CD56 Dim CD16 Research Articles

Cross Talk between the Immune Compartment and the Tumor Cells in Myelodysplastic Syndromes (MDS)

Background: Abnormalities of the immune system and innate immune signaling have been described in patients (pts) with MDS, however the interaction between the MDS tumor cells and the immune compartment is poorly defined. Immune modulatory therapies are being explored in MDS and a better understanding of the interaction and potential cross-talk with immune components and MDS tumor cells is critical in developing new therapeutic strategies. Methods: We developed a multiscale immune profiling strategy to map the immune environment of treatment naïve pts with lower- and higher-risk MDS, using mass cytometry by time of-flight (CyTOF) combined with Olink proteomics analyses. We obtained bone marrow (BM) and peripheral blood (PB) from 33 pts with MDS, (17 higher-risk; 16 lower-) and 10 healthy donors. Pts were representative of the MDS distribution across age, gender, mutational status and histologic subtype. PB and BM were freshly processed and stained with an antibody panel for myeloid cells. For the lymphoid panel, all the pts' samples were stained simultaneously to avoid batch effect. We processed the BM and the PB plasma for Olink. Results: We profiled myeloid cells, NK cells and T cells in pts with MDS to determine differences in their constituents. Phenograph clustering across all pts revealed distinct T lymphoid metaclusters, that corresponded to known immune cell populations. We observed a robust immune response in the BM from MDS patients. T cells and myeloid cells were found in equal proportion in the BM from MDS and healthy patients. However, the B cell population were present at significantly lower frequency in the MDS BM (p<0.05), whereas gamma delta (gd)T cells and NK cells were at significantly higher frequency in the MDS pts (p <0.05). Paired mass cytometric analysis revealed a distinct composition and phenotype of T cell subsets. Among the T cell compartment, regulatory T cells (Treg) (p<0.001), cytolytic CD8 T cells (expressing perforin (p<0.01) or granzyme (p<0.001)) and exhausted CD8 T cells (expressing PD-1 (p= 0.09), LAG-3 (p<0.001) or TIM-3) seemed to accumulate in the MDS BM. Compared to healthy BM Treg in MDS BM expressed higher levels of TIGIT, TIM3, CD27, CD25, CD28 and OX40. Cytolytic NK cells accumulate in MDS BM (p<0.05). In addition to measuring adaptive lymphocyte responses to tumors, we also analyzed the distribution of innate lymphocytes and in particular NK cells in MDS BM. In the NK cell population, the cytotoxic NK cells (CD56dim CD16+) were found in a higher proportion (p<0.05) than the cytokinic counterpart (CD56bright) in the MDS BM. NK cells that accumulate in the MDS BM expressed higher level of CD45RA, CD38, granzyme B, CD56 suggesting their superior cytotoxic potential at a critical developmental stage of retaining enhanced sensitivity to pro-inflammatory cytokines derived from myeloid cells. We also analyzed a panel of 92 unique cytokines, chemokines and inflammatory markers produced in the tumor milieu using OLink Proteomics proximity extension assay. Many chemokines and cytokines were differently expressed in the MDS BM compared to the healthy BM, suggesting an important inflammatory reaction. Conclusion: These data demonstrate new findings suggesting interactions and cross-talk between Tregs, NK cells, CD8 T cells and gdT cells in the BM of pts with untreated MDS. The data also suggest that there is a strong immune response in the MDS BM, particularly an intense cytotoxic response with the accumulation of cytotoxic CD8 T cells, NK cells and gdT cells. These results are are contrary to those observed in most solid tumors. This cytotoxic response seemed to be counteracted by the presence of Treg. Each of the subgroups of T cells (as well as NK cells) have been implicated as possibly promoting immune tolerance and tumor growth in different model systems and other tumor types. NK cells and gdT cells are known to respond to virus-infected and transformed cells rapidly and without the need for prior sensitization. However, anecdotal evidence from numerous independent studies may suggest that a strong but prolonged NK cell- and gdT cell-mediated antiviral response may result in dysregulation and promote tumorigenesis and/or aggravate pathology. Additional studies are underway to further characterize the immune landscape in those untreated and treated and to study the interaction of the immune components with tumor cells. Disclosures Navada: Onconova Therapeutics Inc: Research Funding. Silverman:Onconova Therapeutics Inc: Patents & Royalties, Research Funding; Celgene: Research Funding; Medimmune: Research Funding.

Mass Cytometry Reveals a Sustained Reduction in CD16+ Natural Killer Cells Following Chemotherapy in Colorectal Cancer Patients.

The immune system and inflammation plays a significant role in tumour immune evasion enhancing disease progression and reducing survival in colorectal cancer (CRC). Patients with advanced stages of colorectal cancer will all undergo treatment with cytotoxic chemotherapy which may alter the complexity of immune cell populations. This study used mass cytometry to investigate the circulating immune cell profile of advanced CRC patients following acute and chronic doses of standard cytotoxic chemotherapy and analysed seven major immune cell populations and over 20 subpopulations. Unsupervised clustering analysis of the mass cytometry data revealed a decrease in NK cells following one cycle of cytotoxic chemotherapy. Investigation into the NK sub-population revealed a decline in the CD56dim CD16+ NK cell population following acute and chronic chemotherapy treatment. Further analysis into the frequency of the NK cell sub-populations during the long-term chemotherapy treatment revealed a shift in the sub-populations, with a decrease in the mature, cytotoxic CD56dim CD16+ accompanied by a significant increase in the less mature CD56dim CD16− and CD56bright NK cell populations. Furthermore, analysis of the phosphorylation status of signalling responses in the NK cells found significant differences in pERK, pP38, pSTAT3, and pSTAT5 between the patients and healthy volunteers and remained unchanged throughout the chemotherapy. Results from this study reveals that there is a sustained decrease in the mature CD16+ NK cell sub-population frequency following long-term chemotherapy which may have clinical implications in therapeutic decision making.

