients with advanced leukemia receiving grafts from CMV seropositive donors, rapid surge of functional NKG2C+NK cell subsets, irrespective of CMV reactivation post-HCT, was observed following CTLA4Ig-DLI. CTLA4Ig-DLI probably promotes an early and sustained NKG2C+NK cell surge with functional anti-leukemia potential demonstrated both in-vitro and in-vivo, an absence of which both numerically and functionally, strongly correlated with early DP. Importantly, NKG2C+NK cells remained persistently elevated, even beyond two years post-transplantation and was the predominant factor associated with disease-free survival without GVHD, on long-term follow-up.

A novel population of memory-activated natural killer cells associated with low parasitaemia in Plasmodium falciparum-exposed sickle-cell trait children.

ObjectivesThe sickle‐cell trait phenotype is associated with protection from malaria. Multiple molecular mechanisms have been proposed to explain this protection, but the role of the host immune system has been poorly investigated. We hypothesised that cellular immunity to malaria may develop differently in sickle‐cell trait children (HbAS) and children with normal haemoglobin (HbAA) repeatedly exposed to Plasmodium falciparum (Pf).MethodsPaired samples collected prior to the Pf transmission season and during the first malaria episode of the ensuing transmission season from HbAS and HbAA children were analysed by multiplex bead‐based assay and comprehensive multi‐dimensional flow cytometry profiling.ResultsCellular immune profiles were enriched in HbAS relative to HbAA children before the start of the Pf transmission season, with a distinct NK subset. These cells were identified as a novel subset of memory‐activated NK cells characterised by reduced expression of the ecto‐enzyme CD38 as well as co‐expression of high levels of HLA‐DR and CD45RO. The frequency of this NK subset before the transmission season was negatively correlated with parasite density quantified during the first malaria episode of the ensuing transmission season. Functional assessment revealed that these CD38dim CD45RO+ HLA‐DR+ NK cells represent a important source of IFN‐γ.ConclusionOur data suggest that this novel memory‐activated NK cell subset may contribute to an accelerated and enhanced IFN‐γ‐mediated immune response and to control of parasite density in individuals with the sickle‐cell trait. This distinct cellular immune profile may contribute to predispose HbAS children to a relative protection from malaria.

Systemic antitumor effect by regional hyperthermia combined with low-dose chemotherapy and immunologic correlates in an adolescent patient with rhabdomyosarcoma – a case report

Introduction An abscopal effect is a clinical observation whereby a local treatment is associated with regression of metastatic cancer at a site distant from the primary location of treatment. Here, we describe the clinical systemic effect induced by regional hyperthermia combined with low-dose chemotherapy and provide immunologic correlates. Case presentation A 15-year-old patient had been diagnosed with alveolar rhabdomyosarcoma (ARMS). All previous treatment options failed in the patient including haploidentical stem cell transplantation and donor lymphocyte infusion. The patient presented with local and metastatic disease, and upon admission, underwent regional hyperthermia combined with low-dose chemotherapy. Immediately following therapy severe skin reactions were observed. Skin biopsies revealed an intraepithelial lymphocytic infiltration dominated by CD3+/CD8+ T cells with a regular network of dendritic cells. Clinical images compared before and during sequential treatment cycles showed complete metabolic response of the local tumor for more than 10 months of therapy. In addition, metastases completely regressed although they were not direct targets of regional hyperthermia. The systemic effect was associated with enhanced frequency of NK cells and T cells expressing the lectin-like natural-killer group 2 D activating receptor (NKG2D), an increase of the CD56bright subset of NK cells, as well as an increase of effector/memory and effector CD8+ and CD4+ T cells in the blood while the percentage of CD25+FOXP3+ regulatory T cells declined. Conclusions Regional hyperthermia combined with low-dose chemotherapy had the potential to create a systemic effect which was associated with activation of NK cells and T cells.

Effect of manipulative reduction combined with air enema on intestinal mucosal immune function in children with intussusception.

