NK Cell Subsets Research Articles

Blood immune cells as potential biomarkers predicting relapse-free survival of stage III/IV resected melanoma patients treated with peptide-based vaccination and interferon-alpha.

Despite the recent approval of several therapies in the adjuvant setting of melanoma, tumor relapse still occurs in a significant number of completely resected stage III-IV patients. In this context, the use of cancer vaccines is still relevant and may increase the response to immune checkpoint inhibitors. We previously demonstrated safety, immunogenicity and preliminary evidence of clinical efficacy in stage III/IV resected melanoma patients subjected to a combination therapy based on peptide vaccination together with intermittent low-dose interferon-α2b, with or without dacarbazine preconditioning (https://www.clinicaltrialsregister.eu/ctr-search/search, identifier: 2008-008211-26). In this setting, we then focused on pre-treatment patient immune status to highlight possible factors associated with clinical outcome. Multiparametric flow cytometry was used to identify baseline immune profiles in patients' peripheral blood mononuclear cells and correlation with the patient clinical outcome. Receiver operating characteristic curve, Kaplan-Meier survival and principal component analyses were used to evaluate the predictive power of the identified markers. We identified 12 different circulating T and NK cell subsets with significant (p ≤ 0.05) differential baseline levels in patients who later relapsed with respect to patients who remained free of disease. All 12 parameters showed a good prognostic accuracy (AUC>0.7, p ≤ 0.05) and 11 of them significantly predicted the relapse-free survival. Remarkably, 3 classifiers also predicted the overall survival. Focusing on immune cell subsets that can be analyzed through simple surface staining, three subsets were identified, namely regulatory T cells, CD56dimCD16- NK cells and central memory γδ T cells. Each subset showed an AUC>0.8 and principal component analysis significantly grouped relapsing and non-relapsing patients (p=0.034). These three subsets were used to calculate a combination score that was able to perfectly distinguish relapsing and non-relapsing patients (AUC=1; p=0). Noticeably, patients with a combined score ≥2 demonstrated a strong advantage in both relapse-free (p=0.002) and overall (p=0.011) survival as compared to patients with a score <2. Predictive markers may be used to guide patient selection for personalized therapies and/or improve follow-up strategies. This study provides preliminary evidence on the identification of peripheral blood immune biomarkers potentially capable of predicting the clinical response to combined vaccine-based adjuvant therapies in melanoma.

Immediate major dynamic changes in the T- and NK-cell subset composition after cardiac transplantation.

Early kinetics of lymphocyte subsets involved in tolerance and rejection following heart transplantation (HTx) are barely defined. Here, we aimed to delineate the early alloimmune response immediately after HTx. Therefore, blood samples from 23 heart-transplanted patients were collected before (pre-), immediately (T0), 24 hours (T24), and 3 weeks (3wks) after HTx. Immunophenotyping was performed using flow cytometry. A significant increase was detected for terminally differentiated (TEMRA) CD4+ or CD8+ T cells and CD56dim CD16+ NK cells immediately after HTx linked to a decrease in naïve CD8+ and CM CD4+ T as well as CD56bright CD16- NK cells, returning to baseline levels at T24. More detailed analyses revealed increased CD69+ CD25- and diminished CD69- CD25- CD4+ or CD8+ T-cell proportions at T0 associated with decreasing S1PR1 expression. Passenger T and NK cells were found at low frequencies only in several patients at T0 and did not correlate with lymphocyte alterations. Collectively, these results suggest an immediate, transient shift toward memory T and NK cells following HTx. Opposite migratory properties of naïve versus memory T and NK cells occurring in the early phase after HTx could underlie these observations and may impinge on the development of allo-specific immune responses.

Comparison of NK cell subsets, receptors and functions induced by radiofrequency ablation and microwave ablation in HBV-associated primary hepatocellular carcinoma.

Topical therapy has been shown to induce an immune response in patients with hepatocellular carcinoma (HCC). In this study, a prospective parallel group control experiment was conducted to compare the differences between radiofrequency ablation and microwave ablation in inducing the immune regulation of NK cells. Sixty patients with clinically and pathologically confirmed hepatitis B-associated hepatocellular carcinoma (HCC) were selected for thermal ablation. Patients were randomly assigned into the MWA group (n = 30) and the RFA group (n = 30). Patient's peripheral blood was isolated on days D0, D7, and month M1. NK cell subsets, receptors, and killing function were detected by flow cytometry and LDH. Student t test and rank sum test were used to compare the statistical differences between the RFA (radio frequency) and MWA (microwave) groups. The Kaplan-Meier curve and log-rank test were used to calculate the difference between the two survival curves. Comparison of the frequency of CD3-CD56+ and CD3-CD56+CD16+ in NK cells between the RFA and WMA groups showed that there was no difference in the D0, D7, M1, D7-D0, M1-D0, and M1-D7 groups. The changes of the inhibitory NK cell receptor CD159A were significantly different at D7 (P<0.05). CD107a were compared between the RFA and WMA groups, indicating that CD107a changes induced by NK cells were significantly different at D7-D0 (P<0.05). Comparison of NK cell lysis activity of target K562 cells between the RFA and WMA groups showed that there was no difference at D0, D7, D7-D0. There was no difference in recurrence-free survival (RFS) between the RFA and WMA groups (P=0.11). The difference between MWA and RFA-induced NK cell changes was mainly manifested in the inhibitory receptors CD159a and CD107a 1 week after surgery, with microwave-induced changes being more severe. Comparison of the NK cell lysis activity of the target K562 cells between the RFA and WMA groups showed that there was no difference in D0, D7, D7- D0. Survival analysis showed that these differences did not affect the recurrence-free survival (RFS) in the two groups.