CTLA4Ig-based reduced intensity conditioning and donor lymphocyte infusions for haploidentical transplantation in refractory aggressive B-cell lymphoma relapsing after an autograft: Early results from a pilot study

CTLA4Ig-primed donor lymphocyte infusions (DLIs) have been found to promote natural killer (NK) cell-mediated anti-leukemia effect following haploidentical hematopoietic cell transplantation (HCT). Incorporation of CTLA4Ig in conditioning aided long-term remission in myeloma probably by blocking the CD28-CD86 pro-survival pathway when combined with CTLA4Ig-primed DLI. We explored a similar approach in 12 patients (8-65 years) who had refractory aggressive B-cell lymphoma (R-ABCL) following autologous HCT. They received CTLA4Ig-based reduced-intensity conditioning and sequential CTLA4Ig-primed DLIs on days +7, +21, and +35. None developed acute graft-versus-host disease (GVHD). Two patients developed chronic GVHD. Only 3 patients had disease-progression at 100 days posttransplant with a progression-free and GVHD-free survival at 2 years of 75%. A higher expression of CD80 in tumor cells and a greater proliferation of CD56dim CD16+ NK cells were observed at days +30 and +60 in patients with progression-free survival. We hypothesize that CTLA4Ig, with a greater avidity for CD80, probably interferes with the anti-apoptotic effect mediated through this pathway, and together with early proliferation of mature NK cell when used in conjunction with DLI, this approach might provide a curative option for patients with R-ABCL.

A longitudinal study of the stability, variability, and interdependencies among late-differentiated T and NK cell subsets in older adults.

The stability and variability of older adults' late-differentiated peripheral blood T and natural killer (NK) cells over time remains incompletely quantified or understood. We examined the variability and change over time in T and NK cell subsets in a longitudinal sample of older adults; the effects of sex, cytomegalovirus (CMV) serostatus, and chronic disease severity on immune levels and trajectories; and interdependencies among T and NK cell subsets. Older adults (N = 149, age 64-94 years, 42% male) provided blood every 6 months for 2.5 years (up to 5 waves) to evaluate late-differentiated CD8 T cells (CD28-, CD57+) and CD56dimNK cells (CD57+, NKG2C+, FcɛRIγ-). In multilevel models, most of the variance in immune subsets reflected stable differences between people. However, CD56dimNK cell subsets (CD57+ and FcɛRIγ-) also increased with age, whereas T cell subsets did not. Independent of age, all subsets examined were higher in CMV-positive older adults. Men had higher levels of CD56dim CD57+ than women. Chronic disease was not associated with any immune subset investigated. T and NK cell subsets correlated within each cell type, but interdependencies differed by CMV serostatus. Our results suggest the accumulation of these stable cell populations may be driven less by chronological aging, even less by chronic disease severity, and more by CMV, which may differentially skew T and NK cell differentiation.

Abstract B153: Human NK cell distribution memory and residence in tissue sites

Abstract Natural killer (NK) cells are innate immune cells with the ability to kill tumor cells without prior exposure. NK cells express multiple activating and inhibitory receptors in addition to the low-affinity immunoglobulin G binding receptor CD16. Accumulating evidence implicates a role of NK cells in not only direct killing of tumor cells, but also in cancer immunosurveillance and preventing metastasis to tissues sites. However, at present the distribution, diversity and tissue driven differences in NK cell function are not well characterized which may have an implication on the anti-cancer potential of tissue NK cells. Through collaboration with LiveOnNY our local organ procurement organization, we receive blood, bone marrow (BM), lung, intestines, tonsil and associated lymph nodes (LN) from research consented organ donors. Here we used this unique tissue resource to investigate the distribution, phenotypic, functional and transcriptional diversity of NK cell subsets in human tissues. We found that NK cells are ubiquitously distributed in human tissues comprising up to 40% of the CD45+ CD14/19- cells in blood, BM, spleen and lung, while only a small fraction (up to 2%) in the intestine and the LN. While blood, BM, spleen and lung are dominated by mature NK cells (CD56dim CD16+), majority of the NK cells in intestine and LN are immature (CD56hi CD16-). Age, sex and CMV sero-status do not show any correlation with NK cell distribution or subset frequency in tissues. Notably, NK cell subset distribution seems to drive functional differences between tissue NK cells, with lymphoid site NK cells expressing lower levels of effector molecules granzyme B, TNFα, Prf, Ifnγ and displaying reduced degranulation compared with their counterparts from blood, BM and spleen. For an in-depth analysis of tissue-mediated effects on NK cell subset functionality, we performed whole-transcriptome profiling on immature and mature NK cells isolated from blood, BM, spleen, lung and LN. Our analysis identified several effector molecules and NK cell surface receptors being differentially expressed between immature and mature NK cells. Furthermore, while mature NK cells of blood and tissues have similar transcriptional profiles, the transcriptional profiles of immature blood and tissue NK cells show tissue driven heterogeneity with differential expression of transcription factors, metabolic enzymes and NK cell surface receptors. Interestingly, the transcriptional signature of immature NK cells is reminiscent of the transcriptional signature of tissue resident memory T-cells showing increased expression of CD103, CD49a, CXCR6. We validated the expression of these markers using multiparameter flow cytometry and found that subset of immature NK cells in mucosal sites (lung and intestine) do indeed express markers of tissue residence. Additionally, by applying trajectory projection algorithm on NK cells from tissue sites, we show that resident NK cells comprise a distinct population from immature NK cells. Our study has identified novel, previously unidentified diversity of tissue NK cells. Phenotypic and transcriptional profiling data provide evidence for putative resident NK cells being present in certain tissue sites. Blood NK cells differ from their counterparts in tissues; especially, immature NK cells in tissues may be specifically trained to function in the tissue environment. Finally, due to the uniqueness of phenotypic, functional and transcriptional features of tissue NK cells, they may be more suited to fight cancer in situ in tissues. Citation Format: Pranay Dogra, Takashi Senda, Peter Szabo, Dustin Carpenter, Marta Toth, Puspa Thapa, Mark Snyder, Michelle Miron, Brahma Kumar, Donna L. Farber. Human NK cell distribution memory and residence in tissue sites [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B153.

