Density Of Natural Killer Cells Research Articles

Density of tumor-infiltrating NK and Treg cells is associated with 5 years progression-free and overall survival in resected lung adenocarcinoma

Surgical resection of pulmonary adenocarcinoma is considered to be curative but progression-free survival (PFS) has remained highly variable. Antitumor immune response may be important, however, the prognostic significance of tumor-infiltrating natural killer (NK) and regulatory T (Treg) lymphocytes is uncertain. Resected pulmonary adenocarcinoma tissues (n = 115) were studied by immunohistochemical detection of NKp46 and FoxP3 positivity to identify NK and Treg cells, respectively. Association of cell densities with clinicopathological features and progression-free survival (PFS) as well as overall survival (OS) were analyzed with a follow-up time of 60 months. Both types of immune cells were accumulated predominantly in tumor stroma. NK cell density showed association with female gender, non-smoking and KRAS wild-type status. According to Kaplan-Meier analysis, PFS and OS proved to be longer in patients with high NK or Treg cell densities (p = 0.0293 and p = 0.0375 for PFS, p = 0.0310 and p = 0.0448 for OS, respectively). Evaluating the prognostic effect of the combination of NK and Treg cell density values revealed that PFS and OS were significantly longer in NKhigh/Treghigh cases compared to the other groups combined (p = 0.0223 and p = 0.0325, respectively). Multivariate Cox regression analysis indicated that high NK cell density was independent predictor of longer PFS while high NK and high Treg cell densities both proved significant predictors of longer OS. The NKhigh/Treghigh combination also proved to be an independent prognostic factor for both PFS and OS. In conclusion, NK and Treg cells can be components of the innate and adaptive immune response at action against progression of pulmonary adenocarcinoma.

Hierarchical cluster analysis and nonlinear mixed-effects modelling for candidate biomarker detection in preclinical models of cancer.

BackgroundPreclinical models of cancer can be of translational benefit when assessing how different biomarkers are regulated in response to particular treatments. Detection of molecular biomarkers in preclinical models of cancer is difficult due inter-animal variability in responses, combined with limited accessibility of longitudinal data. MethodsNonlinear mixed-effects modelling (NLME) was used to analyse tumour growth data based on expected tumour growth rates observed 7 days after initial doses (DD7) of Radiotherapy (RT) and Combination of RT with DNA Damage Response Inhibitors (DDRi). Cox regression was performed to confirm an association between DD7 and survival. Hierarchical Cluster Analysis (HCA) was then used to identify candidate biomarkers impacting responses to RT and RT/DDRi and these were validated using NLME. ResultsCox regression confirmed significant associations between DD7 and survival. HCA of RT treated samples, combined with NLME confirmed significant associations between DD7 and Cluster specific CD8+ Ki67 MFI, as well as DD7 and cluster specific Natural Killer cell density in RT treated mice. ConclusionApplication of NLME, as well as HCA of candidate biomarkers may provide additional avenues to assess the effect of RT in MC38 syngeneic tumour models. Additional studies would need to be conducted to confirm association between DD7 and biomarkers in RT/DDRi treated mice.

Skin infiltrating NK cells in cutaneous T-cell lymphoma are increased in number and display phenotypic alterations partially driven by the tumor.

Cutaneous T-cell lymphomas (CTCL) are characterized by focal infiltration of malignant T cell clones in solitary skin lesions. Many CTCL patients experience an indolent disease, but some progress to advanced disease with high fatality. We hypothesized that natural killer (NK) cells participate in local control of tumor growth in CTCL skin. Immunohistochemistry and flow cytometry analysis of the density, localization, phenotype and function of NK cells in twenty-nine fresh or formalin-fixed skin biopsies from twenty-four CTCL patients and twenty-three biopsies from twenty healthy controls highlighted higher numbers of CD56+CD3- NK cells in CTCL skin. A reduced fraction of CTCL skin NK cells expressed the maturation marker CD57, the cytotoxic protein granzyme B and the activation marker CD69, indicating reduced tumor-killing abilities of the NK cells. Retained expression of immune checkpoint proteins or inhibitory proteins including PD1, TIM3, LAG3, CD73 and NKG2A and the activating receptors CD16 and NKp46 indicated maintained effector functions. Indeed, the capacity of NK cells to produce anti-tumor acting IFNγ upon PMA+ionomycin stimulation was similar in cells from CTCL and healthy skin. Co-cultures of primary human NK cells or the NK cell line NKL with CTCL cells resulted in reduced levels of granzyme B and CD69, indicating that close cellular interactions with CTCL cells induced the impaired functional NK cell phenotype. In conclusion, increased numbers of NK cells in CTCL skin exhibit a partially impaired phenotype in terms of activity. Enhancing NK cell activity with NK cell activating cytokines such as IL-15 or immune checkpoint blockade therefore represents a potential immunotherapeutic approach in CTCL.

