Proportion Of Natural Killer Cells Research Articles

Insight into innate immune response in “Yusho”: The impact of natural killer cell and regulatory T cell on inflammatory prone diathesis of Yusho patients

BackgroundIn 1968 in western Japan, polychlorinated biphenyl-contaminated “Kanemi rice oil” was used in cooking, causing food poisoning in many people. More than 50 years have passed since the Yusho incident, and although inflammatory disorders such as suppuration have been observed in Yusho patients, the etiology of this inflammation susceptibility remains obscure. ObjectivesTo investigate the mechanisms of susceptibility to inflammation in Yusho patients, peripheral immune cell fractions and concentrations of inflammatory cytokines were evaluated in blood samples collected from both Yusho patients and age-matched healthy subjects undergoing medical examination in Nagasaki. MethodsTo exclude diagnostic uncertainty, serum levels of polychlorinated biphenyl (PCB), polychlorinated quarterphenyl (PCQ), and polychlorinated dibenzofuran (PCDF) were measured. Immune cell (e.g. natural killer and regulatory T cell) populations were analyzed by flow cytometry. Serum cytokines involved in immune cell activation were measured by ELISA. ResultsThe relative proportion of natural killer cells was higher in Yusho patients than in healthy subjects, while the proportion of regulatory T cells did not differ between groups. Serum concentrations of IL-36 and IFN-γ were significantly lower in Yusho patients than in healthy subjects. Conversely, serum cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), which is a cytokine related to activated NK cells, was higher in Yusho patients than in healthy subjects and was positively correlated with PCDF blood levels. ConclusionIncreased numbers of NK cells in Yusho patients suggests that the innate immune response has been activated in Yusho patients. The seemingly paradoxical results for CTLA-4 and IFN-γ may reflect counterbalancing mechanisms preventing excessive NK cell activation. This dysregulation of innate immunity might contribute to the inflammation observed in Yusho patients.

Peripheral blood natural killer cell proportion and ovarian function in women with recurrent implantation failure

Current knowledge of the association between peripheral natural killer (NK) cell proportion and ovarian function in reproductive-age women is limited. We explored the association between NK cell proportion and ovarian function in women who underwent in-vitro fertilization (IVF) treatment. This was a retrospective cohort study using the data of 20–44-year-old women with recurrent implantation failure (RIF) who were tested for NK cell proportion and anti-Müllerian hormone (AMH). Indicators of ovarian function included AMH, observed-to-(age-appropriate) reference AMH ratio, high FSH, peak E2 and total number of oocytes during the first IVF cycle following the test. We used different model specification controlling for women’s age, and body mass index. Among a total of 936 women, majority showed lower AMH compared to age-appropriate level. Average NK cell proportion was 13.5 ± 5.7%. Number of oocytes showed positive association with NK cell (ß = 0.040, p = .025). In the subgroup with NK ≥ 18%, NK cell proportion was negatively associated with AMH (−0.106, p = .012), AMH ratio (−0.049, p = .014) and number of oocytes (−0.021, p < .001) while the associations with others remain close to null. High NK cell proportion may be harmful to ovarian reserve or function.

Adaptive NKG2C+ natural killer cells are related to exacerbations and nutritional abnormalities in COPD patients

Chronic obstructive pulmonary disease (COPD) is a chronic and often progressive disorder with a heterogeneous presentation and frequent systemic manifestations. Several aspects like persistence in smoking habit, continuous exacerbations, alpha-1-antitrypsin deficiency and inflammatory-immune response, are involved in the pathophysiology and progression of the disease. However, the role of natural killer (NK) cells remains controversial. Otherwise, human cytomegalovirus (HCMV) infection has been reported to induce an adaptive differentiation and expansion of an NK cell subset which carries the CD94/NKG2C receptor, which may contribute to an upset immune defense. For these reasons, our objective is to assess the distribution of NK cells and their subset in COPD patients and some of its phenotypes.MethodsPeripheral blood samples were obtained from 66 COPD patients. HCMV serology and the proportions of total NK cells and the NKG2C+ and NKG2A+ subsets were evaluated by flow cytometry. The NKG2C genotype was also assessed.ResultsEighty-eight per cent of COPD patients were HCMV(+), and the proportions of total NK cells were higher in patients with severe-very severe airway obstruction than in those with only mild-moderate involvement. There were no differences in the proportions of NKG2C+ cells between controls and COPD, either among COPD patients classified by severity of the disease. However, the percentage of NKG2C+ cells were higher in COPD patients with frequent exacerbations than in occasional exacerbators, and higher in cases with reduced lean mass (Fat free mass index) than in those with normal nutritional status.ConclusionThese results suggest a relationship between levels of NKG2C+ cells in COPD patients and clinical variables closely linked to a poor/worse prognosis.

