Intratumoral Natural Killer Cells Research Articles

Cordycepin Augments the Efficacy of Anti-PD1 against Colon Cancer

Colon cancer has a poor clinical response to anti-PD1 therapy. This study aimed to evaluate the effect of cordycepin on the efficacy of anti-PD1 treatment in colon cancer. The viability of CT26 mouse colon carcinoma cells, cell-cycle progression, morphology, and the expression of mRNA and protein were assessed. A syngeneic animal model was established by implanting CT26 cells into BALB/c mice for in vivo experiments. Multi-parameter flow cytometry was used to analyze the splenic cell lineages and tumor microenvironment (TME). The in vitro data revealed that cordycepin, but not adenosine, inhibited CT26 cell viability. The protein, but not mRNA, expression levels of A2AR and A2BR were suppressed by cordycepin but not by adenosine in CT26 cells. The combination of cordycepin, but not adenosine, with anti-PD1 exhibited a greater tumor-inhibitory effect than anti-PD1 alone as well as inhibited the expression of A2AR and A2BR in splenic macrophages. In the TME, the combination of cordycepin and anti-PD1 increased the number of CD3+ T cells and neutrophils and decreased the number of natural killer (NK) cells. Overall, cordycepin augmented the antitumor effects of anti-PD1 against mouse colon carcinoma cells and inhibited the expression of the adenosine receptors A2AR and A2BR in splenic macrophages and intratumoral NK cells.

The E3 ubiquitin ligase FBXO38 maintains the antitumor function of natural killer cells by sustaining IL-15R signaling.

Natural killer (NK) cells are the main innate antitumor effector cells but their function is often constrained in the tumor microenvironment (TME). It has been reported that the E3 ligase FBXO38 accelerates PD-1 degradation in tumor-infiltrating T cells to unleash their cytotoxic function. In this study, we found that the transcriptional levels of FBXO38 in intratumoral NK cells of cancer patients and tumor-bearing mice were significantly lower than in peritumoral NK cells. Conditional knock-out (cKO) of FBXO38 in NK cells accelerated tumor growth and increased tumor metastasis. FBXO38 deficiency resulted in impaired proliferation and survival of tumor-infiltrating NK (TINK) cells. Mechanistically, FBXO38 deficiency enhanced TGF-β signaling, including elevating expression of Smad2 and Smad3, which suppressed expression of the transcription factor Eomes and further reduced expression of surface IL-15Rβ and IL-15Rγc on NK cells. Consequently, FBXO38 deficiency led to TINK cell hyporesponsiveness to IL-15. Consistent with these observations, FBXO38 mRNA expression was positively correlated with the proliferation of TINK cells in multiple human tumors. To study the therapeutic potential of FBXO38, mice bearing human tumors were treated with FBXO38 overexpressed human primary NK cells and showed a significant reduction in tumor size and prolonged survival. In conclusion, our results suggest that FBXO38 sustains NK-cell expansion and survival to promote antitumor immunity, and have potential therapeutic implications as they suggest FBXO38 could be harnessed to enhance NK cell-based cancer immunotherapy.

Blockade of CD300A enhances the ability of human NK cells to lyse hematologic malignancies.

The human cluster of differentiation (CD)300A, a type-I transmembrane protein with immunoreceptor tyrosine-based inhibitory motifs, was investigated as a potential immune checkpoint for human natural killer (NK) cells targeting hematologic malignancies (HMs). We implemented a stimulation system involving the CD300A ligand, phosphatidylserine (PS), exposed to the outer surface of malignant cells. Additionally, we utilized CD300A overexpression, a CD300A blocking system, and a xenotransplantation model to evaluate the impact of CD300A on NK cell efficacy against HMs in in vitro and in vivo settings. Furthermore, we explored the association between CD300A and HM progression in patients. Our findings indicated that PS hampers the function of NK cells. Increased CD300A expression inhibited HM lysis by NK cells. CD300A overexpression shortened the survival of HM-xenografted mice by impairing transplanted NK cells. Blocking PS-CD300A signals with antibodies significantly amplified the expression of lysis function-related proteins and effector cytokines in NK cells, thereby augmenting the ability to lyse HMs. Clinically, heightened CD300A expression correlated with shorter survival and an "exhausted" phenotype of intratumoral NK cells in patients with HMs or solid tumors. These results propose CD300A as a potential target for invigorating NK cell-based treatments against HMs.