Single-cell cloning of NK cells from rhesus macaques: a new methodology for in-depth analysis of MHC-E-dependent adaptive functions

Abstract Natural killer (NK) cells provide rapid and robust responses against a wide range of viral infections. More recently, multiple studies have shown that subsets of NK cells are also capable of antigen-specific recall to eliminate infected cells. In humans and nonhuman primates (NHP), antigen-specific memory NK cell responses can at least be partially delineated by expression of NKG2C, a ligand for MHC-E. Due to MHC-E limited polymorphism, MHC-E-presented viral antigens may induce universally antigen-specific memory NK cells. To study antigen-specific NK cells in NHP, we developed a new in vitromethod to clonally expand single rhesus macaque NK cells (rhNKCL) from blood and spleen. To define the effector functions of rhNKCL, we used a calcein acetoxymethyl ester (CAM) cytotoxicity assay and intracellular staining assay in combination with K562 cell lines or autologous BLCL as targets. We also identified, by in silicomethodology, peptides from influenza, SIV, and SARS-CoV-2 viruses predicted to display strong binding to MHC-E. Our procedure showed for the first-time clonal expansion of single rhesus macaque NK cells. We observed a conserved NHP NK cell phenotype of CD3 −NKG2A/C +CD16 +CD56 +following expansion with expression of additional NK cell surface receptors CD8, NKp80, and NKG2D. Upon stimulation with K562 cells, rhNKCL effector functions (cytotoxicity, CD107a, IFNγ, TNFα) were like primary NK cells. Altogether, this new method of single-cell cloning in rhesus macaques could provide key information about antigen-specific NK cells, including epigenetic, metabolic, transcriptional, and phenotypic profiles, which could be exploited to further enhance antiviral NK cell activity in future vaccine strategies or cell therapies. Supported by grants from NIH (R01 AI158516, R01 DK130472, R01 AI161010, NIH-funded BEAT-HIV Martin Delaney Collaboratory to cure HIV-1 infection by Combination Immunotherapy UM1 AI164570).

The CD16 and CD32b Fc-gamma receptors regulate antibody-mediated responses in mouse natural killer cells

Abstract Natural killer (NK) cells are innate lymphocytes capable of mediating immune responses without prior sensitization. NK cells express Fc-gamma receptors (FcγR) that engage the Fc region of IgG. Studies investigating the role of FcγR on mouse NK cells have been limited due to lack specific reagents. In this study, we characterize the expression and biological consequences of activating mouse NK cells through their FcγR. We demonstrate that most NK cells express the activating CD16 receptor, and a subset of NK cells also express the inhibitory CD32b receptor. Critically, these FcγRs are functional on mouse NK cells and can modulate antibody-mediated responses. We also characterized mice with conditional knock-out alleles of Fcgr3 (CD16) or Fcgr2b (CD32b) in the NK and innate lymphoid cell (ILC) lineage. NK cells in these mice did not reveal any developmental defects and were responsive to cross-linking activating NK receptors, cytokine stimulation, and killing of YAC-1 targets. Importantly, CD16-deficient NK cells failed to induce antibody-directed cellular cytotoxicity (ADCC) of antibody-coated B cell lymphomas in in vitro assays. In addition, we demonstrate the important role of CD16 on NK cells using an in vivo model of cancer immunotherapy using anti-CD20 antibody treatment of B cell lymphomas. This project was funded by fellowships from Burroughs Wellcome Fund (BWF) and the Cancer Research Institute to O.A.A., and grants from the Parker Institute for Cancer Immunotherapy (PICI), and the NIH (AI068129 and AI146581) to L.L.L.