Distribution of Natural Killer Cell in Patients with Newly Diagnosed Myelodysplastic Syndromes and Its Correlation with Prognosis

To investigate the distribution of peripheral blood lymphocytes and natural killer (NK) cells, and its influence on the prognosis of patients with myelodysplastic syndromes (MDS). The lymphocytes proportion, absolute lymphocyte counts (ALC), NK cell proportion and absolute NK cell counts (ANKC) as well as the related data of 95 MDS patients diagnosed between 2013 and 2017 analyzed retrospectively. The correlation of ALC and ANKC with prognosis was also analyzed. As compared with low ALC patients, MDS patients with ALC≥0.885×109/L had a higher overall response rate (66.7% vs 35.8%) (P＜0.01). The ALC of effective patients after treatment significatitly increased in compaison of ALC at diagnosis. Multivariate analysis indicated that patients with ALC≥0.885×109/L had long overall survival (OS) time in comparison with patients with low level (16.4 vs 12.4 months) (P＜0.05). The OS time of patients with ANKC≥0.110×109/L was shorter in comparison with patients with low level (10.9 vs 16.3 months) (P＜0.01). Otherwise, blast, cytogenetic risks and treatment response were also independent risk factors of MDS (P＜0.05). Revised International Prognostic Scoring System (IPSS-R) combined with ANKC could improve predictive accuracy of IPSS-R alone (AUC 0.718 vs 0.674) (P＜0.05). Lymphocytes and NK cells are important for the prognosis evaluation of MDS patients.

Distribution of Natural Killer Cell in Patients with Newly Diagnosed Myelodysplastic Syndromes and Its Correlation with Prognosis

To investigate the distribution of peripheral blood lymphocytes and natural killer (NK) cells, and its influence on the prognosis of patients with myelodysplastic syndromes (MDS). The lymphocytes proportion, absolute lymphocyte counts (ALC), NK cell proportion and absolute NK cell counts (ANKC) as well as the related data of 95 MDS patients diagnosed between 2013 and 2017 analyzed retrospectively. The correlation of ALC and ANKC with prognosis was also analyzed. As compared with low ALC patients, MDS patients with ALC≥0.885×109/L had a higher overall response rate (66.7% vs 35.8%) (P＜0.01). The ALC of effective patients after treatment significatitly increased in compaison of ALC at diagnosis. Multivariate analysis indicated that patients with ALC≥0.885×109/L had long overall survival (OS) time in comparison with patients with low level (16.4 vs 12.4 months) (P＜0.05). The OS time of patients with ANKC≥0.110×109/L was shorter in comparison with patients with low level (10.9 vs 16.3 months) (P＜0.01). Otherwise, blast, cytogenetic risks and treatment response were also independent risk factors of MDS (P＜0.05). Revised International Prognostic Scoring System (IPSS-R) combined with ANKC could improve predictive accuracy of IPSS-R alone (AUC 0.718 vs 0.674) (P＜0.05). Lymphocytes and NK cells are important for the prognosis evaluation of MDS patients.

Compartmentalized cytotoxic immune response leads to distinct pathogenic roles of natural killer and senescent CD8+ T cells in human cutaneous leishmaniasis.