CD56dim CD16- Natural Killer Cell Profiling in Melanoma Patients Receiving a Cancer Vaccine and Interferon-α.

Natural killer (NK) cells are innate cytotoxic and immunoregulatory lymphocytes that have a central role in anti-tumor immunity and play a critical role in mediating cellular immunity in advanced cancer immunotherapies, such as dendritic cell (DC) vaccines. Our group recently tested a novel recombinant adenovirus-transduced autologous DC-based vaccine that simultaneously induces T cell responses against three melanoma-associated antigens for advanced melanoma patients. Here, we examine the impact of this vaccine as well as the subsequent systemic delivery of high-dose interferon-α2b (HDI) on the circulatory NK cell profile in melanoma patients. At baseline, patient NK cells, particularly those isolated from high-risk patients with no measurable disease, showed altered distribution of CD56dim CD16+ and CD56dim CD16− NK cell subsets, as well as elevated serum levels of immune suppressive MICA, TN5E/CD73 and tactile/CD96, and perforin. Surprisingly, patient NK cells displayed a higher level of activation than those from healthy donors as measured by elevated CD69, NKp44 and CCR7 levels, and enhanced K562 killing. Elevated cytolytic ability strongly correlated with increased representation of CD56dim CD16+ NK cells and amplified CD69 expression on CD56dim CD16+ NK cells. While intradermal DC immunizations did not significantly impact circulatory NK cell activation and distribution profiles, subsequent HDI injections enhanced CD56bright CD16− NK cell numbers when compared to patients that did not receive HDI. Phenotypic analysis of tumor-infiltrating NK cells showed that CD56dim CD16− NK cells are the dominant subset in melanoma tumors. NanoString transcriptomic analysis of melanomas resected at baseline indicated that there was a trend of increased CD56dim NK cell gene signature expression in patients with better clinical response. These data indicate that melanoma patient blood NK cells display elevated activation levels, that intra-dermal DC immunizations did not effectively promote systemic NK cell responses, that systemic HDI administration can modulate NK cell subset distributions and suggest that CD56dim CD16− NK cells are a unique non-cytolytic subset in melanoma patients that may associate with better patient outcome.

Cytotoxic Natural Killer Subpopulations as a Prognostic Factor of Malignant Pleural Effusion.

Malignant pleural effusion (MPE) is a sign of advanced disease of poor prognosis. As natural killer (NK) cells are involved in the first line of tumour defence, we aimed to validate a new diagnostic and prognostic indicator for MPE based on NK subpopulations of pleural fluid (PF) and peripheral blood (PB). NK subpopulations were determined in PF and PB in 71 patients with malignant, paramalignant or benign pleural effusion. The receiver operating characteristic(ROC) curves, Kaplan-Meier, multivariable Cox model and decision trees created with theCHAID (Chi-square automatic interaction detector) methodology were employed. We demonstrated that the PF/PB ratios of the CD56 bright CD16- and CD56 dim CD16- NK subpopulations were higher (p = 0.013 and p = 0.003, respectively) in MPEs and paramalignant pleural effusions(PPEs) than in benign ones, with an AUC of 0.757 and 0.741, respectively. The PF/PB ratio of CD16+ NK and CD57+ NK obtained a higher hazard ratio (HR) in the crude Cox's regression analysis. In the adjusted Cox's regression analysis, the PF/PB ratio of CD16+ NK gave the highest HR (HR 6.1 [1.76-21.1]) (p = 0.004). In the decision tree created for the MPE prognosis, we observed that the main predictor variable among the studied clinical, radiological, and analytical variables was lung mass, and that 92.9% of the patients who survived had a PF/PB ratio of the CD56 dim CD16+ NK subpopulation ≤ 0.43. Our data suggest that both the PF/PB ratios of cytotoxic subpopulations CD57+ NK and CD16+ NK are useful as a prognostic factor of MPE. Other subpopulations (CD56 bright CD16- and CD56 dim CD16- NK) could help to diagnose MPE.

Low-Dose Interleukin-2 Therapy Enhances Cytotoxicity of CD56bright NK Cells in Patients with Chronic Gvhd

