Natural Killer Markers Research Articles

Cytotoxic function of human natural killer cells is transiently inhibited by transforming growth factor beta (TGFβ).

e14557 Background: Natural killer (NK) cells can eliminate early neoplastic cells, however, their efficacy as an adoptive therapy for solid tumors is limited. We have shown that NK cells recovered from human kidney tumors are CD56pos/CD16dim/neg, poorly cytotoxic, and produce vascular endothelial growth factor (VEGF). These features are analogous to human decidual NK (dNK) cells that support the development and growth of the placenta during implantation. Transforming growth factor beta (TGFβ) is found at high levels in the decidua and kidney tumor environment, and is known to convert peripheral blood NK (pNK) cells to dNK-like cells in vitro. We hypothesized that TGFβ-mediated inhibition of NK cell proliferation and function is transient and therefore reversible. Methods: pNK cells were isolated from healthy donors (HD) by negative selection or purchased from ATCC (NK-92; CRL-2407). RT-qPCR and flow cytometry were used to assess NK markers: CD56, CD16 NKp46, CD49a, CD69; TGFβ receptors R1, R2, R3; VEGF; and cytokines IFNγ, TNFα. NK cells were cultured for 4-5 days with proliferative cytokines IL2 or IL15 alone and in combination, and without or with TGFβ (2-5 ng/mL). Cytotoxic function of NK cells (effectors) was quantified by lysis of human K562 cells engineered to express firefly luciferase (targets). Statistics (ANOVA/t-test) were performed using GraphPad Prism with p≤0.05 considered significant. Results: pNK cells from HD were CD56pos/CD16pos and NK-92 cells were CD56pos/CD16neg as reported. Both expressed all three TGFβ receptors, required IL2 or IL15 to survive in culture and were potently cytotoxic against K562 targets. TGFβ treatment of NK cells cultured with IL2 or IL2 plus IL15 suppressed proliferation by 20% and potently inhibited cytotoxic ability (85% reduction; p≤0.05). TGFβ caused significant changes in CD49a (35% increase), CD69 (45% increase) and IFNγ (50% decrease) but had no effect on NKp46, VEGF, or TNFα. TGFβ-mediated changes in NK cell phenotype and function reverted to normal when NK cells were returned to standard culture conditions for 4-5 days. Conclusions: Despite differences in CD16 expression, pNK cells and NK-92 cells are potently cytotoxic and represent an effective anti-cancer therapy. TGFβ limits this potential by blocking the proliferative effects of IL2 and IL15 and quelling cytotoxic function. TGFβ caused increased expression of integrin CD49a, a marker of tissue resident immune cells, and the early lymphocyte activation marker CD69. Augmentation of these surface markers was concomitant with diminution of IFN-γ, a cytokine critical for cell-mediated responses to tumors. These collective changes are akin to dNK cells that are not cytotoxic and instead support the implant. Importantly, all alterations were restored if NK cells were no longer exposed to TGFβ. Thus, inhibition of TGFβ signaling could preserve NK function in the tumor environment.

Abstract 5340: NK-humanized mouse models for the in vivo evaluation of NK cell-based therapies in solid tumors

Abstract Novel immunotherapeutic strategies targeting Natural Killer (NK) cells using monoclonal or multi-specific antibodies and immune modulator molecules are under development and suitable mouse models are needed to evaluate these therapeutics. We developed and characterized robust in vivo models that allow the engraftment and long-term maintenance of fully functional human NK (huNK) cells in immunodeficient transgenic mice for human cytokines (IL15). In efficacy studies, we confirmed that huNK cells are recruitable by NK cell-based therapies including therapeutic antibodies or multi-specific biologics and able to control tumor growth in these models.To establish a more relevant model for better clinical translatability for Fc-competent therapeutics evaluation, we optimized the model by validating a mouse strain with a functional knock-out of murine Fcγ receptors (FcgR-KO) to minimize the confounding impact of mouse immune cells. We characterized huNK cells kinetics and profile by flow cytometry compared to NOG-huIL15 mice. In efficacy studies with the FcgR-KO strain, therapeutic antibodies are inactive in non-NK-humanized mice, whereas in NK-humanized mice huNK cells can be recruited by the therapeutic antibody to control tumor growth by ADCC, in B cell lymphoma or multiple myeloma bearing mice.We also report the development of NK-humanized models for the evaluation of NK-based therapies in solid tumors. We characterized huNK intratumoral infiltration in several subcutaneous tumor xenograft models and evaluated the impact of tumor environment on the expression of functional NK markers (NKp46, NKG2D, Tim3, CD69) by flow cytometry and IHC studies.In summary, we have developed and optimized robust murine models sustaining fully functional huNK cells that can be proficiently recruited in vivo by various NK-based therapeutics. These models can also be used to evaluate the efficacy of combination therapies. Citation Format: Pauline Rettman, Samira Benhamouche-Trouillet, Michel Pelat, Katarzyna Franciszkiewicz, Georges Azar, Lydia Blot, Loreley Calvet, Sukhvinder Sidhu, Céline Nicolazzi. NK-humanized mouse models for the in vivo evaluation of NK cell-based therapies in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5340.