Diagnostic and Treatment Challenges in a Pediatric Patient with Progressive Refractory ALPS-like Disease

Introduction: Diagnostic criteria for Autoimmune Lymphoproliferative Syndrome (ALPS) is based on clinical, laboratory and genetic findings. The underlying pathomechanisms are largely related to defects in the lymphocyte apoptosis pathways (FAS, FAS ligand, or CASP10) but other combined immunodeficiencies may also present with ALPS-like features (e.g. CASP8, NFKB1, CTLA4, ALP1). Clinically, patients often present with progressive autoimmune cytopenias and treatment relies on T cell immune modulation. Hereby, we present diagnostic and treatment challenges in a pediatric patient with ALPS-like phenotype with rapid decline during an episode of severe autoimmune hemolytic anemia. MethodsRetrospective chart review for clinical and laboratory data. Deep immune phenotyping was obtained for T, B and NK cells subsets at several timepoints during the acute illness. Genetic evaluation for both germline and somatic mutations were pursued.Case presentation: Patient is an 11-year-old Syrian male of consanguineous parents who presented at age 6 with immune thrombocytopenic purpura (ITP) and met criteria for ALPS with required criteria (chronic lymphadenopathy and increased T Cell Receptor αβ CD4-CD8- double negative T cells (TCRαβDNT) (4.2%, normal <2%), primary accessory criteria (defective lymphocyte apoptosis twice) and secondary accessory criteria (elevated B12, IgG and FAS-L). Panel sequencing (207 genes of inborn errors of immunity) and later Whole Exome Sequencing were both unrevealing. At 11 years of age, he was admitted with a Coombs positive severe autoimmune hemolytic anemia (hemoglobin of 3.5) that was complicated by EBV and parvovirus viremia, and aplastic anemia. Treatment course was complex including steroid, high dose IvIg, rituximab, sirolimus, mycophenolate mofetil, eculizumab, and epoetin alpha. Biomarkers of immune dysregulation, such as TCRαβDNT, follicular helper T cells and CD19hiCD21lo B cells were monitored closely and improved on therapy. Total T cell, CD4, CD8, and Treg populations were all initially low but returned to baseline following combined treatment. Screening for somatic mutations and whole genome sequencing is ongoing. Discussion/ConclusionOur case highlights the diagnostic and treatment challenges in a pediatric patient with ALPS-like Disease. Cellular biomarkers may guide therapy if autoimmune cytopenia recurs. Somatic and germline testing is needed to understand the underpinning of this disorder and may further guide treatment strategies.

Pre-vaccination CD56 dimCD16 +NK cell abundance and T h1/T h17 ratio predict responsiveness to conjugated pneumococcal vaccine in older adults

Abstract Older adults are at high risk of morbidity and mortality from Streptococcus pneumoniae (pneumococcus) infections. There are two available vaccines for pneumococcus: T-cell-independent capsular polysaccharide Pneumovax and T-cell-dependent conjugated Prevnar. However, how older adults respond to these vaccines at the cellular level and whether there are baseline predictors for responsiveness, is not known. To address this, we recruited older adults (60+ yrs), who are vaccinated with Prevnar (n=19) or Pneumovax (n=20). Vaccine responsiveness was quantified using opsonophagocytic assays, which revealed that both vaccines induce strong responses. Interestingly, sex was associated with Prevnar responses, where women mounted stronger responses than men. Pre-vaccination flow cytometry data showed that Th1 cells positively, Th17 cells negatively correlated with Prevnar responses. Furthermore, bulk RNA-seq data from PBMCs showed that baseline expression levels of cytotoxic genes (NCAM1, GNLY, PRF1) are negatively associated with Prevnar responses. scRNA-seq data from top and bottom responders showed that this cytotoxicity signature stems from CD56 dimCD16 +NK cells, where having more of these cells are detrimental to responses. Interestingly, women had significantly higher Th1, lower Th17 and lower CD16+ NK cells compared to men, which explains their stronger Prevnar responses. This is the first study to uncover older adults’ responses to two pneumococcal vaccines; we uncovered an activated immune phenotype of Th and NK cell subsets that impedes responses to Prevnar. Interestingly, this phenotype only affected adjuvanted vaccine responses, providing an opportunity for precision vaccinology for pneumococcus disease. Supported by grants from National Institute of Health (R35 GM124922, R01 AG052608) and JAX cancer center (JAX-CC).

Accurate enumeration of leukocyte subsets in peripheral blood with a 15-color immunoprofiling panel on a Cytek ®spectral flow cytometer

Abstract A white blood cell (WBC) differential is routinely performed on automated hematology analyzers as a general health check to evaluate immune status of patients. The recent advances in high parameter flow cytometry, especially full spectral flow cytometry, enable the analysis of more comprehensive leukocyte subsets to meet the increased demands in clinical research. We designed a 15-color, single-tube panel to identify and enumerate all major subsets of leukocytes on a Cytek full spectral flow cytometer. Human peripheral blood collected in K2EDTA, Heparin, ACD and Cyto-chex blood tubes were stained with 15 cFluor ®conjugated mAbs with a Lyse No Wash method and analyzed on a Cytek spectral flow cytometer. The performance of this assay was evaluated side by side with BD Multitest™ 6-color TBNK assay on BD FACSCanto II system on 13 healthy donor samples. Assay precision was examined on 12 donor samples in triplicates on three consecutive days. This 15-color panel identifies more than 20 leukocyte subpopulations: including neutrophils, eosinophils, basophils, lymphocytes, hematopoietic stem cells, monocyte subsets, T cell subsets, B cells, NK cell subsets. The results are comparable to that from BD 6-Color TBNK assay with R 2=0.961, 0.986, 0.940, 0.954, and 0.930 for CD3+, CD4+, CD8+ T, B and NK cells respectively. The correlations for all subsets are statistically significant with P &amp;lt; 0.001. The coefficient of variations (CV) of intra assay precision for all cell populations of interest are less than 25%. Cytek’s single-tube 15-color pan leukocyte panel demonstrated an effective, high sensitivity flow cytometry approach that can be used for monitoring immune cell subsets in peripheral blood in discovery and translational research.