Introduction: Natural killer (NK) cells play an important role in defense against infections and cancer. Two major subsets of mature NK cells have been described. CD56bright CD16- NK cells, which normally represent only 5-10% of circulating NK cells, are thought to exhibit less cytolytic activity and greater immune regulatory functions than CD56dim CD16+ NK cells. However, recent studies have reported that cytolytic activity of CD56bright NK cells increases when these cells are stimulated with IL-15or the combination of IL-12, IL-15 and IL-18. Previous studies from our center have shown that daily administration of low-dose IL-2 in patients with chronic graft-versus-host disease (cGVHD) induces selective expansion of CD4+FoxP3+Helios+ regulatory T cells and CD56bright NK cells and improves clinical manifestations of cGVHD. The function of CD56bright NK cells expanded by low-dose IL-2 has not previously been studied.Methods: Single cell mass cytometry (CyTOF) with a panel of 35 metal tagged antibodies was performed on cryopreserved peripheral blood mononuclear cells (PBMC) from 10 adult patients with active cGVHD receiving daily low-dose IL-2 therapy. Patients in this clinical trial received extracorporeal photopheresis (ECP) for 8 weeks prior to starting daily low dose IL-2. ECP therapy (twice weekly) was continued when patients began low-dose IL-2 (1x106 IU/M2/day x 8 weeks). The analytic panel included 26 cell surface markers to identify distinct lymphocyte subsets and 9 intracellular markers to measure functional status and activation of specific signaling pathways. viSNE was used to visualize of high-dimensional data on a two-dimensional map and quantify single cell mass cytometry data. NK cytolytic activity was measured in flow cytometry-based cytotoxicity assays. CD56bright and CD56dim NK cells from 8 adult patients were purified from cryopreserved PBMC by cell sorting and incubated with labeled K562 cells for 4 hours followed by staining with 7-AAD and Annexin-V. E:T ratio of 1:1 was used for incubation with K562 targets.Results: No changes in extracellular or intracellular NK cell markers or quantitative changes in NK cells were observed during the initial 8 week ECP treatment period. Selective expansion of CD56bright NK cells was noted after 1 week of IL-2 therapy (9W) and continued during 8 weeks of daily IL-2 therapy. Increased expression of NKp30, Nkp46, NKG2D, HLA-DR and Ki67 occurred in expanded CD56bright NK cells with peak expression at 1 week after starting IL-2 (9W). At later time points during IL-2 therapy, expression of NKG2D, HLA-DR and Ki67 returned to baseline (Figure 1A). Expression of CD56, CD122 and NKG2A continued to increase during IL-2 treatment. In contrast, expression of CD25 by expanded CD56bright NK cells decreased during IL-2 treatment. Expression of phosphorylated signaling proteins did not change in any NK cell subset during IL-2 treatment. Cytolytic activity was measured in CD56bright and CD56dim NK cell subsets at different times during ECP and IL-2 therapy. After thawing, flow-sorted NK cell subsets were cultured for 16-20 hours with IL-2 (100 IU/ml). Cells were then washed and incubated with tumor targets (K562) for 4 hours and % killing was assessed by flow cytometry. Compared to pre IL-2 treatment, cytolytic activity of CD56bright NK cells increased during IL-2 treatment while cytotoxicity of CD56dim NK cells did not change. Notably, cytotoxicity of CD56bright NK cells became significantly higher than CD56dim NK cells during IL-2 therapy (Figure 1B).Conclusion: Single cell mass cytometry revealed that daily low dose IL-2 therapy induces selective expansion, activation and increased expression of activating NK receptors in CD56bright NK cells. CD56dim NK cells were not affected by IL-2 therapy. In vitro assays revealed that cytolytic activity of CD56bright NK cells increased during IL-2 treatment and exceeded the cytotoxicity of CD56dim NK cells. CD56bright NK cells, traditionally considered to be minimally tumor-responsive, are effectively stimulated by daily low dose IL-2 exposure to enable potent cytotoxicity in response to tumor targets. In patients receiving low-dose IL-2 after allogeneic HSCT, expanded CD56bright NK cells may contribute to graft versus leukemia (GVL) and help prevent relapse after transplant. DisclosuresNikiforow:Kite Pharma: Consultancy. Ho:Jazz Pharmaceuticals: Consultancy. Antin:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Soiffer:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Circulating NKp46+ Natural Killer cells have a potential regulatory property and predict distinct survival in Non-Small Cell Lung Cancer

ABSTRACTNatural killer (NK) cells are innate effector lymphocytes widely involved in cancer immunosurveillance. In this study, we described three circulating NK cell subsets in patients with non-small cell lung cancer (NSCLC). Compared to healthy donors (HD), lower rate of the cytotoxic CD56dim CD16+ NK cells was found in NSCLC patients (76.1% vs 82.4%, P = 0.0041). In contrast, the rate of CD56bright NK cells was similar between patients and HD. We showed in NSCLC patients a higher rate of a NK cell subset with CD56dim CD16− phenotype (16.7% vs 9.9% P = 0.0001). The degranulation property and cytokines production were mainly drive by CD56dim CD16− NK cell subset in patients. Analysis of natural cytotoxicity receptors (NCRs) expression identified four distinct clusters of patients with distinct NK cell subset profiles as compared to one major cluster in HD. Notably the cluster characterized by a low circulating level of NKp46+ NK cell subsets was absent in HD. We showed that the rate of circulating NKp46+ CD56dim CD16+ NK cells influenced the patients’ survival. Indeed, the median overall survival in patients exhibiting high versus low level of this NK cell subset was 16 and 27 months respectively (P = 0.02). Finally, we demonstrated that blocking NKp46 receptor in vitro was able to restore spontaneous tumor specific T cell responses in NSCLC patients. In conclusion, this study showed a distinct distribution and phenotype of circulating NK cell subsets in NSCLC. It also supports the regulatory role of NKp46+ NK cell subset in NSCLC patients.

1356P - The level of circulating NKp46+ CD56dim CD16+ natural killer cells predicts distinct survival in non-small cell lung cancer

1356P - The level of circulating NKp46+ CD56dim CD16+ natural killer cells predicts distinct survival in non-small cell lung cancer

Early Everolimus Initiation Fails to Counteract the Cytotoxic Response Mediated by CD8+ T and NK Cells in Heart Transplant Patients.