Recombinant Newcastle disease viruses expressing immunological checkpoint inhibitors induce a pro-inflammatory state and enhance tumor-specific immune responses in two murine models of cancer.

Tumor microenvironments are immunosuppressive due to progressive accumulation of mutations in cancer cells that can drive expression of a range of inhibitory ligands and cytokines, and recruitment of immunomodulatory cells, including myeloid-derived suppressor cells (MDSC), tumor-associated macrophages, and regulatory T cells (Tregs). To reverse this immunosuppression, we engineered mesogenic Newcastle disease virus (NDV) to express immunological checkpoint inhibitors anti-cytotoxic T lymphocyte antigen-4 and soluble programmed death protein-1. Intratumoral administration of recombinant NDV (rNDV) to mice bearing intradermal B16-F10 melanomas or subcutaneous CT26LacZ colon carcinomas led to significant changes in the tumor-infiltrating lymphocyte profiles. Vectorizing immunological checkpoint inhibitors in NDV increased activation of intratumoral natural killer cells and cytotoxic T cells and decreased Tregs and MDSCs, suggesting induction of a pro-inflammatory state with greater infiltration of activated CD8+ T cells. These notable changes translated to higher ratios of activated effector/suppressor tumor-infiltrating lymphocytes in both cancer models, which is a promising prognostic marker. Whereas all rNDV-treated groups showed evidence of tumor regression and increased survival in the CT26LacZ and B16-F10, only treatment with NDV expressing immunological checkpoint blockades led to complete responses compared to tumors treated with NDV only. These data demonstrated that NDV expressing immunological checkpoint inhibitors could reverse the immunosuppressive state of tumor microenvironments and enhance tumor-specific T cell responses.

Characterization of natural killer and cytotoxic T-cell immune infiltrates in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor response to systemic therapies, including immunotherapy. Given the immunotherapeutic potential of natural killer (NK) cells, we evaluated intratumoral NK cell infiltrates along with cytotoxic T cells in PDAC to determine their association with patient outcomes. We analyzed tumors from 93 PDAC patients treated from 2012 to 2020. Predictor variables included tumor-infiltrating lymphocytes (TILs), T-cell markers (CD3, CD8, CD45RO), NK marker (NKp46), and NK inhibitory marker (major histocompatibility complexclass I[MHC-I]) by immunohistochemistry. Primary outcome variables were recurrence-free survival (RFS) and overall survival (OS). Mean TILs, CD3, and NKp46 scores were 1.3 ± 0.63, 20.6 ± 17.5, and 3.1 ± 3.9, respectively. Higher expression of CD3 and CD8 was associated with higher OS, whereas NK cell infiltration was not associated with either RFS or OS. There was a tight positive correlation between MHC-I expression and all T-cell markers, but not with NKp46. Overall NK cell infiltrates were low in PDAC and did not predict clinical outcomes, whereas T-cell infiltrates did. Further characterization of the immune infiltrate in PDAC, including inhibitory signals and suppressive cell types, may yield better biomarkers of prognosis and immune targeting in this refractory disease.

A Pan-Cancer Single-Cell Transcriptomic Atlas of Natural Killer (NK) Cells Reveals Intrinsic and Extrinsic Mediators of NK Cell Anti-Tumor Functions