Особливості лімфоїдного мікрооточення в інвазивному протоковому раку грудної залози

The purpose of the study was to assess the distribution of tumor-infiltrating T-, B-lymphocytes and natural killer cells in various molecular subtypes of invasive ductal breast cancer and to establish their relationship with the degree of tumor differentiation. Materials and methods. The basis of the scientific work was a complex morphological, including immunohistochemical study of 193 cases of invasive ductal breast cancer. General histological processing of the samples was carried out in accordance with the standard technique. Immunohistochemical studies for CD3, CD20, CD56, ER, PR, c-erbB2, Ki-67 were performed according to the manufacturer's protocol with the control of samples. The grade of malignancy was determined according to the modified scheme of P. Scarff, H. Bloom and W. Richardson. The presence, localization, and expression intensity of diagnostic and prognostic biomarkers CD3, CD20, and CD56-positive cells (T-, B-, and natural killer cells, respectively) were determined using the new bioimage analysis software QuPath. Results and discussion. The differences obtained in our study varied depending on the subpopulations of immune cells and their location in the tumor tissue. The density of T and B lymphocytes was higher within the tumor and in the invasive margin in the non-luminal phenotypes compared to the luminal A and B phenotypes. Compared to the density of T-lymphocytes, the B-cell infiltrate was significantly (p <0.01) less pronounced in tumors of both luminal and non-luminal phenotypes. The lowest density of natural killer cells among tumor-infiltrating lymphocytes was found within the tumor of luminal subtypes. The percentage of infiltrates of T and B cells both within the tumor focus and in the invasive margin, as well as natural killer cells in the invasive margin, was significantly lower with G1 and G2 than with the degree of differentiation G3 (p <0.01). As for intratumoral natural killer cells, differences were found only between the degree of differentiation of G2 and G3, and the differences between G1 and G2 were not statistically significant (p> 0.05). Qualitative and quantitative assessment of the intensity of expression of T-, B- and natural killer cells also demonstrated a different degree of expression of lymphocytes, depending on the molecular subtype and location of the infiltrate. The luminal subtype A was characterized by a significant predominance of mild expression (2+) CD3 both within the tumor and in the invasive margin of the tumor (p <0.05), while in the luminal B and Her2 + phenotypes, the intensity of CD3 expression prevailed at the 1+ level. Triple negative tumors were characterized by strong expression of (3+) CD3 within the tumor and in the invasive margin of the tumor in all cases studied. Conclusion. Thus, tumor-infiltrating lymphocytes in different molecular subtypes of invasive ductal breast cancer can be considered a prognostic biomarker. Our results indicate a relationship between lymphoid infiltrate and the degree of differentiation in invasive ductal breast cancer, especially in less favorable molecular subtypes