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.

Combination of NK and Other Immune Markers at Early Phase Stratify the Risk of Sepsis Patients: A Retrospective Study.

Immune dysfunction plays a pivotal role in sepsis pathogenesis. Previous studies have revealed the crucial role of T cells and human leukocyte antigen-DR (HLA-DR) in sepsis. However, the function of natural killer (NK) cells remains unclear. This study aimed to investigate whether NK cells are associated with sepsis prognosis. In addition, we aimed to explore the interrelation and influence between NK and other immunological features in patients with sepsis. This retrospective, observational study included patients with sepsis from two hospitals in mainland China. The clinical characteristics and immune results during the early phase were collected. Patients were classified according to the level of immune cells to analyze the relationship between immunological features and 28-day mortality. A total of 984 patients were included in this study. Non-survivors were older and had lower levels of lymphocytes, monocytes, NK cells, HLA-DR, and T cells. Patients were classified into eight groups according to their levels of NK cells, HLA-DR, and T cells. Only patients with decreased NK and T cell counts showed a significant increase in 28-day mortality. An increase in CD8+ T cells was correlated with the alleviation of 28-day mortality only among patients with high NK cell levels. This study provides novel insights into the association between NK cells and 28-day mortality as well as the interrelation between NK cells and other immune cells in sepsis. The relationship between CD8+ T cells and 28-day mortality in sepsis is dependent on NK cell count.

Pathogenetic significance of CD3+CD56+ T-lymphocytes in hemorrhagic fever with renal syndrome

Aim to evaluate the pathogenetic role of T-lymphocytes carrying the natural killer marker CD56 in hemorrhagic fever with renal syndrome (HFRS).
 Material and methods. The study group included 65 patients with HFRS receiving the inpatient treatment in the Clinics of SamSMU, and 15 healthy people were chosen for the control group. The following examinations were scheduled for all patients in the different stages of the disease: a clinical blood test, flow cytometry with a given set of monoclonal antibodies, enzyme-linked immunosorbent assay of blood serum to determine the cytokine profile. For statistical analysis we used the SPSS v.23 software.
 Results. The percentage of CD3+ CD56+ NKT blood content in comparison with those for cells of innate immunity (neutrophil granulocytes, monocytes, natural killers) and lymphocytes of adaptive immune response, as well as the level of cytokines in the blood serum showed that in polyuric and convalescent periods, the number of these cells significantly increases and remains closely embedded in correlations with other components of the immune system.
 Conclusion. A new hypothesis has been proposed to interpret the role of NKT in the development of the immune response in HFRS.

Identification of natural killer markers associated with fatal outcome in COVID-19 patients.

Increasing evidence has shown that coronavirus disease 19 (COVID-19) severity is driven by a dysregulated immunological response. Previous studies have demonstrated that natural killer (NK) cell dysfunction underpins severe illness in COVID-19 patients, but have lacked an in-depth analysis of NK cell markers as a driver of death in the most critically ill patients. We enrolled 50 non-vaccinated hospitalized patients infected with the initial virus or the alpha variant of SARS-CoV-2 with moderate or severe illness, to evaluate phenotypic and functional features of NK cells. Here, we show that, consistent with previous studies, evolution NK cells from COVID-19 patients are more activated, with the decreased activation of natural cytotoxicity receptors and impaired cytotoxicity and IFN-γ production, in association with disease regardless of the SARS-CoV-2 strain. Fatality was observed in 6 of 17 patients with severe disease; NK cells from all of these patients displayed a peculiar phenotype of an activated memory-like phenotype associated with massive TNF-α production. These data suggest that fatal COVID-19 infection is driven by an uncoordinated inflammatory response in part mediated by a specific subset of activated NK cells.

The roles of different forms of IL-15 in human melanoma progression.