Two developmental pathways generate functionally distinct populations of natural killer cells

Abstract Natural killer (NK) cells function by eliminating virus-infected cells or tumor cells during early defenses. However, the early development of NK cells and lineage relationships between NK cells and helper innate lymphoid cells (ILCs) remain elusive. Common precursors for ILCs (ILCPs) can differentiate into both helper ILCs and NK cells. Here, we identified a NK lineage-restricted progenitor population, early NK progenitor (ENKP), which does not develop from ILCPs, thus ENKP may represent the ILCP-independent pathway of NK cell development. Competitive chimera experiment shows ENKPs generate NK cells more efficiently than ILCPs, suggesting that ENKP-dependent pathway is the major pathway for NK cell development. scRNA-seq shows ENKP-derived NK cells express Ly49 receptors and higher levels of cytotoxic genes whereas ILCP-derived NK cells have very low expression of Ly49 receptors and express higher levels of genes implicated in tissue residency such as CD69 and CD200R. Furthermore, Ly49H+ NK cells which response to MCMV infection mostly develop from ENKPs but not ILCPs. Consistently, ENKP-derived NK cells but not ILCP-derived NK cells expanded dramatically after MCMV infection. Our findings establish the existence of two pathways of NK cell development that generate functionally distinct NK cell subsets.

Imbalanced receptors, gene signatures, and function of NK subsets predicts poor clinical outcomes in AML

Abstract NK cells mediate anti-leukemic immunosurveillance in AML. We hypothesized that impaired NK cells predict poor outcomes in AML. We analyzed the expression of receptors on CD56 NK cell subsets and their corresponding ligands on the CD45dim blast by flow cytometry in 100 AML and 17 healthy bone marrows. Risk stratification, complete remission, relapse, measurable residual disease, overall survival, relapse-free survival, NK cell ligand expression, NK function, and degranulation were evaluated. Additionally, we mapped the transcriptional landscape of AML-NK cells using single-cell RNA-seq. The expression of activating receptors by CD56dim NK was decreased in adverse risk, relapse, and MRD+ patients. Inhibitory receptors were increased in adverse risk, relapse, and MRD+. NK cell activating ligands were decreased in patients with higher expression of the corresponding receptor while inhibitory ligands were increased. Patients with a lower frequency of CD56dim NK showed less degranulation and killing rates. Transcriptionally, we observed an enrichment of inflammatory-associated pathways and an upregulation of inhibitory receptors in AML, especially in patients with the worst prognosis. Our data indicate that NK exhaustion, with a high frequency of inhibitory receptors, leads to impaired cytotoxicity and can predict poor outcomes in AML. Also, a selective pressure in the expression of NK cell ligands on the AML blast together with imbalanced receptors may vary in both NK and leukemic cells. Closing these gaps in the knowledge of NK-mediated immune evasion in leukemia is of significant interest for targeting the leukemic microenvironment by NK cell-mediated immunotherapy. "division funds" - 3117175

Comprehensive single-cell transcriptomic and proteomic analysis reveals NK cell exhaustion and unique tumor cell evolutionary trajectory in non-keratinizing nasopharyngeal carcinoma

BackgroundNonkeratinizing nasopharyngeal carcinoma (NK-NPC) has a strong association with Epstein-Barr virus (EBV) infection. The role of NK cells and the tumor cell evolutionary trajectory in NK-NPC remain unclear. In this study, we aim to investigate the function of NK cell and the evolutionary trajectory of tumor cells in NK-NPC by single-cell transcriptomic analysis, proteomics and immunohistochemistry.MethodsNK-NPC (n = 3) and normal nasopharyngeal mucosa cases (n = 3) were collected for proteomic analysis. Single-cell transcriptomic data of NK-NPC (n = 10) and nasopharyngeal lymphatic hyperplasia (NLH, n = 3) were obtained from Gene Expression Omnibus (GSE162025 and GSE150825). Quality control, dimension reduction and clustering were based on Seurat software (v4.0.2) process and batch effects were removed by harmony (v0.1.1) software. Normal cells of nasopharyngeal mucosa and tumor cells of NK-NPC were identified using copykat software (v1.0.8). Cell-cell interactions were explored using CellChat software (v1.4.0). Tumor cell evolutionary trajectory analysis was performed using SCORPIUS software (v1.0.8). Protein and gene function enrichment analyses were performed using clusterProfiler software (v4.2.2).ResultsA total of 161 differentially expressed proteins were obtained between NK-NPC (n = 3) and normal nasopharyngeal mucosa (n = 3) by proteomics (log2 fold change > 0.5 and P value < 0.05). Most of proteins associated with the nature killer cell mediated cytotoxicity pathway were downregulated in the NK-NPC group. In single cell transcriptomics, we identified three NK cell subsets (NK1-3), among which NK cell exhaustion was identified in the NK3 subset with high ZNF683 expression (a signature of tissue-resident NK cell) in NK-NPC. We demonstrated the presence of this ZNF683 + NK cell subset in NK-NPC but not in NLH. We also performed immunohistochemical experiments with TIGIT and LAG3 to confirm NK cell exhaustion in NK-NPC. Moreover, the trajectory analysis revealed that the evolutionary trajectory of NK-NPC tumor cells was associated with the status of EBV infection (active or latent). The analysis of cell-cell interactions uncovered a complex network of cellular interactions in NK-NPC.ConclusionsThis study revealed that the NK cell exhaustion might be induced by upregulation of inhibitory receptors on the surface of NK cells in NK-NPC. Treatments for the reversal of NK cell exhaustion may be a promising strategy for NK-NPC. Meanwhile, we identified a unique evolutionary trajectory of tumor cells with active status of EBV-infection in NK-NPC for the first time. Our study may provide new immunotherapeutic targets and new sight of evolutionary trajectory involving tumor genesis, development and metastasis in NK-NPC.