MP39-15 YAP EXPRESSION PLAYS IMPORTANT ROLES IN MALIGNANT AGGRESSIVENESS BY REGULATING THE INFILTRATION OF CD57 + NATURAL KILLER CELLS, CD68 + MACROPHAGES, AND MAST CELLS AND THE EXPRESSION OF VEGF-A AND PD-L1 IN CLEAR CELL RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology I (MP39)1 Sep 2021MP39-15 YAP EXPRESSION PLAYS IMPORTANT ROLES IN MALIGNANT AGGRESSIVENESS BY REGULATING THE INFILTRATION OF CD57+ NATURAL KILLER CELLS, CD68+ MACROPHAGES, AND MAST CELLS AND THE EXPRESSION OF VEGF-A AND PD-L1 IN CLEAR CELL RENAL CELL CARCINOMA Kojiro Ohba, Yasuyoshi Miyata, Junki Harada, Tsuyoshi Matsuda, Tsubasa Kondo, Asato Otsubo, Yuta Mukae, Hidenori Ito, Hiroki Kurata, Kensuke Mitsunari, Hideki Sakai, Tomohiro Matsuo, and Yasushi Mochizuki Kojiro OhbaKojiro Ohba More articles by this author , Yasuyoshi MiyataYasuyoshi Miyata More articles by this author , Junki HaradaJunki Harada More articles by this author , Tsuyoshi MatsudaTsuyoshi Matsuda More articles by this author , Tsubasa KondoTsubasa Kondo More articles by this author , Asato OtsuboAsato Otsubo More articles by this author , Yuta MukaeYuta Mukae More articles by this author , Hidenori ItoHidenori Ito More articles by this author , Hiroki KurataHiroki Kurata More articles by this author , Kensuke MitsunariKensuke Mitsunari More articles by this author , Hideki SakaiHideki Sakai More articles by this author , Tomohiro MatsuoTomohiro Matsuo More articles by this author , and Yasushi MochizukiYasushi Mochizuki More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002054.15AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The Hippo signalling pathway is known as a critical regulator of malignant aggressiveness, and Yes-associated protein (YAP) is the most important downstream effector in this pathway. Although, YAP has pro-oncogenic activities, its detailed mechanisms are not fully understood. Recently, YAP has been reported to play important roles in tumour immunity, by regulating tumour-infiltrating lymphocytes and vascular endothelial growth factor (VEGF)-A and programmed cell death ligand 1 (PD-L1). However, the relationships between YAP expression, the densities of macrophages, natural killer (NK) cells, and mast cells; and VEGF-A and PD-L1 expression in human clear cell renal cell carcinoma (ccRCC) are not clear. METHODS: In 157 ccRCC tissues, the expression levels of YAP and VEGF-A were evaluated by immunohistochemistry, according to the percentage of positively stained cells and staining intensity. The densities of CD57+ NK cells, CD68+ macrophages, and mast cells were calculated as the number of positively stained cells per high-power field (HPF). All examinations were performed using immunohistochemical staining. RESULTS: Positive expression of YAP was significantly associated with high tumor grade, high pT stage, the presence of metastasis, and poor cause-specific survival in patients with ccRCC (p<0.001). CD57+ NK cell density was significantly lower (p<0.001) in YAP-positive tissues (mean±SD, 22.1 ± 8.0/HPF) than in YAP-negative tissues (33.1 ± 8.4/HPF). In contrast, CD68+ macrophage density was significantly higher (p<0.001) in YAP-positive tissues (44.2±14.5/HPF) than that in YAP-negative tissues (29.3±9.8/HPF). Similarly, YAP expression was positively correlated with mast cell density (p<0.001). However, H-scores of PD-L1 were significantly lower (p<0.001) in YAP-positive tissues (6.6±9.6) than in YAP-negative tissues (3.1±6.0). In addition, a similar positive correlation was detected between YAP expression and VEGF-A expression (p<0.001). CD57+ NK cell, CD68+ macrophage, and mast cell density and VEGF-A and PD-L1 expression were all significantly associated with pathological features. CONCLUSIONS: YAP expression was positively associated with tumour growth, invasion, and metastasis by stimulating the infiltration of CD58+ macrophages and mast cells into cancer tissues; increasing the expression of VEGF-A and PD-L1 in cancer cells; and suppressing the infiltration of CD57+ NK cells. Source of Funding: None © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e710-e710 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kojiro Ohba More articles by this author Yasuyoshi Miyata More articles by this author Junki Harada More articles by this author Tsuyoshi Matsuda More articles by this author Tsubasa Kondo More articles by this author Asato Otsubo More articles by this author Yuta Mukae More articles by this author Hidenori Ito More articles by this author Hiroki Kurata More articles by this author Kensuke Mitsunari More articles by this author Hideki Sakai More articles by this author Tomohiro Matsuo More articles by this author Yasushi Mochizuki More articles by this author Expand All Advertisement Loading ...

NK Cell Homing and Persistence in Bone Marrow after Adoptive Immunotherapy Correlates with Therapy Response