Melanoma is a lethal skin cancer, and the risk of developing it is increased by exposure to ultraviolet (UV) radiation. The production of cytokines such as interleukin-15 (IL-15), induced by the exposure of skin cells to UV rays, could also promote melanoma development. The aim of this study is to investigate the possible role of Interleukin-15/Interleukin-15 Receptor α (IL-15/IL-15Rα) complexes in melanoma development. The expression of IL-15/IL-15Rα complexes by melanoma cells was evaluated both ex vivo and in vitro by tissue microarray, PCR, and flow cytometry. The presence of the soluble complex (sIL-15/IL-15Rα) in the plasma of metastatic melanoma patients was detected using an ELISA assay. Subsequently, we investigated the impact of natural killer (NK) cell activation after rIL-2 starvation followed by exposure to the sIL-15/IL-15Rα complex. Finally, by analyzing public datasets, we studied the correlation between IL-15 and IL-15Rα expressions and melanoma stage, NK and T-cell markers, and overall survival (OS). Analysis of a melanoma tissue microarray shows a significant increase in the number of IL-15+ tumor cells from the benign nevi to metastatic melanoma stages. Metastatic melanoma cell lines express a phorbol-12-myristate-13-acetate (PMA)-cleavable membrane-bound IL-15 (mbIL-15), whereas cultures from primary melanomas express a PMA-resistant isoform. Further analysis revealed that 26% of metastatic patients present with consistently high plasmatic levels of sIL-15/IL-15Rα. When the recombinant soluble human IL-15/IL-15Rα complex is added to briefly starved rIL-2-expanded NK cells, these cells exhibit strongly reduced proliferation and levels of cytotoxic activity against K-562 and NALM-18 target cells. The analysis of public gene expression datasets revealed that high IL-15 and IL-15Rα intra-tumoral production correlates with the high levels of expression of CD5+ and NKp46+ (T and NK markers) and significantly correlates with a better OS in stages II and III, but not in stage IV. Membrane-bound and secreted IL-15/IL-15Rα complexes are continuously present during progression in melanoma. It is notable that, although IL-15/IL-15Rα initially promoted the production of cytotoxic T and NK cells, at stage IV promotion of the development of anergic and dysfunctional cytotoxic NK cells was observed. In a subgroup of melanoma metastatic patients, the continuous secretion of high amounts of the soluble complex could represent a novel NK cell immune escape mechanism.

Transcriptional reprogramming of natural killer cells by vaccinia virus shows both distinct and conserved features with mCMV.

Natural killer (NK) cells have an established role in controlling poxvirus infection and there is a growing interest to exploit their capabilities in the context of poxvirus-based oncolytic therapy and vaccination. How NK cells respond to poxvirus-infected cells to become activated is not well established. To address this knowledge gap, we studied the NK cell response to vaccinia virus (VACV) in vivo, using a systemic infection murine model. We found broad alterations in NK cells transcriptional activity in VACV-infected mice, consistent with both direct target cell recognition and cytokine exposure. There were also alterations in the expression levels of specific NK surface receptors (NKRs), including the Ly49 family and SLAM receptors, as well as upregulation of memory-associated NK markers. Despite the latter observation, adoptive transfer of VACV-expercienced NK populations did not confer protection from infection. Comparison with the NK cell response to murine cytomegalovirus (MCMV) infection highlighted common features, but also distinct NK transcriptional programmes initiated by VACV. Finally, there was a clear overlap between the NK transcriptional response in humans vaccinated with an attenuated VACV, modified vaccinia Ankara (MVA), demonstrating conservation between the NK response in these different host species. Overall, this study provides new data about NK cell activation, function, and homeostasis during VACV infection, and may have implication for the design of VACV-based therapeutics.

Abstract 4247: Development of NK humanized mice models for the in vivo evaluation of NK cell engagers