Cytotoxic activity mediated by CD8+ T cells is the main signature of the immunopathogenesis of cutaneous leishmaniasis (CL). Here, we performed a broad evaluation of natural killer (NK) cell phenotypic and functional features during cutaneous leishmaniasis. We demonstrate for the first time that CL patients present the accumulation of circulating NK cells with multiple features of replicative senescence including low proliferative capacity and shorter telomeres, elevated expression of CD57, KLRG1 but diminished CD27 stimulatory receptor expression. Moreover, they exhibited higher cytotoxic and inflammatory potential than age-matched controls. The accumulation of circulating senescent NK cells (CD56dim CD57bright ) correlated positively with skin lesion size in the same patients, suggesting that they, like circulating senescent CD8+ T cells, may contribute to the immunopathology of CL. However, this senescent population had lower cutaneous lymphocyte antigen expression and so had diminished skin-homing potential compared with total or senescent CD8+ T cells. This was confirmed in CL skin lesions where we found a predominance of CD8+ T cells (both senescent and non-senescent) that correlated with the severity of the disease. Although there was also a correlation between the proportions of senescent NK cells (CD56+ CD57+ ) in the skin and lesion size, this was less evident. Collectively our results demonstrate first-hand that senescent cytotoxic cells may mediate skin pathology during human cutaneous leishmaniasis. However, as senescent cytotoxic CD8+ T cells predominate in the skin lesions, they may have a greater role than NK cells in mediating the non-specific skin damage in CL.

Alterations in the innate immune system due to exhausting exercise in intensively trained rats

It is known that intensive physical activity alters the immune system’s functionality. However, the influence of the intensity and duration of exercise needs to be studied in more depth. We aimed to establish the changes in the innate immune response induced by two programmes of intensive training in rats compared to sedentary rats. A short training programme included 2 weeks of intensive training, ending with an exhaustion test (short training with exhaustion, S-TE). A second training programme comprised 5-week training including two exhaustion tests and three trainings per week. In this case, immune status was assessed before (T), immediately after (TE) and 24 h after (TE24) an additional final exhaustion test. Biomarkers such as phagocytic activity, macrophage cytokine and reactive oxygen species (ROS) production, and natural killer (NK) cell activity were quantified. S-TE was not enough to induce changes in the assessed innate immunity biomarkers. However, the second training was accompanied by a decrease in the phagocytic activity, changes in the pattern of cytokine secretion and ROS production by macrophages and reduced NK cell proportion but increased NK cytotoxic activity. In conclusion, a 5-week intense training programme, but not a shorter training, induced alterations in the innate immune system functionality.

Reduced proportion and activity of natural killer cells in patients with Graves’ disease

Natural killer cells not only play important roles in protecting against viral infection and cancer but also involved in the pathogenesis of Graves’ disease. Killer Ig-like receptor (KIR) genes encode receptors which are mostly expressed on and regulate the activation of natural killer cells. Our previous research found that the KIR2DS4 gene frequency was lower in patients with Graves’ disease than in controls. Nevertheless, the specific mechanisms by which natural killer cell act is obscure in Graves’ disease. In total, 178 participants including newly diagnosed Graves’ disease patients (n = 95) and healthy individuals (n = 83) were recruited in this study. TSH (thyrotropin), FT3 (free triiodothyronine), and FT4 (free thyroxine) were assayed using electro chemiluminescent immunoassays. The counts of natural killer cell (CD3−CD56+natural killer cell), activated natural killer cell (CD3−CD56+CD69+natural killer cell), and KIR2DS4-expressing natural killer cell (CD3−CD56+CD158i+natural killer cell) in peripheral blood were analyzed using flow cytometry. The proportions of natural killer cells and activated natural killer cells were lower in the newly diagnosed Graves’ disease patients than in the controls; the difference was statistically significant ( P &lt; 0.05). However, the difference in the proportion of KIR2DS4-expressing natural killer cells between the two groups was not statistically significant. In Graves’ disease patients, no relationship was found between the proportion of natural killer cells and the blood FT3 level, the blood FT4 level, or the blood TSH level; however, the proportion of activated natural killer cells was negatively correlated with FT3 and FT4 and positively correlated with TSH. Our research findings revealed that a reduction in the counts of natural killer cell and activated natural killer cell might be involved in Graves’ disease pathogenesis.