Background: Natural killer (NK) cells are innate lymphoid cells capable of killing leukemic cells without prior sensitization. Adoptive immunotherapy with allogeneic NK cells has been used at the University of Minnesota and was shown to be effective in a subset of patients with acute myeloid leukemia refractory to therapy. The factors determining clinical success of adoptive NK cell immunotherapy are largely unknown. We hypothesized that NK cell homing and persistence in the bone marrow niche may be predictive for the clinical outcomes.Methods: We retrospectively analyzed bone marrow biopsy findings 14 days after NK cell infusion in 109 patients treated at our institution between 2003-2014. To evaluate the homing and persistence of NK cell in the bone marrow, multiple methods were used. Immunohistochemical staining for the NK cell marker CD56 was performed on the core biopsy in available cases. Flow cytometry data performed on aspirate material at the time of the biopsy was re-evaluated to enumerate the NK cell percentage. Finally the percentage of Large Granular Lymphocytes was determined in a semi-quantitative manner from Wright-Giemsa stained aspirate smears. CD56 immunohistochemical stain was quantified by counting the number of CD56 positive cells per microscopic field of view (High Power Field, HPF) (400 x magnifications) and averaging over the 10 microscopic fields with the highest density of CD56 positive cells. Cases with obvious CD56 staining of blasts on the trephine core were excluded from further analysis.Results: 95 bone marrow biopsy cases could be stained for CD56, but 6 cases were excluded from analysis due to CD56 staining leukemic blast population. For the whole study group the average CD56+ cell density per High Power Field (HPF) was 10.1 (SD=17). A control group composed of 10 patients with bone marrow biopsy shortly after chemotherapy, but who did not receive NK cell infusion had an average of 3.8 CD56+ cells per HPF (SD=3.4).Patients who had a higher density of NK cells in the bone marrow compared to the control group (more than 3.8 cells/HPF) were less likely to have persistence of acute leukemia as defined by ≥5% blasts, than patients with an NK cell density similar to the control group (Figure 1, Chi-square test, p=0.037). As well, patients with <5% leukemic blasts had more CD56 positive cells per HPF (Median 5.6, range 0.1-95.8 Versus median 1.8, range 0.1-28.1, p=0.009, Mann-Whitney test) and had a greater proportion of NK cells by flow cytometry (p=0.004, Mann-Whitney test).The clinical factors which may affect NK cell homing and persistence were also evaluated. Total Body Irradiation (TBI) at a dose 200 cGy for 2 days was applied as part of pre-infusion conditioning in a subset of the patients. Patients who received TBI (32 with TBI Vs 55 without) had a significantly higher density of CD56 positive cells (Figure 2, Median 4.75Vs 2.8, p=0.04, Mann-Whitney test). Among those patients who did not receive TBI conditioning, a subset received an anti-CD25 immunotoxin agent to reduce regulatory T cells (Denileukin Difitox, Ontak). The density of NK cells was significantly higher in the patients who received anti-CD25 immunotoxin (Figure 3, Median 9.8 Vs 2.5, p=0.02, Mann-Whitney test).Conclusions: Homing and survival/expansion of adoptively transferred NK cells in the bone marrow 14 days after infusion correlates with morphologic remission of acute leukemia. While the factors influencing NK cell homing and persistence in the BM niche remain to be elucidated, our results suggest a role for TBI as part of the conditioning and anti-CD25 immunotoxin in promoting the persistence of adoptively transferred NK cells in the bone marrow niche. [Display omitted] DisclosuresMiller:Oxis Biotech: Consultancy; Fate Therapeutics: Consultancy, Research Funding; Celegene: Consultancy.

Low Abundance of Lactococcus lactis in Human Colorectal Cancer Is Associated with Decreased Natural Killer Cells

A limited number of studies have demonstrated the role of Lactococcus lactis (L. lactis) in human colorectal cancers (CRCs). The association of L. lactis abundance with the density of natural killer (NK) cells has not been investigated before. In this study, the L. lactis abundance in 60 CRC specimens, 20 adenoma (AD) specimens, and 29 normal colorectal tissues (NCs) specimens was investigated using the fluorescence in situ hybridization of 16S ribosomal RNA. The density of NK cells was detected using immunofluorescence in 28 CRC specimens, 12 AD specimens, and 22 NC specimens. The presence of L. lactis in NCs (48.28%) was detected significantly higher than that in the AD (20.00%, P = .044) and CRC (23.33%, P = .018) specimens. The abundance of L. lactis in NCs (32.73 ± 7.24) was also found to be significantly higher than that in AD (8.91 ± 5.89, P = .029) and CRC (5.63 ± 1.67, P = .003) specimens. In addition, the density of NKp30+ NK cells in NCs (51.14 ± 4.84) was significantly higher than that in the AD (6.10 ± 1.31) and CRC (1.72 ± 0.40) specimens (P < .001). Moreover, a positive association of L. lactis abundance with NKp30+ NK cells density in the colorectal samples (P < .001) was observed. The low abundance of L. lactis in the CRC tissues was associated with the decreased NK cells, which suggested that this might contribute to the progression of CRC by decreasing the number of NK cells. Supplemental data for this article is available online at https://doi.org/10.1080/01635581.2021.1944649

Do endometrial natural killer and regulatory T cells differ in infertile and clinical pregnancy patients? An analysis in patients undergoing frozen embryo transfer cycles.