Background: Natural killer (NK) cells are critical mediators of anti-tumor immunity, engaging in direct cytotoxic responses and promoting pro-inflammatory immune microenvironments. The molecular mechanisms underlying these functions, however, have yet to be fully elucidated. This is due in part to a lack of understanding of the phenotypic and functional diversity of NK cells across cancer types. Here, we present a single-cell transcriptomic atlas of >250,000 NK cells spanning 340 patients across 17 cancer types to advance our understanding of tumor-infiltrating NK cells. Methods: We re-analyzed publicly available single-cell RNA-sequencing (scRNA-seq) datasets of primary, therapy-naïve tumors, as well as matching peripheral blood samples and healthy donor blood samples. We employed systematic computational analyses to deconvolute the phenotypic and functional diversity of tumor-infiltrating NK cells and define key regulators of NK cell functions in both a cancer type-specific and pan-cancer manner. Feature expression module scoring was used to infer phenotypic and functional characteristics of NK cell clusters in each cancer type. Transcription factor regulon and cell-cell communication prediction algorithms were applied to understand the mechanistic drivers of NK cell populations within tumors. Results: Tumor-infiltrating NK cells display distinct patterns of phenotypic and functional features across cancer types. Nonetheless, we identify consensus NK cell populations across cancers that demonstrate the utility and performance of our gene module-based phenotypic and functional annotations. Interestingly, we observe the enrichment of adaptive-like and CD56 bright NK cell populations in tumors that exhibit features of tissue residency, including the upregulation of ITGA1, ITGAE, and CD69 alongside the downregulation of FCG3RA. Notably, these tissue-resident populations display enhanced chemokine and cytokine activity compared to their recruited counterparts, suggesting the significance of tissue-resident NK cell populations in immune recruitment to tumors. We find the phenotypic composition of intra-tumoral NK cells is vastly different than that of circulating NK cells which harbor predominantly CD56 dim populations, as well as a significant proportion of adaptive-like NK cells in several cancer types. Leveraging the paired nature of several tumor and peripheral blood samples, we performed temporal comparative analyses that revealed the robust expression of KLF2 and S1PR5 in circulation which is then lost in paired tumor tissues. While KLF2 and S1PR5 have been previously implicated in the homing and migration of immune cells, their roles in the trafficking of NK cells to tumors have yet to be defined. In modeling transitions from canonical to stressed NK cell states, we not only identify well-established NK cell checkpoints ( TIM-3, LAG3, and CISH) but also identify novel putative checkpoints yet to be investigated in NK cells, such as the AP-1 family of transcription factors. Additionally, we examine extrinsic factors contributing to intra-tumoral NK cell phenotypes, detecting significant crosstalk between NK cells and immunomodulatory macrophages and CD4+ T cells mediated by galectin-9 and TGFβ interactions. Conclusions: We define the phenotypic and functional pan-cancer landscape of NK cells and uncover both intrinsic and extrinsic factors influencing their anti-tumor functions. We envision our NK cell atlas will facilitate the development of strategies to enhance NK cell-based immunotherapies against cancer.

S1914 Salmonella Abundance Associated with Disease Progression in Stomach Adenocarcinoma and Decreased Intratumoral Natural Killer Cell Infiltrate

S1914 Salmonella Abundance Associated with Disease Progression in Stomach Adenocarcinoma and Decreased Intratumoral Natural Killer Cell Infiltrate

Intratumoral NKp46+ natural killer cells are spatially distanced from T and MHC-I+ cells with prognostic implications in soft tissue sarcoma.

Soft tissue sarcomas (STS) are rare, heterogenous malignancies with an unmet need for novel immunotherapies. Tumor infiltrating lymphocytes (TILs) have been linked with favorable outcomes in STS patients, though the contribution of natural killer (NK) cells and spatial relationships of TILs with MHC-I expressing cells lacks detailed characterization. Using archived and prospectively collected specimens, we evaluated intratumoral NK cells by immunohistochemistry (IHC), flow cytometry, and immunofluorescence (IF). We assessed spatial localization of NK and T cells by multiplex IF, analyzing the effects of MHC-I expression status on NK and T cell clustering. Both intratumoral NKp46 and CD56dim expression were associated with significantly improved overall survival (P=0.05), while higher infiltrates of CD56bright NK cells predicted a worse prognosis (P=0.05). The presence of intratumoral NK cells was inversely proportional to CD3+ T cells. Spatial analyses showed NK cells preferentially clustering close to other NK cells with sparse CD3+ T and CD8+ T cells in range (P<0.0001). Additionally, CD3+ T and CD8+ T cells showed significantly greater co-localization with MHC-I+ cells, compared to NK cells (P<0.0001). After neoadjuvant radiotherapy, there was greater CD8 clustering, while after neoadjuvant chemotherapy, there was overall lower TIL clustering. Intratumoral NK cells are prognostic in STS and localize closer to MHC-I- cells than T cells. Although both NK and T cells are associated with improved survival in STS, their differential distribution in the TME based on MHC-I expression status may serve as a biomarker for improved immunotherapy treatment selection.