Inhaled Kathon may induce eosinophilia-mediated disease in the lung.

In 2011, a link between humidifier disinfectants and patients with idiopathic pulmonary fibrosis was identified in Korea, and Kathon was suggested as one of the causative agents. In this study, Kathon induced apoptotic cell death along with membrane damage at 24 h post-exposure. Additionally, on day 14 after a single instillation with Kathon, the total number of pulmonary cells and the levels of TNF-α, IL-5, IL-13, MIP-1α, and MCP-1α clearly increased in the lung of mice. The proportion of natural killer cells and eosinophils were significantly elevated in the spleen and the bloodstream, respectively, and the level of immunoglobulin (Ig) A, but not IgG, IgM, and IgE, dose-dependently increased. Therefore, we suggest that inhaled Kathon may induce eosinophilia-mediated disease in the lung by disrupting homeostasis of pulmonary surfactants. Considering that eosinophilia is closely related to cancer and fibrosis, further studies are needed to understand the relationship between them.

Accumulation of Tumor-Infiltrating CD49a+ NK Cells Correlates with Poor Prognosis for Human Hepatocellular Carcinoma

The discovery of CD49a+ liver-resident natural killer (NK) cells in mice alters our view of NK cells and provides another opportunity to study NK cells. Although evidence has suggested roles for NK cells in liver diseases, whether and how CD49a+ NK cells contribute to liver diseases remain unclear. In this study, we observed that accumulation of CD49a+ tissue-resident NK cells in human hepatocellular carcinoma (HCC) was higher than in peritumoral tissues. We studied the exhausted and regulatory phenotypes of CD49a+ tissue-resident NK cells by analysis of protein and mRNA. The proportion of CD49a+ NK cells was positively correlated to the proportion of NK cells expressing inhibitory receptors. In addition, CD49a+ NK cells expressed more of checkpoint molecules PD-1, CD96, and TIGIT. Transcriptomic analysis implicated CD49a+ tissue-resident NK cells in the negative regulation of immune responses. Comparison of murine and human CD49a+ NK cells revealed their distinct characteristics and functions. Finally, accumulation of tissue-resident CD49a+ NK cells in liver tumor was correlated to deteriorating disease condition and poor prognosis. Our findings show that CD49a+ NK cells accumulate in liver tumor and suggest a role for CD49a+ NK cells in the negative regulation of immune responses and the development of HCC.

Cytotoxic effects of ex vivo-expanded natural killer cell-enriched lymphocytes (MYJ1633) against liver cancer

BackgroundAdoptive transfer of immune cells such as T cells and natural killer (NK) cells has emerged as a targeted method of controlling the immune system against cancer. Despite their significant therapeutic potential, efficient methods to generate adequate numbers of NK cells are lacking and ex vivo-expansion and activation of NK cells is currently under intensive investigation. The primary purpose of this study was to develop an effective method for expansion and activation of the effector cells with high proportion of NK cells and increasing cytotoxicity against liver cancer in a short time period.MethodsExpanded NK cell-enriched lymphocytes (NKL) designated as “MYJ1633” were prepared by using autologous human plasma, cytokines (IL-2, IL-12 and IL-18) and agonistic antibodies (CD16, CD56 and NKp46) without an NK cell-sorting step. The characteristics of NKL were compared to those of freshly isolated PBMCs. In addition, the cytotoxic effect of the NKL on liver cancer cell was examined in vitro and in vivo.ResultsThe total cell number after ex vivo-expansion increased about 140-fold compared to that of freshly isolated PBMC within 2 weeks. Approximately 78% of the expanded and activated NKL using the house-developed protocol was NK cell and NKT cells even without a NK cell-sorting step. In addition, the expanded and activated NKL demonstrated potent cytotoxicity against liver cancer in vitro and in vivo.ConclusionThe house-developed method can be a new and effective strategy to prepare clinically applicable NKL for autologous NK cell-based anti-tumor immunotherapy.