Effects of ocrelizumab on NK cell activation in Relapsing Multiple Sclerosis (P2-3.004)

<h3>Objective:</h3> To evaluate the effects of ocrelizumab on circulating immune asset in Relapsing Multiple Sclerosis (RMS) subjects, with a specific focus on NK cell activation profile. <h3>Background:</h3> Ocrelizumab, a humanized monoclonal anti-CD20 antibody, has shown pronounced effects in reduction of disease activity in RMS. Although the clinical response to ocrelizumab is related to a specific B cell depletion, other circulating lymphocytes may be affected by this treatment. Concerning NK lymphocytes, several studies have shown potential regulatory properties of these cells in MS. Some activator cytotoxicity receptors (CD94/NKG2C, NKG2D, NKp46, and DNAM) were suggested to mediate NK cytotoxic regulatory function. Anti-CD20 agents showed to drive the selective in vitro expansion of CD94/NKG2C+ memory NK cells and to enhance their cytotoxic function. However, the phenotypic changes induced by ocrelizumab on NK cell subsets in vivo and their pathogenetic implication are still unexplored. <h3>Design/Methods:</h3> In this prospective longitudinal study, we enrolled 30 RMS patients. RMS patients were treated with ocrelizumab and followed up for twelve months. Frequency and phenotype of several circulating T cell subsets as well as NK cell phenotypic profile were evaluated by flow cytometry, before and after the treatment. <h3>Results:</h3> Six and twelve months after the treatment, a reduction of absolute lymphocyte count was observed. We also noticed increased percentage of T cell subsets with naïve phenotype (CD4+CD45RA+ and CD8+CD45RA+ cells). In parallel, a reduction of activated/memory T cell percentage was observed (CD4+CD45RO+ and CD8+CD45RO+ cells). Further, flow cytometry phenotypic analysis revealed an increased proportion of NK cells expressing the activating receptors, CD94/NKG2C, NKG2D, NKp46 and DNAM upon ocrelizumab treatment. <h3>Conclusions:</h3> Ocrelizumab affects T cell compartment, favoring reduction of activated/memory T cells in RMS patients. Further, ocrelizumab induces phenotypical changes in NK cells toward a higher cytotoxic/regulatory profile in RMS patients. <b>Disclosure:</b> Dr. Abbadessa has nothing to disclose. Dr. Bruzzaniti has nothing to disclose. Dr. Miele has nothing to disclose. Dr. Piemonte has nothing to disclose. Dr. Lepore has nothing to disclose. Elisabetta Signoriello has nothing to disclose. Giacomo Lus has nothing to disclose. Giuseppe Matarese has nothing to disclose. Mario Galgani has nothing to disclose. Prof. Bonavita has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for Novartis, Biogen, Mylan, Sanofi. Prof. Bonavita has received personal compensation in the range of $5,000-$9,999 for serving on a Speakers Bureau for Mylan, Novartis.

Phenotypic and functional analysis in HER2+ targeted therapy of human NK cell subpopulation according to the expression of FcεRIγ and NKG2C in breast cancer patients.