The positive long-term effects of conversion to everolimus (EVL) after heart transplantation (HT) have been evaluated in several studies. However, the timing of EVL initiation, the best way to combine it with other immunosuppressive treatments, and the impact of these combinations on the immune response are poorly understood aspects. Here, we analyzed the immune phenotype and function of HT patients (n = 56) at short and long terms (prospective and retrospective cohorts), taking into account the time of EVL initiation: early (3 months post-transplant, EVL-E group) or late (>1 year post-transplant, EVL-L group) compared with mycophenolate mofetil treatment (MMF group). We show that early EVL conversion from MMF allows the increase of cytotoxic (CD56dim CD16+) NK and effector-memory (EM, CD45RA− CCR7−) CD8+ T cell subsets, which show a significantly higher level of expression of cytotoxic molecules, IFN-γ production and degranulation ability under activation. NK cell expansion is accompanied by an altered balance of receptor expression, increasing the activation state, and lytic activity of those cells. Those changes are detected after as little as 1 month after EVL conversion in association with the expansion of regulatory T cells and the decrease in B cell frequency. However, no changes in the immune cells subsets were observed after late EVL initiation (EVL-L) compared with the MMF group. Our results imply that only early EVL conversion induces key changes in the post-transplant immune response, preserving an efficient anti-viral response, but simultaneously showing a limited ability to counteract the cytotoxic response to the allograft.

Everolimus Failure to Counteract the Cytotoxic Response Mediated by CD8+ T and NK Cells in Heart-Transplant Patients

The negative effects of immunosuppression after heart transplantation (HT) are hard to avoid, but they can be partially overcome using new drugs such as the everolimus (EVL) mTOR (mammalian target of rapamycin) inhibitor. Initial studies have shown that EVL administration in combination with low-doses of calcineurin inhibitors and corticosteroids is effective in reducing the acute rejection and cytomegalovirus infection, two of the main causes of the development of cardiac allograft vasculopathy (CAV), and induce the expansion of regulatory T cells (Tregs). However, the impact of EVL introduction after HT on the in vivo immune response remains poorly understood. Here, we analyzed the changes in the immune phenotype on peripheral blood samples of 56 HT recipients who were treated with mycophenolate mofetil (MMF) or switched from MMF to EVL, and low-dose tacrolimus. We studied two cohorts; a “late cohort” of recipients of more than 1 year post-HT, who were converted from MMF to EVL (EVL group, n=20) following clinical criteria or maintained on MMF (MMF group, n=20) and an “early cohort” (n=16), who were switched from MMF to EVL at 3 months post-HT. Blood samples were taken at a single time one year post-EVL initiation and at different post-transplant times (before EVL initiation 1, 3 and 9 months after EVL conversion) in the early cohort. Immune phenotype was analyzed by flow cytometry and DNA methylation profiles in immune genes by pyrosequencing. Our results showed that EVL conversion from MMF induced the expansion of cytotoxic NK (CD56dim CD16+) and effector-memory (CD45RA- CCR7-) CD8+ T populations, showing significantly higher levels of expression of cytotoxic molecules (granzyme, perforin and IFN-γ) and greater degranulation ability under activation. Treatment with EVL modifies the NK cell receptors repertoire increasing the activation state and cytolytic activity of these cells. These changes are already evident at 1 month after EVL initiation and gradually increase during the first year post-transplant. Moreover, only one month after EVL initiation, HT patients showed a significant expansion of Tregs and decrease of B cells. We observed that these phenotypic alterations were associated with changes in the DNA methylation patterns of key immune genes (IFNG, PRF, FASL and PD1). Thus, quantification of the methylation levels in these genes could help to predict the alterations in the immune phenotype and its functionality after EVL conversion. In short, we observed that both early and late EVL conversion from MMF induces changes in the immune system which could condition the post-transplant immune response. The consequences of EVL initiation after HT can be considered as a double-sided coin; on the one hand induces the Tregs expansion and reduces the B cells number contributing to the immune tolerance, but at the same time failure to blockage the cytolytic ability mediated by CD8+ T and NK cells. According to that, EVL conversion as a general strategy after HT must be taken with caution.

Reduced frequency of natural killer cell on siglec-7(+) is associated with progression of hepatitis B virus-related cirrhosis

Objective: To explore the relationship between sialic-acid-binding immunoglobulin-like lectin 7 (Siglec-7) expressed on NK cells and hepatitis B virus-related cirrhosis. Methods: Peripheral venous blood samples were collected from 23 healthy controls and 31 patients with hepatitis B virus-related cirrhosis (Child-Pugh A, n = 7; Child-Pugh B, n = 12; Child-Pugh C, n = 12). Peripheral blood mononuclear cells (PBMCs) were obtained by using Ficoll-Hypaque density gradient centrifugation and the expression of Siglec-7 and NK cells phenotype and their subpopulations were detected by flow cytometry. Comparisons between various groups were performed using t -test, one-way analysis of variance (ANOVA), and correlations between variables were analyzed using Pearson's-correlation coefficient. Results: (1) There was no significant difference in the percentage of NK cells and in their subpopulations with HBV-related cirrhosis and healthy controls. (2) Siglec-7 expression on NK cells in patients with HBV-related cirrhosis(62.44±13.45%)was significantly down-regulated than that to healthy controls(75.39±12.19%)while the frequency of Siglec-7(+) NK cells were negatively correlated with Child-Pugh score. (3) Subpopulation analysis showed that Siglec-7 expression on CD56(bright)CD16(-)NK cells(66.99±15.93%)was significantly lower than CD56(dim)CD16(+)NK cells(76.54±13.9%) in HBV-related cirrhosis. However, the expression of Siglec-7 in healthy controls showed no difference in these two NK cell subsets. (4) Phenotypic analysis showed that Siglec-7(+) NK cells express higher levels of activating receptor CD16, CD38, NKp46 and lower levels of inhibitory receptor CD158b. Indeed, the frequency of CD16 and CD38 on Siglec-7(+) NK cells in HBV-related cirrhosis was lower than that in healthy controls. Conclusion: The disease progression in patients with hepatitis B virus-related cirrhosis is associated to decreased frequencies of Siglec-7(+)NK cells.