Clinical significance of endometrial and peripheral blood natural killer (NK) and regulatory T cells (Tregs) during frozen embryo transfer (FET) cycles has not been well characterized. Retrospective cohort study. Endometrial tissue was collected from infertility patients prior to a frozen embryo transfer cycle as part of an endometrial receptivity analysis (ERA® ) biopsy or endometrial scratch test. Uterine NK (uNK) and Treg cell density was compared based on pregnancy status in the subsequent frozen embryo transfer cycle. Peripheral blood was also collected from a separate cohort of patients undergoing frozen embryo transfer. Treg cell density was compared by the presence or the absence of a clinical pregnancy in each phase of the cycle. In the 33 luteal phase biopsies there were more endometrial Tregs, similar uNK and a trend toward lower CD16+ uNK cells in women with a future ongoing clinical pregnancy compared to non-pregnant women. There were no differences in uNK and Treg density in natural scratch cycles vs programmed cycles or in non-receptive vs receptive endometrium (ERA® cycles). In the peripheral blood analysis, the pregnant group had higher peripheral blood Tregs on the day of serum β-hCG time point when compared to the non-pregnant group. Higher levels of endometrial Tregs and lower levels of CD16+ uNK cells are positive prognostic factors for infertile women prior to frozen embryo transfer. Our work on phenotypic and proportional analyses of endometrial immune cells may complement the ERA® in predicting improved pregnancy rates in patients with implantation failure.

DO ENDOMETRIAL NATURAL KILLER AND REGULATORY T CELLS DIFFER IN INFERTILE AND CLINICAL PREGNANCY PATIENTS? AN ANALYSIS IN PATIENTS UNDERGOING FROZEN EMBRYO TRANSFER CYCLES

Problem Clinical significance of endometrial and peripheral blood natural killer (NK) and regulatory T cells (Tregs) during frozen embryo transfer (FET) cycles has not been well characterized. Design Retrospective cohort study METHOD OF STUDY: Endometrial tissue was collected from infertility patients prior to a frozen embryo transfer cycle as part of an endometrial receptivity analysis (ERA® ) biopsy or endometrial scratch test. Uterine NK (uNK) and Treg cell density was compared based on pregnancy status in the subsequent frozen embryo transfer cycle. Peripheral blood was also collected from a separate cohort of patients undergoing frozen embryo transfer. Treg cell density was compared by the presence or the absence of a clinical pregnancy in each phase of the cycle. Results In the 33 luteal phase biopsies there were more endometrial Tregs, similar uNK and a trend toward lower CD16+ uNK cells in women with a future ongoing clinical pregnancy compared to non-pregnant women. There were no differences in uNK and Treg density in natural scratch cycles vs programmed cycles or in non-receptive vs receptive endometrium (ERA® cycles). In the peripheral blood analysis, the pregnant group had higher peripheral blood Tregs on the day of serum β-hCG time point when compared to the non-pregnant group. Conclusion Higher levels of endometrial Tregs and lower levels of CD16+ uNK cells are positive prognostic factors for infertile women prior to frozen embryo transfer. Our work on phenotypic and proportional analyses of endometrial immune cells may complement the ERA® in predicting improved pregnancy rates in patients with implantation failure.

The use of multiplex staining to measure the density and clustering of four endometrial immune cells around the implantation period in women with recurrent miscarriage: comparison with fertile controls.

Serval studies showed an increased uterine natural killer cell density in women with recurrent miscarriage. However, no study has previously investigated the density and clustering of major immune cells simultaneously in precisely timed endometrial specimen section of this group of women. This study aimed to investigate the profile of endometrial immune cells populations and clustering level simultaneously in women with recurrent miscarriage and compare the results to fertile controls. A total of 30 women with unexplained recurrent miscarriage and 30 fertile controls were included in this study. Endometrial biopsy was performed precisely 7days after LH surge. The cells density was expressed as percentage of positive immune cell/total stromal cells and the clustering of different endometrial cells was measured by R language toolbox 'spatstat'. Multiplex immunohistochemical method was employed to stain a panel of human endometrium samples simultaneously with antibodies against CD3 for T cells, CD20 for B cells, CD68 for macrophages and CD56 for uterine natural killer cells. The median CD3+, CD68+ and CD56+ cell density in the miscarriage group were significantly higher than those of the fertile controls. In addition, the clustering between CD56+ uterine natural killer cells and CD68+ macrophages in the miscarriage group was significantly increased compared with fertile controls. In conclusion, the significant change in numbers of three out of four endometrial immune cell density and a significant increase in clustering between CD68+ and CD56+ cells suggest that several immune cells and their interactions may be important in the function of the endometrium; abnormal interactions may predispose to recurrent miscarriage.