Effects of regulatory T cells, natural killer cells, and natural killer T cells on immunosuppression therapy in patients with recurrent embryo implantation failure

Objective: This retrospective study aims to investigate the effects of immunosuppressive therapy on the expression of regulatory T (Treg) cells, natural killer (NK) cells, and natural killer T (NKT) cells in patients with recurrent embryo implantation failure (RIF). Materials and Methods: In vitro fertilization and embryo transplantation (IVF-ET) at Jinhua Municipal Central Hospital from July 2013 to November 2015 of 30 RIF patients were enrolled into RIF group, and peripheral blood Treg, NK, and NKT were detected before entering the embryo transfer cycle. Meanwhile, 20 normal non-pregnant women were enrolled into control group. Twenty-four patients in RIF group were administered prednisone; Treg, NK, and NKT cell contents were detected twice before and after the embryo transfer cycle, respectively. The remaining six patients with high NK cell contents were treated with gamma globulin via intramuscular injection, and Treg, NK, and NKT cell contents were detected twice before the embryo transfer cycle and after treatment. Results: Treg cell content was significantly lower in RIF group than in control group, while NK cell proportion was significantly higher in RIF group than in control group before treatment. There was no statistical difference in NKT cells. After treatment, expression rate of Treg in RIF group was 7.1 ± 1.8%, which compared with that before treatment (2.8±1.6%) was significantly higher (p &lt; 0.01). In addition, NK cell proportion was significantly lower than pretherapy (20.9 ± 3.6% vs. 38.6 ± 8.1%, p &lt; 0.05) and NKT cell percentage did not present the obvious difference before and after treatment. Conclusion: The occurrence of RIF may be related to the decrease in Treg expression and increase of NK cells. Immunotherapy can upregulate the expression of Treg and decrease NK cell proportion, thereby regulating maternal fetal immune tolerance and reducing the rejection effect of NK cells on fetal foreign bodies, which are conducive to embryo implantation.

Enrichment of Cytomegalovirus-induced NKG2C+ Natural Killer Cells in the Lung Allograft.

In lung transplant recipients, immunosuppressive medications result in impaired antiviral immunity and a propensity for cytomegalovirus (CMV) reactivation within the lung allograft. Natural killer (NK) cells play a key role in immunity to CMV, with an increase in the proportion of NK cells expressing activating CD94-NKG2C receptors in the blood being a strong correlate of CMV infection. Whether a similar increase in NKG2C NK cells occurs in lung transplant recipients following CMV reactivation in the allograft and if such cells contribute to viral control remains unclear. In this pilot study, we longitudinally assessed the frequency and phenotype of NKG2C NK cells in the blood and bronchoalveolar lavage (BAL) of lung transplant recipients and stratified recipients based on their risk of developing CMV disease. We observed an increase in the proportion of NKG2C NK cells in the blood and BAL of CMV high-risk patients, coincident with both the cessation of antiviral prophylaxis and subsequent detection of actively replicating CMV in the blood and lung allograft. Additionally, these NKG2C NK cells expressed killer-cell immunoglobulin-like receptors distinct from those of other NK subsets and BAL NKG2C NK cells possessed an activated phenotype. Finally, the frequency of NKG2C NK cells in the BAL may be inversely correlated with CMV blood titers. Monitoring the phenotype of NK cells postlung transplant may be a useful biomarker for monitoring patient levels of CMV immunity.

Histidine-rich glycoprotein augments natural killer cell function by modulating PD-1 expression via CLEC-1B.