Predicting NK cell subsets using gene expression levels in peripheral blood and endometrial biopsy specimens.

In molecular analysis of tissue biopsy specimens, one crucial aspect is characterization of immune cell populations. This is especially important for evaluation of uterine receptivity by assessing levels of lymphocyte populations including CD56bright CD16- uterine NK cells and CD56dim CD16+ conventional NK cells. Our objective was to investigate whether measuring total RNA transcripts from a tissue specimen would accurately reflect immune cell levels and be a new technique to assess immune cell subsets. Peripheral blood mononuclear cells (PBMCs) and endometrial tissues were used. Flow cytometry was utilized for the analysis of lymphocyte subsets in PBMCs, and RT-qPCR was applied to quantify RNA transcripts indicative of lymphocyte and granulocyte populations. In PBMC specimens, there were significant correlations between gene expression levels and cell subsets. NK cells correlated with CD16A, NKp46, and CD56 transcripts, B cells correlated with EBF1, and CD8+ T cells correlated with CD8β. Finally, endometrial tissues displayed high CD56 expression and very low CD3ε, CD16A, and NKp30, reflecting the characteristic endometrial NK cell subsets. Strong correlations between RT-qPCR data and levels of lymphocyte subsets indicate that gene expression analysis will be a useful technique for characterizing levels of CD56+ cells in endometrial tissues.

Transcriptome analysis reveals similarities between human blood CD3\u2212 CD56bright cells and mouse CD127+ innate lymphoid cells

For many years, human peripheral blood natural killer (NK) cells have been divided into functionally distinct CD3− CD56bright CD16− and CD3− CD56dim CD16+ subsets. Recently, several groups of innate lymphoid cells (ILC), distinct from NK cells in development and function, have been defined in mouse. A signature of genes present in mouse ILC except NK cells, defined by Immunological Genome Project studies, is significantly over-represented in human CD56bright cells, by gene set enrichment analysis. Conversely, the signature genes of mouse NK cells are enriched in human CD56dim cells. Correlations are based upon large differences in expression of a few key genes. CD56bright cells show preferential expression of ILC-associated IL7R (CD127), TNFSF10 (TRAIL), KIT (CD117), IL2RA (CD25), CD27, CXCR3, DPP4 (CD26), GPR183, and MHC class II transcripts and proteins. This could indicate an ontological relationship between human CD56bright cells and mouse CD127+ ILC, or conserved networks of transcriptional regulation. In line with the latter hypothesis, among transcription factors known to impact ILC or NK cell development, GATA3, TCF7 (TCF-1), AHR, SOX4, RUNX2, and ZEB1 transcript levels are higher in CD56bright cells, while IKZF3 (AIOLOS), TBX21 (T-bet), NFIL3 (E4BP4), ZEB2, PRDM1 (BLIMP1), and RORA mRNA levels are higher in CD56dim cells.

Research on the negative immune regulation of NK cells in patients with primary immune thrombocytopenia

目的探讨原发免疫性血小板减少症（ITP）患者外周血中NK细胞及其相关细胞因子IFN-γ、IL-10、TGF-β的变化。方法以22例初诊ITP患者（初诊组）、20例治疗后完全缓解ITP患者（完全缓解组）为研究对象，以20名健康志愿者为对照组。采用ELISA法检测三组受试者血清IFN-γ及IL-10水平；用流式细胞术检测NK细胞（CD3−CD56+）及其Bright亚群（CD3−CD56bright CD16−）、Dim亚群（CD3−CD56dim CD16+）水平；采用免疫磁珠法分离NK细胞，实时荧光定量PCR检测IFN-γ、IL-10、TGF-β基因mRNA的表达，并将以上测得结果做相关性分析。结果①初诊组ITP患者血清IFN-γ浓度[（653.0±221.6）ng/L]高于完全缓解组[（484.4±219.5）ng/L]和对照组[（390.9±253.5）ng/L]（P值分别为0.022、0.001），血清IL-10浓度低于对照组[（52.09±26.66）ng/L对（79.44±38.43）ng/L，P=0.007]。②初诊组、完全缓解组患者外周血NK细胞比例[（9.53±3.93）%、（9.03±3.78）%]均低于对照组[（13.72±7.42）%]（P=0.013，P=0.007）；初诊患者外周血Bright亚群占NK细胞的比例高于对照组[（6.85±4.43）%对（4.05±2.81）%，P=0.032]；初诊组外周血Dim亚群占NK细胞的比例低于对照组[（93.14±4.43）%对（95.94±2.81）%，P=0.032]。③初诊组、完全缓解组及对照组NK细胞IFN-γ基因mRNA表达差异无统计学意义（P>0.05），初诊组NK细胞IL-10、TGF-β基因mRNA表达高于对照组（1.82±1.32对1.02±1.03，P=0.023；2.80±2.31对1.46±1.37，P=0.028）。外周血Bright细胞占NK细胞的比例与NK细胞IL-10及TGF-β基因mRNA表达呈正相关（r=0.424，P=0.001；r=0.432，P<0.001）。结论NK细胞可能通过加强分泌免疫负调控因子来代偿其数量的不足，在疾病中起保护作用。

IL-15, TIM-3 and NK cells subsets predict responsiveness to anti-CTLA-4 treatment in melanoma patients