Abstract Novel immunotherapeutic strategies targeting Natural Killer (NK) cells using monoclonal or multi-specific antibodies and immune modulator molecules are under development and there is a pressing need to have suitable mouse models for the evaluation of these therapeutics. To date, there are no validated in vivo mouse models that support the development of fully functional human NK cells. In recent years, the adoptive transfer of human peripheral mononuclear blood cells or CD34+ stem cells into highly immunodeficient mouse strains mostly allowed the evaluation of T cell-based therapies. However, these humanized mouse models have limitations and do not show an optimal development of innate immune cells such as NK, dendritic or myeloid cells. This is mostly due to the poor interspecies cross-reactivity of key factors necessary for cell development and maturation, like cytokines and growth factors.Here, we report the development and characterization of robust in vivo models that allow the engraftment and maintenance of fully functional human NK (huNK) cells, using several strains of immunodeficient transgenic mice for human cytokines (IL-15). The optimal parameters (source of immune cells, pre-conditioning regimen, route of administration) were identified. These models were characterized and validated by flow cytometry, and in vivo efficacy studies using an anti-CD20 Ab.In summary, we developed several murine models that exhibit significant numbers of functional huNK cells maintained over time in vivo. Different sources of human immune cell (CD34+ HSC, huNK cells) were used to implant immunodeficient huIL15 transgenic mice. HuNK cells were monitored by flow cytometry in all tested models. We showed huNK engraftment in both irradiated and non-irradiated models and confirmed the maintenance of expression of important functional NK markers on these cells. Efficacy studies performed in disseminated models showed promising results as efficacy of an anti-CD20 Ab, rituximab, was improved in presence of huNK cells, thus confirming the engagement of huNK cells in vivo in these models. Overall, we have developed several models that can sustain fully functional huNK cells over a period of several weeks that can be proficiently recruited in vivo by various NK-based therapeutics. Citation Format: Pauline Rettman, Dorothée Bourges, Laure-Marie Meyer, Ravi Rangara, Anna Ponchet-Lac, Nicolas Moindrot, Sukhvinder Sidhu, Céline Nicolazzi. Development of NK humanized mice models for the in vivo evaluation of NK cell engagers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4247.

Tissue-Resident Clonal Expansions of Rhesus Macaque NK Cells

Recent phenotypic, functional and transcriptomic analyses of natural killer (NK) cells in human and animals have established the presence of tissue resident NK(trNK) cells with specific characteristics and a central role in NK memory. The lack of endogenous clonal markers on NK cells impedes understanding the clonal genesis of trNKs. Transplantation of lentivirally-barcoded autologous hematopoietic stem and progenitor cells (HSPCs) has allowed tracking of NK cells at a clonal level in rhesus macaques (RM). We reported large KIR-restricted clonal expansions of mature CD56 -CD16 +NK in the peripheral blood (PB) of RM, clonally distinct from myeloid, T, B and CD56 +16 -NK, persisting for months to years, suggesting self-renewal independent of ongoing production from HSPCs (Wu et al, Cell Stem Cell, 2014 and Science Imm, 2018).We have now used this model to investigate the clonal distribution of NK cell populations in bone marrow (BM), liver, spleen, lymph nodes (LN), jejunum, colon and bronchoalveolar lavage (BAL). Serial of tissue biopsies were obtained from 3 barcoded monkeys by endoscopy and laparotomy overtime at steady state post transplantation , as well as tissues from necropsy. We compared clonal patterns between various trNKs and PB NKs collected from barcoded RM. Tissue or PB NK were defined as CD3-CD14-CD20-NKG2+ (NKG2A+ and NKG2C+), and NK subsets were further sorted for CD16, CD56, CD49a (putative liver tissue memory-like NK marker), and CXCR3 (critical for NK cell migration into tumor or normal tissues). The same expanded CD56 -CD16 +NK clones found in the PB were also detected at high abundance within BM, LN, liver and/or spleen CD56 -CD16 +NK, but not found in tissue CD56 +16 -NK and CD56 -16 -NK subsets. The liver and spleen bulk NK clonal patterns were highly correlated, and distinct from other tissues. We also observed tissue specific barcoded NK clones in BAL, jejunum and colon samples with no or very low abundance in other tissues and PB. Strikingly, a group of markedly expanded trNK clones, distinct from the expanded CD56 -CD16 +NK clones present concurrently or previously in PB, were present and shared across all tissues examined. These clones were enriched in CD56 -16 + trNK and absent in CD56 +16 - trNK. Notably, in both tissue and PB these clones were expanded in NKG2+ CD56-CD16- NK. These common expanded trNK cells were specifically enriched in both tissue and PB CD56 -16 -CXCR3 +NK, suggesting a role for this chemokine receptor and the ability of these clones to move between tissues. In contrast, CD49a expression did not enrich for these expanded clones.Clonally-expanded and persistent mature trNK cells, shared across multiple tissues but not present within PB mature CD56 -CD16 +NK subsets, combined with prior functional data suggesting NK memory is restricted to liver or other trNK cells, suggests these clonally-expanded trNK cells may be of interest. The pattern of shared clones across tissues, together with identification of a rare PB CD56-CD16-NK subpopulation harboring the clones, suggests preferential hematogenous homing of these clones to multiple tissues. Further analyses of gene expression and clonal dynamics are ongoing and should shed light on the ontology of trNK cells, with implications for the development of NK based immunotherapies and NK memory. DisclosuresNo relevant conflicts of interest to declare.