Augmentation of natural killer (NK) cell cytotoxicity is one of the greatest challenges for cancer immunotherapy. Although histidine‐rich glycoprotein (HRG), a 75‐kDa glycoprotein with various immunomodulatory activities, reportedly elicits antitumor immunity, its effect on NK cell cytotoxicity is unclear. We assessed NK cell cytotoxicity against K562 cells. We also measured concentrations of cytokines and granzyme B in the cell supernatant. The proportion of CD56bright NK cells and NK cell surface PD‐1 expression was assessed with flow cytometry. The neutralizing effects of anti‐C‐type lectin‐like receptor (CLEC) 1B against HRG were also measured. NK cell morphological changes were analyzed via confocal microscopy. HRG significantly increased NK cell cytotoxicity against K562 cell lines. HRG also increased the release of granzyme B and the proportion of CD56bright NK cells. Further, HRG was able to decrease NK cell surface PD‐1 expression. The effects of HRG on NK cells were reversed with anti‐CLEC‐1B antibodies. Additionally, we confirmed NK cell nuclear morphology and F‐actin distribution, which are involved in the regulation of cytotoxic granule secretion. Because both PD‐1 and CLEC‐1B are associated with prognosis during malignancy, HRG incorporates these molecules to exert the antitumor immunity role. These facts indicate the potential of HRG to be a new target for cancer immunotherapy.

Adaptive NK cells in people exposed to Plasmodium falciparum correlate with protection from malaria.

How antibodies naturally acquired during Plasmodium falciparum infection provide clinical immunity to blood-stage malaria is unclear. We studied the function of natural killer (NK) cells in people living in a malaria-endemic region of Mali. Multi-parameter flow cytometry revealed a high proportion of adaptive NK cells, which are defined by the loss of transcription factor PLZF and Fc receptor γ-chain. Adaptive NK cells dominated antibody-dependent cellular cytotoxicity responses, and their frequency within total NK cells correlated with lower parasitemia and resistance to malaria. P. falciparum-infected RBCs induced NK cell degranulation after addition of plasma from malaria-resistant individuals. Malaria-susceptible subjects with the largest increase in PLZF-negative NK cells during the transmission season had improved odds of resistance during the subsequent season. Thus, antibody-dependent lysis of P. falciparum-infected RBCs by NK cells may be a mechanism of acquired immunity to malaria. Consideration of antibody-dependent NK cell responses to P. falciparum antigens is therefore warranted in the design of malaria vaccines.

The number and cytotoxicity and the expression of cytotoxicity-related molecules in peripheral natural killer (NK) cells do not predict the repeated implantation failure (RIF) for the in vitro fertilization patients

Natural killer (NK) cells are thought to play a key role in the successful establishment of a pregnancy by facilitating immunological adaptation of the semi-allogeneic developing embryo. The aim of this study was to explore the cell number, immunophenotypic characteristics, and activities of peripheral blood NK cells in women with repeated implantation failure (RIF). Peripheral blood was obtained from 27 women with RIF and 11 healthy, fertile controls during the middle luteal phase of the menstrual cycle. CD3- CD56+ NK cells were quantified and analyzed by flow cytometry for the expression of cytolytic molecules (granzyme B, granulysin, and perforin) as well as cell surface receptors responsible for NK cell activation or inhibition (NKG2D, NKp30, NKp46, CD158a, CD158b). NK cytotoxicity was measured at three effector-to-target cell ratios. Women with RIF and fertile controls did not differ significantly in the percentage of circulating CD3−CD56+ NK cells, or in the proportions of these cells that expressed granzyme B, granulysin, or perforin. The two groups also did not differ significantly in the proportions of NK cells expressing the receptors NKG2D, NKp30, NKp46, CD158a or CD158b. General linear model analysis showed that NK cytotoxicity increased with effector-to-target cell ratio. However, NK cytotoxicity did not differ significantly between patients with RIF and fertile controls. These results suggest that RIF is not associated with significant alterations in the number or function of peripheral blood NK cells.

Circulating NK cell subsets and NKT‑like cells in renal transplant recipients with acute T‑cell‑mediated renal allograft rejection.