Tumors evade immune cytotoxicity by altering the surface topology of NK cells.

The highly variable response rates to immunotherapies underscore our limited knowledge about how tumors can manipulate immune cells. Here the membrane topology of natural killer (NK) cells from patients with liver cancer showed that intratumoral NK cells have fewer membrane protrusions compared with liver NK cells outside tumors and with peripheral NK cells. Dysregulation of these protrusions prevented intratumoral NK cells from recognizing tumor cells, from forming lytic immunological synapses and from killing tumor cells. The membranes of intratumoral NK cells have altered sphingomyelin (SM) content and dysregulated serine metabolism in tumors contributed to the decrease in SM levels of intratumoral NK cells. Inhibition of SM biosynthesis in peripheral NK cells phenocopied the disrupted membrane topology and cytotoxicity of the intratumoral NK cells. Targeting sphingomyelinase confers powerful antitumor efficacy, both as a monotherapy and as a combination therapy with checkpoint blockade.

A new natural killer cell-specific gene signature predicting recurrence in colorectal cancer patients.

The protective role of Natural Killer (NK) cell tumour immunosurveillance has long been recognised in colorectal cancer (CRC). However, as most patients show limited intra-tumoral NK cell infiltration, improving our ability to identify those with high NK cell activity might aid in dissecting the molecular features which underlie NK cell sensitivity. Here, a novel CRC-specific NK cell gene signature that infers NK cell load in primary tissue samples was derived and validated in multiple patient CRC cohorts. In contrast with other NK cell gene signatures that have several overlapping genes across different immune cell types, our NK cell signature has been extensively refined to be specific for CRC-infiltrating NK cells. The specificity of the signature is substantiated in tumour-infiltrating NK cells from primary CRC tumours at the single cell level, and the signature includes genes representative of NK cells of different maturation states, activation status and anatomical origin. Our signature also accurately discriminates murine NK cells, demonstrating the applicability of this geneset when mining datasets generated from preclinical studies. Differential gene expression analysis revealed tumour-intrinsic features associated with NK cell inclusion versus exclusion in CRC patients, with those tumours with predicted high NK activity showing strong evidence of enhanced chemotactic and cytotoxic transcriptional programs. Furthermore, survival modelling indicated that NK signature expression is associated with improved survival outcomes in CRC patients. Thus, scoring CRC samples with this refined NK cell signature might aid in identifying patients with high NK cell activity who could be prime candidates for NK cell directed immunotherapies.

The Latest Approach of Immunotherapy with Endosomal TLR Agonists Improving NK Cell Function: An Overview.

Toll-like receptors (TLRs) are the most well-defined pattern recognition receptors (PRR) of several cell types recognizing pathogens and triggering innate immunity. TLRs are also expressed on tumor cells and tumor microenvironment (TME) cells, including natural killer (NK) cells. Cell surface TLRs primarily recognize extracellular ligands from bacteria and fungi, while endosomal TLRs recognize microbial DNA or RNA. TLR engagement activates intracellular pathways leading to the activation of transcription factors regulating gene expression of several inflammatory molecules. Endosomal TLR agonists may be considered as new immunotherapeutic adjuvants for dendritic cell (DC) vaccines able to improve anti-tumor immunity and cancer patient outcomes. The literature suggests that endosomal TLR agonists modify TME on murine models and human cancer (clinical trials), providing evidence that locally infused endosomal TLR agonists may delay tumor growth and induce tumor regression. Recently, our group demonstrated that CD56bright NK cell subset is selectively responsive to TLR8 engagement. Thus, TLR8 agonists (loaded or not to nanoparticles or other carriers) can be considered a novel strategy able to promote anti-tumor immunity. TLR8 agonists can be used to activate and expand in vitro circulating or intra-tumoral NK cells to be adoptively transferred into patients.