NK Cell Diversity Influences Outcomes after Cord Blood Transplantation

Natural killer (NK) cells are the first subset of lymphocytes to reconstitute, and likely play an important role in the graft-versus-leukemia response early after allogeneic hematopoietic stem cell transplantation. NK cells are highly diverse, and express an array of activating and inhibiting receptors, which delicately regulate their activation status.To provide a framework to better understand NK cell development and maturation after cord blood transplantation (CBT) and its implication on clinical outcomes, we designed a mass cytometry (also known as cytometry by time of flight) panel of 40 monoclonal antibodies recognizing lineage markers, NK cell receptors and transcription factors to interrogate the NK cell repertoire in prospectively collected peripheral blood (PB) samples from 60 CBT recipients. The resultant high-dimensional single cell data were analyzed by t-Distributed Stochastic Neighbor Embedding (t-SNE) dimension reduction algorithm to generate expression profile graphs.With this mass cytometry platform, we first interrogated and compared the NK cell repertoire in CB (n=10) and adult peripheral blood (PB) (n=20) and used inverse Simpson index to measure the diversity of the NK cell populations. NK cells from CB donors had a significantly lower diversity (median 537, range 311-891) compared to NK cells from healthy adult PB donors (median 2858, range 1228-5630; p<0.0001). Since CMV infection has been shown to drive NK cell maturation, we next asked if CMV infection also alters NK cell diversity by comparing the diversity of the NK cell repertoire in CB (n=10), and in PB from adult healthy donors who were either CMV-seropositive (n=10), or CMV-seronegative (n=10). The diversity of the NK cell repertoire was significantly greater in CMV seropositive healthy individuals (median 4217, range 2672-5630) compared to CMV-seronegative healthy adults (median 2247 , range 2026-2468; P=0.0002) and cord blood donors (median 537, range 311-891; P<0.0001), pointing to an important role for CMV infection in driving NK diversity.Harnessing mass cytometry, we undertook an in-depth examination of the NK repertoire in prospectively collected PB samples from 60 CBT recipients. We first sought to determine if CMV infection early after CBT can drive NK cell maturation and accelerate the acquisition of a diverse repertoire. The median time to CMV reactivation for the cohort was 71 days. CMV reactivation in the first 100 days after CBT (n=25) was associated with significantly greater NK diversity (median 844, range 198-1196) when compared to patients without CMV reactivation (median 405, range 110-889; p=0.0003). Notably, CMV augmented NK diversity mainly in the KIR+ population (median 743, range 207-1500) compared to the KIR- population (median 361, range 92-1038; p=0.007); while in the absence of CMV reactivation, NK cell diversity in the KIR+ and KIR- populations remained similar (median 305, range 102-702 vs. median 332, range 54-830; p=0.98). These data indicate that CMV infection augments NK diversity after CBT, and that this effect is most pronounced within the KIR+ population.We next determined if NK cell diversity can influence outcomes by comparing the diversity of the NK cell repertoire in PB samples collected in the first 100 days after CBT from 15 patients who went on to relapse compared to 45 patients who remained in remission for 2 years. NK cell diversity was significantly higher (median 839, range 416-1069) in patients who remained in remission compared to those who progressed and relapsed (median 446, range 328-925; p=0.03). Moreover, the greatest difference in diversity in the two groups was noted within the KIR+ population (median 788, range 101-1500 for patients in remission vs median 320, range 177-857 for patients who relapsed; p=0.002).In summary, we have used multiparametric mass cytometry to examine expression of 40 NK markers, and to gain insights into the diversity of the NK cell repertoire after CBT. We have shown that CB NK cells have a limited NK cell diversity, which increases in breadth and subsets after transplant. Moreover, we observed that CMV infection reconfigures the NK cell compartment and drives the diversity of the NK cell repertoire, especially within the KIR+ population. These data may provide the basis to rationally design therapeutic strategies that can modulate NK diversity in the settings of infection and transplant. DisclosuresNo relevant conflicts of interest to declare.

PET Imaging of the Natural Killer Cell Activation Receptor NKp30.