Emerging evidence indicates that natural killer (NK) cells and NKT-like cells may affect allograft outcomes following solid organ transplantation. However, the roles of these cells in allograft acceptance and dysfunction are controversial. To assess the changes in NK cell and CD3+CD56+ NKT-like cell frequency and phenotype in renal allograft recipients and to explore their associations with acute T-cell-mediated renal allograft rejection (ACR), longitudinal changes in NK and NKT-like cell frequency and phenotype were characterized using flow cytometry and immunohistochemistry in the peripheral blood and kidney allograft tissues in 142 recipients undergoing kidney transplantation. The serum concentrations of NK cell-associated cytokines were also detected by cytokine multiplex immunoassay. In contrast to the healthy controls, recipients with stable graft function exhibited increased proportions of CD56brightCD16dim subsets and decreased proportions of NKT-like cells in their peripheral blood mononuclear cells (PBMCs). Patients with ACR demonstrated increased proportions of NK cells, which were associated with increased CD3−CD56bright subsets and decreased CD3−CD56dim subsets, an increase in the CD56bright/CD56dim ratio in PBMCs and increased CD56+ NK cell infiltration in the kidney allograft, compared with the stable controls. In addition, there was a decreased proportion of NKT-like cells in patients with ACR, and an increased ratio of CD56bright/NKT-like cells compared with the stable controls. These differences appeared to be consistent with the increase in the serum concentrations of C-C motif chemokine 19 and the decrease in the serum concentrations of interleukin-15. These data indicate that CD56bright NK cells may promote the development of ACR, and that NKT-like cells may have immunoregulatory function. The results also imply that the CD56bright/CD56dim ratio may affect the ACR signatures.

Heterogeneity of NK Cells and Other Innate Lymphoid Cells in Human and Murine Decidua.

Innate lymphoid cells (ILCs) represent a heterogeneous group of cells lacking genetically rearranged antigen receptors that derive from common lymphoid progenitors. Five major groups of ILCs have been defined based on their cytokine production pattern and developmental transcription factor requirements: namely, natural killer (NK) cells, ILC1s, ILC2s, ILC3s, and lymphoid tissue-inducer (LTi) cells. ILC1s, ILC2s, and ILC3s mirror the corresponding T helper subsets (Th1, Th2, and Th17, respectively) and produce cytokines involved in defense against pathogens, lymphoid organogenesis, and tissue remodeling. During the first trimester of pregnancy, decidual tissues contain high proportion of decidual NK (dNK) cells, representing up to 50% of decidual lymphocytes, and ILC3s. They release peculiar cytokines and chemokines that contribute to successful pregnancy. Recent studies revealed that ILCs display a high degree of plasticity allowing their prompt adaptation to environmental changes. Decidual NK cells may derive from peripheral blood NK cells migrated when pregnancy establishes or from in situ differentiation of hematopoietic precursors. Previous studies showed that human and murine decidua contain dNK cells, tissue resident NK cells, and ILC3s, all characterized by unique phenotypic and functional properties, most likely induced by decidual microenvironment to favor the establishment and the maintenance of pregnancy. Thus, during the early phase of pregnancy, the simultaneous presence of different ILC subsets further underscores the complexity of the cellular components of decidual tissues as well as the role of decidual microenvironment in shaping the plasticity and the function of ILCs.

Abstract A072: Calreticulin exposures by malignant blasts correlate with robust anticancer immunity and improved clinical outcome in AML patients

Abstract Cancer cell death can be perceived as immunogenic by the host only when malignant cells emit immunostimulatory signals (so-called “damage-associated molecular patterns,” DAMPs), as they die in the context of failing adaptive responses to stress. Accumulating preclinical and clinical evidence indicates that the capacity of immunogenic cell death to (re-)activate an anticancer immune response is key to the success of various chemo- and radiotherapeutic regimens. Malignant blasts from patients with acute myeloid leukemia (AML) exposed multiple DAMPs, including calreticulin (CRT), heat-shock protein 70 (HSP70), and HSP90 on their plasma membrane irrespective of treatment. In these patients, high levels of surface-exposed (ecto)-CRT correlated with an increased proportion of natural killer cells and effector memory CD4+ and CD8+ T-cells in the periphery. Moreover, CRT exposure on the plasma membrane of malignant blasts positively correlated with the frequency of circulating T-cells specific for leukemia-associated antigens, indicating that ecto-CRT favors the initiation of anticancer immunity in patients with AML. Finally, although the levels of ecto-HSP70, ecto-HSP90, and ecto-CRT were all associated with improved relapse-free survival, only CRT exposure significantly correlated with superior overall survival. Thus, CRT exposure represents a novel powerful prognostic biomarker for patients with AML, reflecting the activation of a clinically relevant AML-specific immune response. Citation Format: Jitka Fucikova. Calreticulin exposures by malignant blasts correlate with robust anticancer immunity and improved clinical outcome in AML patients [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 A072.