Adaptive NK cells constitute an NK cell subpopulation, which expands after human cytomegalovirus (HCMV) infection. This subpopulation has stronger production of cytokines after CD16 stimulation, longer life and persistence than conventional NK cells and are, therefore, interesting tools for cancer immunotherapy. Since there is limited information on adaptive NK cells in cancer patients, we described this population phenotypically and functionally, by flow cytometry, in the context of HER2 + breast cancer (BC) directed therapy. We assessed HCMV status in 78 patients with BC. We found that, similarly to healthy donors (HD), a high proportion of BC patients were HCMV-positive, and nearly 72% of them had an adaptive NK cell subpopulation characterized by the loss of FcεRIγ intracellular adaptor protein or the presence of NKG2C receptor. However, in BC patients, FcεRIγ- and NKG2C + NK cell populations overlapped to a lesser extent than in HD. Otherwise, no profound phenotypic differences were found between BC patients and HD. Although FcεRIγ- or NKG2C + NK cell subsets from BC patients produced more IFN-γ than their FcεRIγ + or NKG2C- NK cell counterparts, IFN-γ production increased only when NK cells simultaneously expressed FcεRIγ- and NKG2C + , whereas in HD the presence of NKG2C marker was sufficient to display greater functionality. Furthermore, in a group of patients treated with chemotherapy and Trastuzumab plus Pertuzumab, FcεRIγ-NKG2C + and FcεRIγ-NKG2C- NK cells retained greater functionality after treatment than FcεRIγ + NKG2C- NK cells. These results suggest that the presence or magnitude of adaptive NK cell subsets might serve as a key determinant for therapeutic approaches based on antibodies directed against tumor antigens.

Abstract 1351: Uterine natural killer cells display a unique functional dichotomy in human endometrial cancer

Abstract Endometrial Cancer (EC) is the most common gynecological malignancy in the United States, with striking heterogeneity of risk factors, molecular identities, and outcomes. Two common subtypes of EC are Type 1, which display endometrioid histology and include those with MSI status, and Type 2, which arise from an atrophic background and are more severe in their morbidity. Despite significant research on checkpoint blockade therapies, such as PD-1/PD-L1 blockade to treat Type 1 ECs, variable response rates underscore the need for further immunomodulation. NK cells are cytotoxic innate lymphocytes that cooperate with other cells of the immune system. Despite years of investigation into basic NK cell biology, our understanding of the role(s) of NK cells in human solid tumors is still in its infancy. For example, in head and neck squamous cell carcinoma (HNSCC) indicate that a subset of NK cells has strong anti-tumor function, while a separate subset of NK cells lacks direct cellular cytotoxicity. Considering these findings, we used flow cytometry to evaluate NK cell phenotypes and frequencies in fresh EC patient tumor samples. Our data suggest a functional dichotomy of NK cell subsets with distinct phenotypes, present in both Type 1 and Type 2 EC tumors (Type 1: 35.5 +/- 10.65 % CD16+ vs 73.5 +/- 10.41 % CD16-, n=6, p=0.10; Type 2: 16.23+/- 3.61 CD16+ vs 83.77 +/- 3.65 CD16-, n=3, p&amp;lt;0.05). The first subset expresses CD16, which mediates antibody dependent cellular cytotoxicity, along with T-BET and EOMES, which are required for direct killing through Perforin and Granzymes and thus reminiscent of conventional NK (cNK) cells found in blood. The second subset is CD16- with decreased expression of T-BET and EOMES, and upregulated expression of tissue resident NK cell (trNK)-associated markers including CD200R1, CD103 and CD49a. To understand frequencies of these NK cell subsets in EC, we used immunohistochemistry for CD3, CD56, CD103, and Granzyme B on formalin-fixed, paraffin-embedded tissues from 10 patients: 4 Type 1, 4 Type 2, and 2 benign controls. We utilized CD56 to denote NK cells and found that NK cells infiltrated the Type 1 EC cases more compared to Type 2 (22.1 +/- 7.3% NK cells in Type 1 infiltrate vs 14.8 +/- 2% NK cells in Type 2 infiltrate, n=5, p=0.17). Further, NK cells expressing CD103, denoting trNK cells, could primarily be found in the glands of EC tumors, while NK cells expressing Granzyme B, denoting the cNK cells, were primarily in the stroma and largely absent in the glands (Granzyme B+: 78.6 +/- 3.7% Stroma vs 21.4 +/- 3.7% Glands, n=3, p&amp;lt;0.05; CD103+: 15.6 +/- 4.5% Stroma vs 84.3 +/- .64% Glands, n=3, p=0.076). This work suggests that an NK cell dichotomy exists within EC, with distinct phenotypes and localizations. Ongoing work will include functional studies to assess killing potential and cytokine production of the two subsets, along with single cell RNA sequencing to unravel which NK cells may be friend or foe in EC. Citation Format: Caprice D. Eisele, Courtney J. Riedinger, Jesse J. Kowalski, Bethany L. Mundy-Bosse, Paige M. Porrett, Patrick L. Collins, Casey M. Cosgrove, Aharon G. Freud. Uterine natural killer cells display a unique functional dichotomy in human endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1351.