Redirecting the immune system in cancer treatment has led to remarkable responses in a subset of patients. Natural killer (NK) cells are innate lymphoid cells being explored as they engage tumor cells in different mechanisms compared with T cells, which could be exploited for treatment of nonresponders to current immunotherapies. NK cell therapies are monitored through measuring peripheral NK cell concentrations or changes in tumor volume over time. The former does not detect NK cells at the tumor site, and the latter is inaccurate for immunotherapies because of pseudoprogression. Therefore, new imaging methods are required as companion diagnostics for optimizing immunotherapies. Methods: In this study, we developed and completed preclinical in vivo validation of 2 antibody-based PET probes specific for NKp30, an activation natural cytotoxicity receptor expressed by human NK cells. Quantitative, multicolor flow cytometry during a variety of NK cell activation conditions was completed on primary human NK cells and the NK92MI cell line. Human renal cell carcinoma (RCC) tumors were stained for the NK cell receptors CD56, NKp30, and NKp46 to determine expression on tumor-infiltrating NK cells. An NKp30 antibody was radiolabeled with 64Cu or 89Zr and evaluated in subcutaneous xenografts and adoptive cell transfer mouse models. Results: Quantitative flow cytometry showed consistent expression of the NKp30 receptor during different activation conditions. NKp30 and NKp46 costained in RCC samples, demonstrating the expression of these receptors on tumor-infiltrating NK cells in human tumors, whereas tumor cells in one RCC sample expressed the peripheral NK marker CD56. Both PET tracers showed high stability and specificity in vitro and in vivo. Notably, 89Zr-NKp30Ab had higher on-target contrast than 64Cu-NKp30Ab at their respective terminal time points. 64Cu-NKp30Ab delineated NK cell trafficking to the liver and spleen in an adoptive cell transfer model. Conclusion: The consistent expression of NKp30 on NK cells makes it an attractive target for quantitative imaging. Immunofluorescence staining on human RCC samples demonstrated the advantages of NKp30 targeting versus CD56 for detection of tumor infiltrating NK cells. This work advances PET imaging of NK cells and supports the translation of imaging agents for immunotherapy monitoring.

Abstract A67: Augmented antitumor activity of NK-92 MI cells expressing a chimeric antigen receptor targeting human mesothelin

Abstract Adoptive natural killer (NK) cell immunotherapy using primary NK cells has been challenging, to date. The use of an ”off-the -shelf” NK cell line where multiple genetic changes can be introduced has advantages. In this study, we explored the use of the NK-92MI cell line expressing a chimeric antigen receptor (CAR) targeting human mesothelin, the current target in our human CAR-T cell therapy trials. The NK-92MI cell line (which makes its own IL-2) was purchased from ATCC and transduced with a lentivirus encoding GFP (vector control) or an anti-human mesothelin scFv-41BB-CD3 ζ second-generation CAR (MesoCAR). Addition of IL-21 enhanced transduction. The transduced cells (MesoCar-NK-92MI) were sorted by flow cytometry to achieve an expression level of &amp;gt;98%. The NK markers NKp30, NKp44, NKG2D, 2B4, and CD56 were highly expressed on the Car-NK-92MI cell line (from 75% to 99.9%). To test the antitumor effects in vitro, three types of human tumor lines transduced to express human mesothelin were tested. Lung cancer (A549Meso), mesothelioma (EMMeso), and head and neck cancer (SSC15Meso) cells were exposed to vector control NK-92MI or MesoCar-NK-92MI cells for 22 hours at varying E:T ratios. The supernatants were collected for detection of cytokines and granzyme B. We also determined the killing function of MesoCar-NK-92MI cells after irradiation of the cells at 10 Gy. To test in vivo activity, NSG mice were injected in the flank with 5 million EMMeso cells. After tumors were established, each mouse was intravenously injected with 10 million vector control NK-92MI cells or MesoCar-NK-92MI cells with a boost injection of 5 million effectors on days 4, 8, and 11. Tumor volumes were assessed over time and mice were sacrificed on day 18. In vitro killing results showed augmented antitumor effect in MesoCar-NK-92MI compared to vector control NK-92MI. Irradiated MesoCar-NK-92MI cells still possessed good antitumor activity with only 32% loss of activity on average. Increased cytokine and granzyme B secretion was also seen with the MesoCar-NK-92MI cell line (with or without irradiation) compared to NK-92MI cells. In animal studies, irradiated MesoCar-NK-92MI cells significantly inhibited tumor growth by an average of 59% with no obvious toxicity or tumor growth in other organs. The non-CAR-expressing NK-92MI cells had significantly less antitumor activity. In conclusion, use of an irradiated NK-92MI cell line expressing a CAR targeting human mesothelin could become a safe and possible “off-the-shelf” adoptive cell therapy that could be used alone or in combination with CAR-T cells or other treatments. Citation Format: Jing Sun, Patrick Woodruff, Soyeon Kim, Estela Noguera-Ortega, Evguenia Arguiri, Maria Liousia, Steven Maceko, Edmund Moon, Steven Albelda. Augmented antitumor activity of NK-92 MI cells expressing a chimeric antigen receptor targeting human mesothelin [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A67.