Abstract A085: High infiltration of NK cells expressing elevated LAG-3 in a subgroup of renal cell carcinoma patients

Abstract Background: Renal cell carcinoma (RCC) is considered one of the most immunogenic cancers with the highest number of indel mutations, frequent infiltration of T-cells, presence of many antigen-specific T-cell clones, and high immuno-oncologic (IO) sensitivity. Since less is known about natural killer (NK) cells in RCC, our primary aim was to investigate the intratumoral phenotype of NK cells as well as further assess the overall immune landscape of the tumors, peripheral blood (PB) and adjacent healthy kidney tissue, which may be critical for patient prognosis and predictions to targeted immunotherapies. Methods: We used multi-parameter flow cytometry together with a comprehensive immunostaining panel containing a total of 56 fundamental markers to cancer immunology to immunophenotype the tumor, adjacent healthy kidney tissue and presurgical peripheral blood (PB) samples from 31 RCC patients who underwent partial or radical nephrectomies. To study the intratumoral T-cell clonalities, T-cell receptor beta (TCRβ) deep sequencing was carried out with eight tumors. Results: Using hierarchical clustering (Spearman correlation distance and Ward linkage) and correlation analyses (Spearman correlation), we discovered that our patient cohort clustered into two distinct subgroups defined by a high (NKhigh, n=11; mean 29.7%) and low (NKlow, n=20; mean 9.4%) percentage of NK cells among the intratumoral lymphocyte population. Accordingly, the NKhigh subgroup had a lower percentage of T-cells (mean 36.9%) than the NKlow group (mean 65.7%), and overall, a significant negative correlation between T and NK cells was discovered. Our TCRβ sequencing results revealed a positive correlation between T-cell clonality and the intratumoral T-cell percentage, whereas the higher proportion of tumor NK cells associated with low T-cell clonality, possibly due to a polyclonal T-cell population. When we compared the expressions of the most clinically relevant IO markers (LAG-3 and PD-1) on the NK cells, LAG-3 was more expressed in the NKhigh group than in the NKlow group (21.3% vs 9.8%; p=0.08). In contrast, no differences were observed with PD-1. Clinical parameters such as tumor grade (Fuhrman), weight, size (diameter), the presence of necrosis, gender, or age of the patients did not differ between the two subgroups. To examine the overall immune landscape of RCC, we compared the cells from the tumor, PB, and healthy kidney tissue of seven patients. Our results showed that tumors have more NK cells compared to their corresponding T-cell-rich PB and healthy tissue counterparts, supporting our findings that some tumors accumulate NK cells. Compared to the adjacent healthy tissue, PD-1 and LAG-3 expressions were higher in the intratumoral CD8 cells. The expressions of PD-1 and LAG-3 on PB CD8+ T-cells or NK cells did not correlate with their intratumoral counterparts, whereas a positive correlation was found between the PB and tumor CD4+ T-cells for both LAG-3 and PD-1. Conclusions: Our study has led to the discovery of two distinct RCC tumor subgroups with differential expressions in the clinically leading molecules. These results suggest that immunophenotyping RCC patients may effectively aid in selecting those who will benefit the most from immune checkpoint inhibition therapies such as anti-PD1 and -LAG3. Prospective analyses on spatial immunoprofiling by multiplexed immunohistochemistry and mutational load by exome sequencing will be assessed next in order to find answers to why some tumors are NK-dominant, with expectations of understanding further the biologic differences between the two tumor types. Citation Format: Moon Hee Lee, Petrus Järvinen, Harry Nisén, Oscar Brück, Mette Ilander, Satu Mustjoki, Kreutzman Anna. High infiltration of NK cells expressing elevated LAG-3 in a subgroup of renal cell carcinoma patients [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 A085.