Intralipid treatment for women with reproductive failures.

Does intravenous intralipid treatment for reproductive failure enhance live births? The answer to this question is yes, BUT only in patients that have a diagnosis of recurrent implantation failure (RIF) or recurrent pregnancy loss (RPL) loss AND demonstrate elevated NK (natural killer) cell density in their endometrial biopsy. Live birth rates have been reported between 33% and 42% among women displaying elevated NK activity with a diagnosis of RIF and 75% and 91% among women experiencing RPL after intralipid infusion. When the pregnancy outcomes of women with a history of reproductive failure and elevated NK cells treated with intralipid were evaluated, the overall live birth rate per cycle of treatment was 61%. The results of published studies suggest that intralipid can be used successfully as a therapeutic option to modulate abnormal NK activity in women with reproductive problems.

Association between chronic endometritis and uterine natural killer cell density in women with recurrent miscarriage: clinical implications.

This aim of this study was to determine the association between uterine natural killer (uNK) cell density and chronic endometritis (CE). Endometrial biopsies from 135 women with recurrent miscarriage were obtained precisely 7 days after luteinizing hormone surge in natural cycles. Endometrial sections were immunostained for CD56 for uNK cells and CD138 for plasma cells, respectively. Uterine NK cell counting was performed according to a standardized protocol and results were expressed as percentage of CD56+ cells/ total stromal cells. High uNK cell density was defined as >4.5% and CE was diagnosed when the plasma cell density > 5.15 cells/ 10 mm2 . The uNK cells density in women with CE (median, 5.1%; range, 3.4-8.8%) was significantly (P < 0.05) higher than that of those without CE (median, 3.8%; range, 1.2%-7.3%). The prevalence of high uNK cell density in women with CE (11/29, 37.9%) was significantly (P < 0.05) higher than that of women without CE (8/106, 7.5%). To conclude, there was a significant association between high uNK cell density and CE. In women with high uNK cell density, plasma cell should be examined to determine if the underlying cause is associated with CE.

Influence of the Tumor Microenvironment on NK Cell Function in Solid Tumors.

Natural killer (NK) cells are a population of innate lymphoid cells playing a pivotal role in host immune responses against infection and tumor growth. These cells have a powerful cytotoxic activity orchestrated by an intricate network of inhibitory and activating signals. The importance of NK cells in controlling tumor growth and in mediating a robust anti-metastatic effect has been demonstrated in different experimental mouse cancer models. Consistently, high density of tumor-infiltrating NK cells has been linked with a good prognosis in multiple human solid tumors. However, there are also tumors that appear to be refractory to NK cell-mediated killing for the presence of an immunosuppressive microenvironment affecting NK cell function. Immunotherapeutic strategies aimed at restoring and increasing the cytotoxic activity of NK cells in solid tumors, including the adoptive transfer of NK and CAR-NK cells, are currently employed in preclinical and clinical studies. In this review, we outline recent advances supporting the direct role of NK cells in controlling expansion of solid tumors and their prognostic value in human cancers. We summarize the mechanisms adopted by cancer cells and the tumor microenvironment to affect NK cell function, and finally we evaluate current strategies to augment the antitumor function of NK cells for the treatment of solid tumors.

CD56+CD57+ infiltrates as the most predominant subset of intragraft natural killer cells in renal transplant biopsies with antibody-mediated rejection

Little is known about the characteristics and clinical implications of specific subsets of intragraft natural killer (NK) cells in kidney transplant recipients. We analyzed 39 for-cause renal transplant biopsies performed at our center from May 2015 to July 2017. According to histopathologic reports, 8 patients (20.5%) had no rejection (NR), 11 (28.2%) had T cell-mediated rejections (TCMR) only, and 20 (51.3%) had antibody-mediated rejection (ABMR). NK cells were defined as CD3–CD56+ lymphocytes that are positive for CD57, CD49b, NKG2A, or KIR. The density of NK cells was significantly higher in the ABMR group (2.57 ± 2.58/mm2) than in the NR (0.12 ± 0.22/mm2) or the TCMR (0.25 ± 0.34/mm2) group (P = 0.002). Notably, CD56+CD57+ infiltrates (2.16 ± 1.89) were the most frequently observed compared with CD56+CD49b+ (0.05 ± 0.13), CD56+NKG2A+ (0.21 ± 0.69), and CD56+KIR+ (0.15 ± 0.42) cells in the ABMR group (P < 0.001). Death-censored graft failure was significantly higher in patients with NK cell infiltration than those without (Log-rank test, P = 0.025). In conclusion, CD56+CD57+ infiltrates are a major subset of NK cells in kidney transplant recipients with ABMR and NK cell infiltration is significantly associated with graft failure post-transplant.