Abstract P3-09-18: Immunization of breast cancer patients in combination with standard-of-care chemotherapy: Results of a phase Ib clinical trial

Abstract We have developed a peptide mimotope-based cancer vaccine, P10s-PADRE, targeting tumor associated carbohydrate antigens (TACAs). We have previously shown safety, feasibility and induction of immune response in a phase I clinical trial in stage IV breast cancer patients. In the current clinical trial, immunization with P10s-PADRE vaccine is combined with the standard-of-care chemotherapy in neo-adjuvant setting in subjects diagnosed with early stage ER-positive breast cancer. Various schedules (timing of vaccination doses relative to chemo) for combination therapy were tested. Immunological endpoint variable include antibody response and activation of natural killer (NK) cells as tested in peripheral blood mononuclear cells. Tumor size before treatment and after surgery was measured for each subject. The secondary endpoint was pathologic complete response (pCR). We observed induction of antibody response and activation of NK cells independent of each other. Vaccination affected the expression of several NK markers including CD94 and NKp46 as we observed a significant upregulation of NKp46 (median = 1.79, P&amp;lt;0.0001), and CD94 (median = 1.50, P&amp;lt;0.0001) in response to immunization. The data indicate a significant increase in serum IFN-γ (Median=1.74, P=0.0016) upon vaccination. Increase in IFN-γ was positively correlated with increase in both CD69 (r = 0.46, P=0.0321) and NKp46 (r = 0.39, P=0.0763) on CD56-dim NK cells. Tumor shrinkage was associated with an increase in NK cells, upregulation of NKp46 and CD94 molecules, and increase in frequency of NKp46-positive and CD94-positive CD56-dim NK cells. Among 25 subjects enrolled in 5 different immunization schedules, 5 experienced complete eradication of their primary tumors that was associated with vaccine induced increase in the frequency of CD94-positive CD56-dim NK cells (p= 0.0473). The results are intriguing and warrant expansion of the immunologically effective combination schedule to further establish the clinical efficacy along with potential biomarkers of response. Citation Format: Issam Makhoul, Fariba Jousheghany, Saddam Mohammed, Eric Siegel, Angela Pennisi, J Thaddeus Beck, Laura Hutchins, Thomas Kieber-Emmons, Behjatolah Monzavi-Karbassi. Immunization of breast cancer patients in combination with standard-of-care chemotherapy: Results of a phase Ib clinical trial [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-09-18.

Supplementation with tocotrienols from Bixa orellana improves the in vivo efficacy of daptomycin against methicillin-resistant Staphylococcus aureus in a mouse model of infected wound

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) is a common cause of skin and soft-tissue infection worldwide. An adequate immune response acts as a first line of defence against infections and therefore plays an essential role in the maintenance of health. Tocotrienols (T3s), the lesser known isomers of vitamin E, possess many biological properties and have been recognized as immunomodulators. PurposeThe aim of this study was to investigate whether the in vivo supplementation with a mixture of 87.1% δ- and 12.9% γ-T3s extract from seeds of Bixa orellana, (T3s) could be effective in increasing the effect of daptomycin (DAP) in a mouse model of wound infection due to MRSA. Study design/methodsBacteria were inoculated onto full-thickness wound on the dorsal side of BALB/c mice at 5 × 106 CFU per mouse. Mice were randomized into five groups: an uninfected group, an infected-untreated group, a T3s-pretreated group with no antibiotics given after challenge, a T3s-pretreated group plus DAP given after challenge, a group only given DAP after challenge. Main outcome measures were: bacterial load on the wounds, analysis of Natural Killer (NK) cytotoxicity, immunological phenotype and markers of tissue repair. ResultsOur results showed that bacterial load in wounds from mice receiving T3s or DAP alone was 1- or 3-log10 lower, respectively, compared with the infected-untreated group. T3s plus daptomycin showed the highest efficacy, achieving a 4-log10 decrease in bacterial load. This higher antimicrobial effect was associated with increased levels of NK cytotoxicity and markers of wound repair. ConclusionThese data suggest that treatment with T3s may be useful for the management of infected wounds as immune adjuvants in combination with DAP.