Downregulation of AT-rich interaction domain 2 underliesnatural killer cell dysfunction in oral squamouscell carcinoma.

The immune system plays a significant role in controlling oral squamous cell carcinoma (OSCC) initiation and progression. Natural killer (NK) cells actively participate in antitumor immunity but become dysfunctional or exhausted in the tumor microenvironment. To explore the mechanisms of NK cell dysfunction in OSCC, we characterized the expression and function of AT-rich interaction domain 2 (ARID2) in NK cells in a murine OSCC model. ARID2 was downregulated in tongue NK cells compared with splenic NK cells. Notably, ARID2 was significantly decreased in NK cells with an exhausted phenotype and weakened antitumor function. ARID2 knockdown resulted in the upregulation of programmed cell death protein 1 (PD-1) and downregulation of interferon-gamma (IFN-γ), tumor necrosis factor (TNF), granzyme B and perforin in NK cells. As a result, ARID2 knockdown impaired NK cell cytotoxicity. Besides, ARID2 overexpression suppressed the expression of PD-1 and lymphocyte-activation gene 3, and promoted the expression of IFN-γ, TNF, granzyme B and perforin in NK cells which were adoptively transferred into OSCC-bearing mice. Taken together, our study implies that the OSCC microenvironment triggers ARID2 downregulation in intratumoral NK cells. In turn, ARID2 downregulation results in PD-1 upregulation on NK cells and subsequently impairs NK cell cytotoxicity. Therefore, we uncovered a novel mechanism of NK cell dysfunction in OSCC.

Transcriptome Analysis of Tumor-Infiltrating Lymphocytes Identifies NK Cell Gene Signatures Associated With Lymphocyte Infiltration and Survival in Soft Tissue Sarcomas.

PurposeClinical successes using current T-cell based immunotherapies have been limited in soft tissue sarcomas (STS), while pre-clinical studies have shown evidence of natural killer (NK) cell activity. Since tumor immune infiltration, especially tumor-infiltrating lymphocytes, is associated with improved survival in most solid tumors, we sought to evaluate the gene expression profile of tumor and blood NK and T cells, as well as tumor cells, with the goal of identifying potential novel immune targets in STS.Experimental DesignUsing fluorescence-activated cell sorting, we isolated blood and tumor-infiltrating CD3-CD56+ NK and CD3+ T cells and CD45- viable tumor cells from STS patients undergoing surgery. We then evaluated differential gene expression (DGE) of these purified populations with RNA sequencing analysis. To evaluate survival differences and validate primary DGE results, we also queried The Cancer Genome Atlas (TCGA) database to compare outcomes stratified by bulk gene expression.ResultsSorted intra-tumoral CD3+ T cells showed significant upregulation of established activating (CD137) and inhibitory genes (TIM-3) compared to circulating T cells. In contrast, intra-tumoral NK cells did not exhibit upregulation of canonical cytotoxic genes (IFNG, GZMB), but rather significant DGE in mitogen signaling (DUSP4) and metabolic function (SMPD3, SLC7A5). Tumors with higher NK and T cell infiltration exhibited significantly increased expression of the pro-inflammatory receptor TLR4 in sorted CD45- tumor cells. TCGA analysis revealed that tumors with high TLR4 expression (P = 0.03) and low expression of STMN1 involved in microtubule polymerization (P < 0.001) were associated with significantly improved survival.ConclusionsUnlike T cells, which demonstrate significant DGE consistent with upregulation of both activating and inhibiting receptors in tumor-infiltrating subsets, NK cells appear to have more stable gene expression between blood and tumor subsets, with alterations restricted primarily to metabolic pathways. Increased immune cell infiltration and improved survival were positively correlated with TLR4 expression and inversely correlated with STMN1 expression within tumors, suggesting possible novel therapeutic targets for immunotherapy in STS.