Objective:To explore the effect of manipulative reduction combined with air enema on intestinal mucosal immune function in children with intussusception.Methods:This is a prospective randomized controlled study in which 60 children with primary intussusception admitted to Hebei Children’s Hospital from October 2018 to October 2019 were selected for this study. They were randomly divided into two groups. The 30 patients in the experimental group underwent manipulative reduction and air enema reduction, and 30 patients in the control group underwent only air enema reduction. Pain scores and pressure during enema were recorded and analyzed. Fasting blood of children in the experimental group were drawn to test the serum T lymphocyte subsets CD3+, CD4+, CD8+ levels, B lymphocyte subsets CD19+ level, and NK cell subsets CD56+ levels before reduction. Among them, fasting blood of 28 children with successful reduction were drawn again in the morning after reduction, and the indicators of each immune cell subgroup before and after reduction were analyzed. Two children with unsuccessful reduction were no longer tested for these indicators.Results:Twenty-Eight children in the experimental group had successful reduction, and two children with unsuccessful reduction were changed to open surgery (28/30). Twenty five Children in the control group had successful reduction, and five were changed to open surgery (25/30). There was no significant difference in the success rate of reduction between two groups (p>0.05). Close observation for 12~24h after reduction found that none of the children had signs of peritonitis. The pain score and reduction pressure of the observation group were lower than those of the control group, and the difference was statistically significant (p<0.05). The levels of serum CD3+, CD4+, and CD8+ after reduction in the experimental group were significantly higher than before reduction, and the difference was statistically significant (p<0.05). CD19+ level was significantly lower than before reduction, and the difference was statistically significant (p<0.05). There was no significant difference in changes of other indicators.Conclusions:Manipulative reduction combined with air enema reduction can relieve pain and air injection pressure during enema, reduce reperfusion injury caused by intestinal ischemia, and protect intestinal mucosal immune function, which is a favored treatment.

Visualization and Quantification of High-Dimensional Cytometry Data using Cytofast and the Upstream Clustering Methods FlowSOM and Cytosplore.

The complexity of data generated by mass cytometry has necessitated new tools to rapidly visualize analytic outcomes. Clustering methods like Cytosplore or FlowSOM are used for the visualization and identification of cell clusters. For downstream analysis, a newly developed R package, Cytofast, can generate a rapid visualization of results from clustering methods. Cytofast takes into account the phenotypic characterization of cell clusters, calculates the cell cluster abundance, then quantitatively compares groups. This protocol explains the applications of Cytofast to the use of mass cytometry data based on modulation of the immune system in the tumor microenvironment (i.e., the natural killer [NK] cell response) upon tumor challenge followed by immunotherapy (PD-L1 blockade). Demonstration of the usefulness of Cytofast with FlowSOM and Cytosplore is shown. Cytofast rapidly generates visual representations of group-related immune cell clusters and correlations with immune system composition. Differences are observed in the clustering analysis, but separation between groups are visible with both clustering methods. Cytofast visually shows the patterns induced by PD-L1 treatment that include a higher abundance of activated NK cell subsets, expressing a higher intensity of activation markers (i.e., CD54 or CD11c).

Efficacy of third-party chimeric antigen receptor modified peripheral blood natural killer cells for adoptive cell therapy of B-cell precursor acute lymphoblastic leukemia.

We developed an innovative and efficient, feeder-free culture method to genetically modify and expand peripheral blood-derived NK cells with high proliferative capacity, while preserving the responsiveness of their native activating receptors. Activated peripheral blood NK cells were efficiently transduced by a retroviral vector, carrying a second-generation CAR targeting CD19. CAR expression was demonstrated across the different NK-cell subsets. CAR.CD19-NK cells display higher antileukemic activity toward CD19+ cell lines and primary blasts obtained from patients with B-cell precursor ALL compared with unmodified NK cells. In vivo animal model data showed that the antileukemia activity of CAR.CD19-NK cell is superimposable to that of CAR-T cells, with a lower xenograft toxicity profile. These data support the feasibility of generating feeder-free expanded, genetically modified peripheral blood NK cells for effective "off-the-shelf" immuno-gene-therapy, while their innate alloreactivity can be safely harnessed to potentiate allogeneic cell therapy.