Integrated molecular and immunophenotypic analysis of NK cells in anti-PD-1 treated metastatic melanoma patients

ABSTRACTPurpose: Anti-PD-1 therapy has revolutionized the treatment and improved the survival of stage IV melanoma patients. However, almost half of the patients fail to respond due to immune evasive mechanism. A known mechanism is the downregulation of major histocompatibility complex (MHC) class I expression, which prevents T cell recognition of the tumor. This study determined the relationship between natural killer (NK) cell numbers and clinical response to anti-PD-1 therapy in metastatic melanoma.Experimental Design: Twenty-five anti-PD-1 treated metastatic melanoma patients were categorized into responders (complete response (CR)/partial response (PR)/stable disease (SD) ≥ 6 mo, n = 13) and non-responders (SD < 6 days/progressive disease (PD), n = 12) based on RECIST response. Whole transcriptome sequencing and multiplex immunofluorescent staining were performed on pre-treatment and on a subset of early during treatment tumor samples. Spatial distribution analysis was performed on multiplex immunofluorescent images to determine the proximity of NK cells to tumor cells. Flow cytometry was used to confirm NK phenotypes in lymph node metastases of treatment naïve melanoma patients (n = 5). Cytotoxic assay was performed using NK cells treated with anti-PD-1 or with isotype control and co-cultured with 3 different melanoma cell lines and with K562 cells (leukemia cell line).Results: Differential expression analysis identified nine upregulated NK cell specific genes (adjusted p < 0.05) in responding (n = 11) versus non-responding patients (n = 10). Immunofluorescent staining of biopsies confirmed a significantly higher density of intra- and peri-tumoral CD16+ and granzyme B + NK cells in responding patients (p < 0.05). Interestingly, NK cells were in closer proximity to tumor cells in responding PD-1 treated patients compared to non-responding patients. Patients who responded to anti-PD-1 therapy, despite MHC class I loss had higher NK cell densities than patients with low MHC class I expression. Lastly, functional assays demonstrated PD-1 blockade induces an increase in NK cells’ cytotoxicity.Conclusions: A higher density of tumoral NK cells is associated with response to anti-PD-1 therapy. NK cells may play an important role in mediating response to anti-PD-1 therapy, including in a subset of tumors downregulating MHC class I expression.

Uterine natural killer cell density as a predictor for implantation success or failure in fertile surrogates and in women with implantation failure

Uterine natural killer cell density as a predictor for implantation success or failure in fertile surrogates and in women with implantation failure

PO-414 Stage IV melanoma patients with tumoural MHC class i loss only respond to anti-PD-1 therapy in the presence of high NK cell density

IntroductionPrior to 2011 there were no effective systemic therapies for advanced stage melanoma patients and the overall survival was 7.5 months. Recently, immune checkpoint inhibitors, anti-PD-1 and anti-CTLA-4 inhibitors, have produced response rates as high as 50% and doubled overall survival, but only 30% of patients have a durable response. One important resistance mechanism to immunotherapies is the downregulation of MHC Class I, resulting in the expansion of melanoma cells resistant to CD8 +T cell killing. Natural Killer (NK) cells monitor MHC Class I expression and eliminate cells that fail to express it; thus, NK cells are likely critical in preventing resistance to anti-PD-1 therapy. This study sought to investigate whether tumour-infiltrating NK cells in the presence of MHC class I loss improved survival outcome of patients treated with anti-PD-1.Material and methodsTwenty-five stage IV metastatic melanoma patients treated with anti-PD-1 therapy were categorised into responders (CR/PR/SD >6 mo, n=13) and non-responders (SD <6 mo/PD, n=12) based on RECIST response. Whole transcriptome sequencing and multiplex immunofluorescent staining were performed on formalin-fixed-paraffin embedded pre-treatment tumour samples. Flow cytometry was used to confirm novel NK phenotypes in melanoma lymph node metastases of treatment naïve stage IV melanoma patients (n=5).Results and discussionsDifferential expression analysis identified nine up-regulated NK cell specific genes in responders when compared to non-responders (adjusted p<0.05). Immunofluorescent staining of biopsies confirmed a significantly higher density of intratumoural and peritumoural CD16+ (intratumoural p=0.0015 and peritumoural p=0.0039) and granzyme B+ (intratumoural p=0.019 and peritumoural p=0.011) NK cells in responding patients. Flow cytometry demonstrated 46%±8% of the NK cells were identified as expressing PD-1. When stage IV melanoma patients were further stratified by MHC class I loss, responding patients with MHC class I loss, had a higher NK cell density and better survival outcome, compared to non-responders (p=0.012).ConclusionThis study showed that the presence of higher numbers of NK cells are associated with improved responses to anti-PD-1 therapy. Most importantly, responding patients with MHC class I loss had higher NK cell densities, suggesting that NK cells play an important role in mediating response to anti-PD-1 in patients whose tumour down regulates MHC class I expression.