Oncolytic Orf virus licenses NK cells via cDC1 to activate innate and adaptive antitumor mechanisms and extends survival in a murine model of late-stage ovarian cancer

BackgroundNovel therapies are needed to improve outcomes for women diagnosed with ovarian cancer. Oncolytic viruses are multifunctional immunotherapeutic biologics that preferentially infect cancer cells and stimulate inflammation with the potential...

Lirilumab and Avelumab Enhance Anti-HPV+ Cervical Cancer Activity of Natural Killer Cells via Vav1-Dependent NF-κB Disinhibition.

BackgroundWe investigated the efficacy and mechanism of the anti-KIR immunotherapy lirilumab and anti-PD-L1 immunotherapy avelumab on natural killer (NK) cell activity against HPV+ cervical cancer.MethodsNK cell-mediated lysis of autologous biopsy-derived malignant cervical squamous cells and normal cervical squamous cells were measured by europium-release cytotoxicity assays. Cytokine and granzyme B release were measured by ELISPOT effector-cell-based assays and ELISA. Murine cervical cancer tumor models were constructed to assess implanted tumor volumes over time and intratumoral immune cell infiltration. Receptor-crosslinking and plate-immobilized antibody stimulation studies, with or without p65 and Vav1 silencing, were used to investigate NF-κB pathway disinhibition in NK cells.ResultsLirilumab and avelumab each enhanced NK cell disinhibition and NK cell-mediated lysis of autologous cervical cancer cells in vitro while reducing HPV+ tumor volumes and increasing intratumoral NK cell infiltration and cytolysis in vivo. Moreover, lirilumab and avelumab each promoted NK cell NF-κB disinhibition as well as stimulated cytokine and granzyme B expression in a NF-κB-dependent manner. Lirilumab+avelumab enhanced all aforementioned effects compared to either monotherapy. Vav1 silencing eliminated disinhibition of NF-κB signaling by lirilumab and avelumab, indicating their disinhibiting effects are Vav1-dependent.ConclusionsThis study supports a novel approach to enhancing NK cell lysis against HPV+ cervical cancer cells through combining lirilumab and avelumab.

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 &lt;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 &lt;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&gt; 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 &lt;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

Type 1 Innate Lymphoid Cells Limit the Antitumoral Immune Response

Natural killer (NK) cells are known to be able to kill established tumor cell lines, but important caveats remain regarding their roles in the detection and elimination of developing primary tumors. Using a genetic model of selective ILC1 and NK cell deficiency, we showed that these cells were dispensable for tumor immunosurveillance and immunoediting in the MCA-induced carcinogenesis model. However, we were able to generate primary cell lines derived from MCA-induced tumors with graded sensitivity to NK1.1+ cells (including NK cells and ILC1). This differential sensitivity was associated neither with a modulation of intratumoral NK cell frequency, nor the capacity of tumor cells to activate NK cells. Instead, ILC1 infiltration into the tumor was found to be a critical determinant of NK1.1+ cell-dependent tumor growth. Finally, bulk tumor RNAseq analysis identified a gene expression signature associated with tumor sensitivity to NK1.1+ cells. ILC1 therefore appear to play an active role in inhibiting the antitumoral immune response, prompting to evaluate the differential tumor infiltration of ILC1 and NK cells in patients to optimize the harnessing of immunity in cancer therapies.