Abstract 4688: Comprehensive analysis of natural killer cell-associated markers using MultiOmyxTM immunofluorescence assay

Abstract As a promising alternative platform for cellular immunotherapy, natural killer cells (NK) have recently gained attention as an important type of innate immune regulatory cell. NK cell immunotherapy approaches have been translated into clinical applications, and clinical trials of NK cell infusion in patients with hematological malignancies (HM) and solid tumors have thus far yielded many encouraging clinical results. Understanding the pattern of NK expression and the relationship to different states of NK cells may have direct relevance for immune responses in cancer. Approaches capable of simultaneously detecting NK cells with detailed information on NK differentiation state, however, remain limited. In this study, we used MultiOmyx hyperplexed immunofluorescence (IF) assay to classify and characterize the spatial arrangement of NK cell markers in a pan-cancer cohort including 9 tissue microarrays (TMAs) from breast, head and neck, prostrate, colon, bladder, lung, kidney, ovarian and melanoma indications. The panel includes CD3, CD4, CD8, CD16, CD45, CD56, CD57, CD137, FoxP3, Granzyme B, HLA-E, NKG2A, NKP46 and tumor segmentation marker. The panel enables the detection of NK cells expressing CD56 and/or NKP46 in the 9 different tumor indications. We also studied the expression of activating and inhibiting receptors, such as CD16 and NKG2A, in NK cell population. The active cytotoxic subsets of mature NK cells were evaluated using co-expression of NK cell surface markers with CD57 and the cytotoxic molecule expression in NK cells was assessed using co-expression with Granzyme B. Using proprietary deep-learning-based image analysis, we were able to quantify the densities of these different NK cell population and study the prevalence of these NK cells in different cancer indications included in this study. Many strategies have been developed for exploiting NK-mediated anti-tumor activities. CAR-NK cell therapy and antibodies that directly target NK cell inhibitory receptors such as NKG2A and TIGIT, are currently being evaluated in the clinical trials. The MultiOmyx NK cell panel reported in this study enables the comprehensive profiling of the NK population and can provide greater understanding of NK cell biology during cancer progression. The panel can be further used to explore the efficacy of the NK cell-based immune therapy. Citation Format: Erinn A. Parnell, Jiong Fei, Harry Nunns, Eric Leones, Elaine Yeung, Blair Russell, Flora Sahafi, Qingyan Au. Comprehensive analysis of natural killer cell-associated markers using MultiOmyxTM immunofluorescence assay. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4688.

Redistribution and Activation of CD16brightCD56dim NK Cell Subset to Fight against Omicron Subvariant BA.2 after COVID-19 Vaccination.

With the alarming surge in COVID-19 cases globally, vaccination must be prioritised to achieve herd immunity. Immune dysfunction is detected in the majority of patients with COVID-19; however, it remains unclear whether the immune responses elicited by COVID-19 vaccination function against the Omicron subvariant BA.2. Of the 508 enrolled patients infected with Omicron BA.2, 102 were unvaccinated controls, and 406 were vaccinated. Despite the presence of clinical symptoms in both groups, vaccination led to a significant decline in nausea or vomiting, abdominal pain, headache, pulmonary infection, and overall clinical symptoms and a moderate rise in body temperature. The individuals infected with Omicron BA.2 were also characterised by a mild increase in both serum pro- and anti-inflammatory cytokine levels after vaccination. There were no significant differences or trend changes between T- and B-lymphocyte subsets; however, a significant expansion of NK lymphocytes in COVID-19-vaccinated patients was observed. Moreover, the most effective CD16brightCD56dim subsets of NK cells showed increased functional capacities, as evidenced by a significantly greater IFN-γ secretion and a stronger cytotoxic potential in the patients infected with Omicron BA.2 after vaccination. Collectively, these results suggest that COVID-19 vaccination interventions promote the redistribution and activation of CD16brightCD56dim NK cell subsets against viral infections and that they could facilitate the clinical management of patients infected with Omicron BA.2.

Cross-Talk between Mucosal-Associated Invariant T, Natural Killer, and Natural Killer T Cell Populations is Implicated in the Pathogenesis of Placenta Accreta Spectrum

The study included 32 women with PAS and 20 with normally implanted placenta as a control group. Vascular endothelial cell growth factor (VEGF), Soluble FMS Like Tyrosine Kinase (sFLT-1/sVEGFR1), and Endoglin (ENG) were measured in placenta tissue by ELISA. Granzyme B (GrzB) expression in trophoblastic and stromal mesenchymal cells was evaluated by immunohistochemistry. MAIT, NK, and NKT cells were assessed in blood and placenta by flow cytometry. Alterations were observed in levels of MAIT cells, NK cell subsets, and NKT cells in patients compared with controls. Several significant correlations were detected between these cells and GrzB scores, VEGF, ENG, and sFLT-1 levels. This is the first study analysing these cells in PAS patients and correlating their levels with changes in some angiogenic and antiangiogenic factors implicated in trophoblast invasion and with GrzB distribution in trophoblast and stroma. Interrelation between these cells probably plays an important role in pathogenesis of PAS.