Complex role of NK cells in regulation of oncolytic virus–bortezomib therapy

In the present work, we investigated the role of natural killer (NK) cells in combination therapy with oncolytic virus (OV) and bortezomib, a proteasome inhibitor. NK cells display rapid and potent immunity to metastatic and hematological cancers, and they overcome immunosuppressive effects of tumor microenvironment. We developed a mathematical model to address the question of how the density of NK cells affects the growth of the tumor. We found that the antitumor efficacy increases when the endogenous NKs are depleted and also when exogenous NK cells are injected into the tumor. These predictions were validated by our in vivo and in vitro experiments.

Detailed analysis of inflammatory cell infiltration and the prognostic impact on nasopharyngeal carcinoma.

One of the most striking characteristics of nasopharyngeal carcinoma (NPC) is the presence of a very abundant immune cells infiltrate containing mainly T-lymphocytes. The purpose of this study was to present our analysis providing a comprehensive characterization of antitumor inflammatory response in NPC. The densities of 9 types of inflammatory cells were assessed in 197 patients with NPC, including CD3 + T-lymphocytes, CD8 + cytotoxic T-lymphocytes, CD20 + B-lymphocytes, CD56 + natural killer (NK) cells, FOXP3 + regulatory T-lymphocytes, CD1a + immature dendritic cells, CD83 + mature dendritic cells, neutrophil elastase + neutrophils, and tryptase + mast cells. We characterized the inflammatory infiltrate in relation to clinical stage and patient survival. The expression of programmed death-1 (PD-1) on tumor-infiltrating lymphocytes (TILs) was also detected. The correlations between PD-1 expression and clinical characteristics and posttreatment outcome were analyzed. The patients with NPC with a low density of tumor-infiltrating FOXP3+, CD8 + T-lymphocytes, neutrophils, and mast cells showed a significantly longer overall survival (OS) and progression-free survival (PFS). However, patients with a high density of NK cells showed a better OS and PFS. The densities of NK cells and mast cells could be served as biomarkers for predicting recurrence or distant metastasis in patients with NPC. Moreover, PD-1 positivity predicted poor prognosis in patients with NPC. The densities of inflammatory cells are correlated with the prognosis of patients with NPC.

Activated human primary NK cells efficiently kill colorectal cancer cells in 3D spheroid cultures irrespectively of the level of PD-L1 expression

ABSTRACT Haploidentical Natural Killer (NK) cells have been shown as an effective and safe alternative for the treatment of haematological malignancies with poor prognosis for which traditional therapies are ineffective. In contrast to haematological cancer cells, that mainly grow as single suspension cells, solid carcinomas are characterised by a tridimensional (3D) architecture that provide specific surviving advantages and resistance against chemo- and radiotherapy. However, little is known about the impact of 3D growth on solid cancer immunotherapy especially adoptive NK cell transfer. We have recently developed a protocol to activate ex vivo human primary NK cells using B lymphoblastic cell lines, which generates NK cells able to overcome chemoresistance in haematological cancer cells. Here we have analysed the activity of these allogeneic NK cells against colorectal (CRC) human cell lines growing in 3D spheroid culture and correlated with the expression of some of the main ligands regulating NK cell activity. Our results indicate that activated NK cells efficiently kill colorectal tumour cell spheroids in both 2D and 3D cultures. Notably, although 3D CRC cell cultures favoured the expression of the inhibitory immune checkpoint PD-L1, it did not correlate with increased resistance to NK cells. Finally, we have analysed in detail the infiltration of NK cells in 3D spheroids by microscopy and found that at low NK cell density, cell death is not observed although NK cells are able to infiltrate into the spheroid. In contrast, higher densities promote tumoural cell death before infiltration can be detected. These findings show that highly dense activated human primary NK cells efficiently kill colorectal carcinoma cells growing in 3D cultures independently of PD-L1 expression and suggest that the use of allogeneic activated NK cells could be beneficial for the treatment of colorectal carcinoma.