Abstract C135: Notch as an immunologic basis of cancer disparity

Abstract Identification of the host immune factors that influence naturally occurring lymphocyte responses is an important prerequisite to successfully design a successful immunotherapeutic modality. Lymphocytes, such as T-cells and NK cells differ significantly in their ability to mediate antitumor responses depending on the genetic and immunophenotypic constitution of the individuals. The major histocompatibility and the leukocyte receptor complexes are the two most polymorphic regions of the human immune genome. Individuals with increasingly diverse repertoires of MHC class-I molecules have a greater potential for their polyclonal T cells and natural killer (NK) cells to be responsive. Our work suggested that lymphocyte repertoire varies with host backgrounds and that tumor antigen-reactive activated CD8+T cells could enhance intratumoral NK cell function. Furthermore, our data indicate that by modulating the expression of hematopoietic and dendritic cell (DC) Notch ligands, tumors negatively direct lymphocyte differentiation away from their effector phenotype. In human lung tumor infiltrates, we noted a significant correlation between Notch ligand Jag1 or Jag2-expressing dendritic cells with the programmed cell death protein PD-1-expressing CD8+T-effector-memory cells (p = 0.0005). PD-1 expression in T-cells signifies their exhaustion, thus diminishing their survival and antitumor function. These differences in Notch components could underlie poor prognosis in African-Americans (AA). In the clinical trial NCT00774176, the expression of mastermind-like protein-1 that affects Notch-dependent angiogenesis correlated with COPD exacerbation in AA. Also, Notch2 N-terminal-like protein (N2N) was upregulated in various carcinomas as per the TCGA database, with increased N2N expression in AA relative to Caucasians (p = 0.0037). N2N represses the transcriptional activities of Notch2 and Notch1 intracellular domains, which we showed are important for antitumor T-cell function and memory. The comparative analysis of Caucasians and AA lung cancer patient tumor samples show appreciable ethnicity-specific differences in T-cell Notch parameters. Correspondingly, disparities in the expression of Notch components in ethnic minorities can affect antitumor lymphocyte immunity and impact cancer relapse. Citation Format: Anil Shanker, Pierre P Massion, David P Carbone, Mikhail M Dikov. Notch as an immunologic basis of cancer disparity [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr C135.

Plasticity and Polarization of Human NK Cells in the Tumor Microenvironment

Abstract The presence and function of innate lymphoid cells (ILC), including natural killer (NK) cells, within human tumors has been poorly characterized. Here, we have assessed the heterogeneity of NK and ILC populations by single-cell RNA sequencing (scRNAseq) of hundreds of individual NK cells and ILCs within human head and neck squamous cell carcinoma (HNSCC), matched lymph node metastases, matched peripheral blood, and blood from healthy donors. Fresh tumor specimens and blood were obtained from 8 patients undergoing surgical resection of HNSCC. Unsupervised clustering revealed 8 different clusters of NK cells and ILC subsets. We observed significant heterogeneity, with distinct subsets showing profiles consistent with that of conventional NK cells, ILC1-like cells and ILC3-like cell. Importantly, peripheral NK cells were distinct from intratumoral NK cells. The presence of these different cell subsets within primary HNSCC tumors was confirmed by flow cytometry. Further, plasticity between the NK cell subsets was supported by in vitro and in vivo experimental data. Given the ability of NK and ILCs to polarize the immune responses through the secretion of cytokines and the ability of certain subsets to kill target cells, we hypothesize that the differences observed in NK and ILC populations and their plasticity may result in different immune responses, and may influence clinical outcomes following therapy.

PD-1+ natural killer cells in human non-small cell lung cancer can be activated by PD-1/PD-L1 blockade.

Natural killer (NK) cells are critically involved in anti-tumor immunity by targeting tumor cells. In this study, we show that intratumoral NK cells from NSCLC patients expressed elevated levels of the immune checkpoint receptor PD-1 on their cell surface. In contrast to the expression of activating receptors, PD-1+ NK cells co-expressed more inhibitory receptors compared to PD-1- NK cells. Intratumoral NK cells were less functional compared to peripheral NK cells, and this dysfunction correlated with PD-1 expression. Tumor cells expressing PD-L1 inhibited the functionality of PD-1+ NK cells in ex vivo models and induced PD-1 clustering at the immunological synapse between NK cells and tumor cells. Notably, treatment with PD-1 blockade was able to reverse PD-L1-mediated inhibition of PD-1+ NK cells. Our findings highlight the therapeutic potential of PD-1+ NK cells in immune checkpoint blockade and could guide the development of NK cell-stimulating agents in combination with PD-1 blockade.