CD8αα expression on NK cells is associated with different K562 and MOLT4 killing capabilities of PBMC and different involvement CD8pos and CD8neg subsets in anti-viral response

Introduction. The CD8αα are present in a subset of T cells and NK cells, but its function is mostly unknown, as well as the role of CD8+ and CD8- NK cell subsets in physiological and pathological environments. Methods. We investigated 130 healthy individuals’ blood samples for the NK cell cytotoxicity against K562 and MOLT4 cell lines. We also analyzed patients after SarsCov2 infection and compared to healthy control. The NK cell phenotype and cytotoxicity were studied by the FACScan flow cytometer using BD monoclonal antibodies. Results. We confirmed that MOLT4 is significantly more resistant to the NK cell cytotoxicity compared to the “classical” K562. CD8+ NK cells are more effective at K562 killing compared to CD8- subsets. The correlation of lymphocyte levels with the specific K562 lysis was weaker for CD8- NK cell subsets (r = 0.37) than CD8+ NK cells (r = 0.45) or whole NK cells population (r = 0.46). However, we found that CD8+ NK cells mostly did not participate in the MOLT-4 killing. CD8- NK cells frequency correlates with MOLT4 lysis more significantly (r = 0.49) than CD8+ NK cells lymphocytes levels (r = 0.27) or whole NK cells population (r = 0.44). Also, we showed that HLA-DR and CD158a positive NK cell levels did not correlate with the MOLT4 and K562 killing, while HLA-DR and CD158a negative subsets levels did with the same significance as the whole NK cells population. Decreased of NK lymphocytes after SarsCov2 infection results to decrease NK population owing to CD8+NK decreased but not CD8neg. Conclusion. NK cell numbers determine NK cell cytotoxicity indirectly through the surface phenotype. CD8 expression on the NK cells is associated with the effective cytotoxicity against K562 but at the same time obstructs a response to MOLT4. CD8αα on NK cells might participate in HLA recognition or enhance response to HLA class-I negative target cells.

Chronic Comorbidities in Middle Aged Patients Contribute to Ineffective Emergency Hematopoiesis in Covid-19 Fatal Outcomes

Mexico is among the countries with the highest estimated excess mortality rates due to the COVID-19 pandemic, with more than half of reported deaths occurring in adults younger than 65 years old. Although this behavior is presumably influenced by the young demographics and the high prevalence of metabolic diseases, the underlying mechanisms have not been determined. The age-stratified case fatality rate (CFR) was estimated in a prospective cohort with 245 hospitalized COVID-19 cases, followed through time, for the period October 2020-September 2021. Cellular and inflammatory parameters were exhaustively investigated in blood samples by laboratory test, multiparametric flow cytometry and multiplex immunoassays. The CFR was 35.51%, with 55.2% of deaths recorded in middle-aged adults. On admission, hematological cell differentiation, physiological stress and inflammation parameters, showed distinctive profiles of potential prognostic value in patients under 65 at 7 days follow-up. Pre-existing metabolic conditions were identified as risk factors of poor outcomes. Chronic kidney disease (CKD), as single comorbidity or in combination with diabetes, had the highest risk for COVID-19 fatality. Of note, fatal outcomes in middle-aged patients were marked from admission by an inflammatory landscape and emergency myeloid hematopoiesis at the expense of functional lymphoid innate cells for antiviral immunosurveillance, including NK and dendritic cell subsets. Comorbidities increased the development of imbalanced myeloid phenotype, rendering middle-aged individuals unable to effectively control SARS-CoV-2. A predictive signature of high-risk outcomes at day 7 of disease evolution as a tool for their early stratification in vulnerable populations is proposed.

The human cytomegalovirus-specific and UL40-mediated imprint in the natural killer cell repertoire is associated with antibody-mediated rejection in lung transplant recipients.

CD16+ natural killer (NK-) cells play, together with donor-specific antibodies (DSA) and via antibody-dependent cellular cytotoxicity (ADCC), an important role in the pathogenesis of antibody-mediated rejection (ABMR) in lung-transplant recipients (LTRs). Cytotoxic CD16+NKG2C+ NK cells proliferate in response to human Cytomegalovirus (HCMV) infections via the presentation of HCMV-encoded and highly polymorphic UL40 peptides. In our study, we aimed to clarify whether infections with HCMV-strains carrying different UL40 peptide variants are associated with the shift of the NK cell repertoire and the development of ABMR in LTRs. We included 30 DSA+ABMR+, 30 DSA+ABMR- and 90 DSA-ABMR- LTRs. In all patients, 1 episode of high-level HCMV-replication occurred. In all DSA+ABMR+ LTRs, HCMV-replication occurred prior to ABMR diagnosis. The association of HCMV UL40 variants with the expansion of CD16+ NK cell subsets and ABMR was assessed in NK cell proliferation and ADCC assays. Our study revealed that the VMAPRTLIL and VMTPRTLVL UL40 variants were significantly overrepresented in DSA+ABMR+ LTRs. Both peptides were associated with a pronounced proliferation of cytotoxic and proinflammatory CD16+NKG2C+ NK cells. The stimulation with both peptides led to a shift of the NK cell repertoire towards CD16+NKG2C+ NK cells, which was associated with strong ADCC responses after stimulation with endothelial cells and plasma from DSA+ABMR+ LTRs. Distinct UL40 peptide variants of the infecting HCMV-strain are associated with the development of ABMR after lung transplantation, due to a shift towards a highly cytotoxic CD16+NKG2C+ NK cell population. These peptides are thus potential prognostic markers for ABMR.