ABSTRACTDespite the success of immune checkpoint blockade in melanoma, the majority of patients do not respond. We hypothesized that the T and NK cell subset frequencies and expression levels of their receptors may predict responses and clinical outcome of anti-CTLA-4 treatment. We thus characterized the NK and T cell phenotype, as well as serum levels of several cytokines in 67 melanoma patients recruited in Italy and Sweden, using samples drawn prior to and during treatment. Survival correlated with low expression of the inhibitory receptor TIM-3 on circulating T and NK cells prior to and during treatment and with the increased frequency of mature circulating NK cells (defined as CD3−CD56dim CD16+) during treatment. Survival also correlated with low levels of IL-15 in the serum. Functional experiments in vitro demonstrated that sustained exposure to IL-15 enhanced the expression of PD-1 and TIM-3 on both T and NK cells, indicating a causative link between high IL-15 levels and enhanced expression of TIM-3 on these cells. Receptor blockade of TIM-3 improved NK cell-mediated elimination of melanoma metastasis cell lines in vitro. These observations may lead to the development of novel biomarkers to predict patient response to checkpoint blockade treatment. They also suggest that induction of additional checkpoints is a possibility that needs to be considered when treating melanoma patients with IL-15.

HETEROGENEITY OF NK AND NKT LYMPHOCYTE POPULATIONS IN HEALTHY DONORS

Natural killer(NK) and NKT lymphocytes are important components of innate immunity, and compose a first-line defense against cancer. These populations are characterized by high heterogeneity and are divided into several subpopulations, by differences in functional activity as well as CD56 and CD16 expression. Studying heterogeneity for these lymphocyte populations in healthy donors is important, due to imbalance between different lymphocyte subsets in cancer patients. Changes in the ratio of these subpopulations may be of prognostic and clinical significance in malignant diseases. The present study was conducted with peripheral blood lymphocytes in 50 healthy donors. When analysing population of NK lymphocytes we have identified 18.0±11.3% of antigen-positive cells, their fluctuations ranged from 7.6 to 29.2%, whereas average number of cells with CD3 - CD56 + and CD3 - CD16 + phenotypes was equal to 16,2±8.1%, and 11,0±6.7%, respectively. The subpopulation analysis showed that the primary pool of NK cells was presented by CD56 dim CD16 dim cells by 52.3±19.9 percent. We detected minor subpopulations, e.g., CD56 dim CD16 bright , CD56 - CD16 + , CD56 bright CD16 - (0.3±0.2%, 1.7±0.9%, and 1.3±0.6%, respectively). Search for intracellular perforin has revealed that the number of CD56 + Perf + cells comprized 25.1±14.8%, CD16 + Perf + , 23.8±16.0%. Cytometric analysis showed that perforin is found, predominantly, in CD56 dim CD16 dim NK lymphocytes, whereas the cells with CD56 dim CD16 bright , CD56-CD16 + , CD56 bright CD16 - immunophenotypes did not produce perforin. For the first time, we have discovered a subpopulation of NK cells with the СD56dimCD16dim immunophenotype that did not contain intracellular perforin (2.0%). The NKT cell population with СD3 + CD16/СD56 + phenotype was detected in 7.1% (25% – 3.45; 75% – 8.75) antigen-positive cells, within a range of 2.5 to 11.9%. Analysis with a combination of monoclonal antibodies CD3/CD56/CD16 has shown that the number of CD3 + CD56 + cells was 4.33% (25% – 2.25; 75% – 7.3), whereas the number of CD3 + CD16 + was 3.087% (25% – 0.9; 75% – 6.2). These data demonstrate that the differences in results between the CD3/CD16/CD56, and CD3/CD16 test systems are statistically significant (p &lt; 0.05). It was shown that the primary-pool NKT-cells are CD56 + CD16 - cells, whose number is about 5.45% (25% – 2.95; 75% – 7.3) among total CD3 + lymphocyte population. Two minor subpopulations were also detected which differed in expression of CD56 and CD16 antigens. Hence, the level of CD56 - CD16 + cells was 3% (25% – 0.25; 75% – 3.05), and the number of CD56 + CD16 + was equal to 0.67% (25% – 0.25; 75% – 0.9). Hence, the observed wide phenotypic diversity of NK and NKT-cells reflects their ability to exert various functional activities. This study, showing high heterogeneity of NK and NKT lymphocytes, may serve as a basis for the study of imbalances between different subpopulations of these cells in cancer patients.

Alteration in Leukocyte Subsets and Expressions of FcγR and Complement Receptors among Female Ragpickers in Eastern India.

BackgroundThere are a million ragpickers in India who gather and trade recyclable municipal solid wastes materials for a living. The objective of this study was to examine whether their occupation adversely affects their immunity.MethodsSeventy-four women ragpickers (median age, 30 years) and 65 age-matched control housemaids were enrolled. Flow cytometry was used to measure leukocyte subsets, and leukocyte expressions of Fcγ receptor I (CD64), FcγRIII (CD16), complement receptor 1 (CD35) and CR3 (CD11b/CD18), and CD14. Serum total immunoglobulin-E was estimated with enzyme-linked immunosorbent assay.ResultsCompared with the controls, ragpickers had significantly (p < 0.0001) higher levels of CD8+T-cytotoxic, CD16+CD56+natural killer, and CD4+CD45RO+memory T-cells, but depleted levels of CD19+B-cells. The percentage of CD4+T-helper-cells was lower than the control group (p < 0.0001), but their absolute number was relatively unchanged (p = 0.42) due to 11% higher lymphocyte counts in ragpickers. In ragpickers, the percentages of CD14+CD16+intermediate and CD14dim CD16+nonclassical monocyte subsets were elevated with a decline in CD14+CD16-classical monocytes. The expressions of CD64, CD16, CD35, and CD11b/CD18 on both monocytes and neutrophils, and CD14 on monocytes were significantly higher in ragpickers. In addition, ragpickers had 2.7-times more serum immunoglobulin-E than the controls (p < 0.0001). After controlling potential confounders, the profession of ragpicking was positively associated with the changes.ConclusionRagpicking is associated with alterations in both innate (neutrophils, monocytes, and natural killer cell numbers and expression of complement and Fcγ receptors) and adaptive immunity (numbers of circulating B cells, helper, cytotoxic, and memory T cells).

Enhanced Natural Killer and Cytotoxic T Lymphocyte Responses, with Decreased Monocytic Myeloid Derived Suppressor Cells May Promote Treatment Free Remission in Chronic Myeloid Leukaemia Patients Following Tyrosine Kinase Inhibitor Cessation

Introduction:An attempt at tyrosine kinase inhibitor (TKI) withdrawal in deep molecular remission leads either to treatment free remission (TFR) or early molecular relapse (MolR) in chronic myeloid leukaemia (CML) patients. We hypothesise that immune responses promote sustained TFR and immunological markers may predict response following TFR attempt.Methodology: We studied 54 CML patients (from ALLG trials CML 8, median follow-up 66 mo, and CML 10, median follow-up 24 mo) at baseline on TKI (minimum 24 mo MR4.5) and 3 mo and 6 mo following TKI discontinuation. MolR was defined as any single sample on follow-up with BCR-ABL1 >0.1% or two consecutive BCR-ABL1 positive samples at any value. Effector immune responses of CD56dim natural killer (NK) cells and NK cell receptor repertoire were characterised by flow cytometry and cytotoxic T lymphocyte (CTL) responses to leukaemia-associated-antigens (LAAs) WT1, BMI-1, PR3 and PRAME by interferon-gamma ELISPOT. Immune suppressor regulatory T cells (Treg; CD4+CD25brightCD127-FoxP3+), Granulocytic and Monocytic Myeloid-Derived Suppressor Cells (MDSCs; HLA-DR-Lin-CD11b+CD33+CD66b+CD15+ and HLA-DR-Lin-CD11b+CD33+CD66b-CD14+, respectively), Programmed cell death-1 (PD-1) expression on T cells, NK cells, B cells and Monocytes, and major B cell subsets were characterized by flow cytometry.Results: TFR patients displayed increased CD3-CD56dimCD16bright cytolytic NK cells as a proportion of total lymphocytes at baseline (n=23, 27.1% ± 2.9) vs MolR (n=23, 19.1% ± 2.0, p=0.02). TFR patients displayed a more mature CD56dim CD57+ NK cell phenotype at baseline (74.5% ± 2.2 of total NK cells) vs MolR (66.3% ± 2.7, p=0.04). Extensive characterisation of NK cell receptor repertoire revealed NKG2D activating receptor expression was increased in TFR patients (baseline= 56.8% ± 3.8, 3 mo= 61.4% ± 5.0, 6 mo= 49.9% ± 5.8) vs MolR (baseline= 44.2% ± 3.7, 3 mo= 42.2% ± 5.5, 6 mo= 22.0% ± 8.3, all p=0.02). KIR2DL2/DL3/DS2-positive NK cells were increased in MolR patients at 3 and 6 mo vs TFR. (MolR; 3 mo= 44.8% ± 4.6, 6 mo= 48.8% ± 4.9. TFR; 3 mo= 31.5% ± 4.0 p=0.05, 6 mo= 31.1% ± 2.1, p=0.001).No significant differences were observed in CD56brightCD16-/dim immunoregulatory NK cells, C-type lectin receptor expression (CD94/NKG2A/NKG2C, CD161, CD69), Natural cytotoxicity receptors (NKp30, NKp44, NKp46), CD62L (on T cells and NK cells) and KIR2DL5 expression. No difference in NK Cell-mediated K562 degranulation as a surrogate marker of NK cell function was observed between TFR and MolR patients.Functional CTL immune responses were observed in TFR and MolR patients. BMI-1 CTL responses were increased at baseline in TFR (23%) vs MolR (9%). PR3 CTL responses were not detected in TFR at baseline, 3 mo or 6 mo (0%) vs MolR (baseline= 18%, 3 mo= 50%, 6 mo= 50%). No difference was observed in WT1 or PRAME CTLs.Quantification of immune suppressor cell types revealed decreased Monocytic MDSCs in TFR patients at baseline (10.0% ± 2.3) vs MolR (17.7% ± 3.1, p=0.02). There was no difference in granulocytic MDSCs or Treg between TFR and MolR. No difference in PD-1 expression was observed on NK cells, T cells, B cells and Monocytes. Extensive characterisation of B cell subsets revealed no difference in TFR vs MolR (Table 1).Conclusion: In keeping with STIM and EURO-SKI trials, a threshold level of particular NK cell subsets may be important in maintaining TFR. We found additionally that enhanced NK and CTL effector responses and decreased inhibitory NK KIR2DL2/DL3/DS2 expression, in combination with reduced monocytic MDSC may promote sustained TFR. Methods to enhance nett immune effector responses, such as mature CD56dimCD57+ NK cells and BMI-1 CTL responses or targeting inhibitory KIR may increase TFR success rates. [Display omitted] DisclosuresWhite:Ariad: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Ross:Novartis Pharmaceuticals: Honoraria, Research Funding; BMS: Honoraria. Hughes:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Honoraria. Yong:Celgene: Research Funding; BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.