Expression of cd35 and cd57 in placenta of women with asthma

Background. Prevalence of asthma (BA) among pregnant women reaches 12%. Obstetric complications (preeclampsia, placental insufficiency, IUGR, etc.) mainly occur in the absence of control of asthma. Aspects of the placenta status in women with chronic allergic respiratory disease are not well understood.&#x0D; Purpuses and tasks — studying the expression of markers of immature dendritic cells (CD35) and natural killer (CD57) cells in the placenta of patients with asthma, depending on the of severity, control and the type of therapy.&#x0D; Materials and methods. The study involved 200 pregnant women, including 170 with asthma (44 patients with mild intermittent, 52 with mild persistent, 61 with moderate and 13 with severe asthma), and 30 patients without pulmonary and allergic diseases. We analized complications and pregnancy outcomes, conducted the immunohistochemical study of placental samples of 134 patients for the expression of CD35 (marker of immature dendritic cells) and CD57 (marker of NK cells) on cryostat sections, immunomorphological study of the placenta with determination of the fixation of pathogenic immune complexes (PIC), IL-6, IL- 4, IL-10 in the placenta.&#x0D; Results. CD35 expression in the placenta of women with asthma is increased, while in patients undergoing preeclampsia CD35 expression is lower, especially in cases of uncontrolled asthma in the 3rd trimester. In all cases with persistent and incompletely controlled asthma, the expression of the natural killer marker (CD57) in the placenta correlates with the presence of PIC, IL-6, IL-4, IL-10 and inflammatory changes in the placenta. Conclusions. The results of the study suggest that a decrease in CD35 expression in cases with the combination of uncontrolled persistent asthma and preeclampsia seems to deplete the mechanisms of the immune response. Increased NK cell marker expression (CD57) in the placenta of women with uncontrolled and persistent asthma indicates on the cytotoxic role of this subpopulation of cells, leading to morphological and immune changes in the placenta that adversely affect the fetus and newborn.

Abstract 3244: Tumor infiltrating (TINKs) and tumor-associated (TANKs) natural killer cells (TINKs): A new paradigm in colorectal cancer

Abstract Tumor infiltrating immune cells often show a skewed phenotype that reflects attenuation of anti-tumor activity and enhancement of pro-tumor activities, including angiogenesis. Natural Killer (NK) cells are effectors lymphocytes of innate immunity, primarily involved in immunosurvelliance against tumors through their cytotoxic activity. We previously reported that NKs from Non Small Cell Lung Cancer (NSCLC) show the decidual-like CD56brightCD16-VEGFhighPlGFhighIL-8+IFNγlow phenotype [Bruno et al, Neoplasia, 2014; Bruno et al, JNCI, 2014]. Here we investigated whether tumor associated (TANKs) and tumor infiltrating (TINKs) NK cells undergo an angiogenic-switch in colorectal cancer (CRC). NK subset distribution and cytokine profiling were performed by multicolor flow cytometry, using peripheral blood and tissue samples from CRC patients. Conditioned media (CM) from FACS-sorted NKs were used either for secretomic profiling, by antibody membrane array or angiogenesis functional assay on human umbilical endothelial vein cells (HUVECs) We found that CD56brightCD16− NK cells predominate in CRC adjacent and tumor tissues, produce VEGF, PlGF, IL-8 and show impaired cytotoxicity. Further, TINK/TANKs from CRC patients express the decidual NK markers CD9 and CD49a. Secretomic analysis on CRC peripheral blood NKs revealed up regulation for several pro angiogenic factors, including Angiogenin, Angiopoietin-1/2, TIMP-1/2, Tie-2, MMP1, MMP9. Media conditioned by FACS sorted NK cells from peripheral blood and tumor tissue of CRC patients were able to induce HUVEC proliferation, migration and the formation of capillary like structures. Our data demonstrate that TINK/TANKS from CRC patients are switched toward a pro-angiogenic/pro-tumor phenotype and function. We propose that TINK/TANKs could represent the hallmark for a new paradigm in CRC inflammation. 1. Bruno A, Focaccetti C, Pagani A, Imperatori AS, Spagnoletti M, Rotolo N, Cantelmo AR, Franzi F, Capella C, Ferlazzo G, Mortara L, Albini A, Noonan DM. The proangiogenic phenotype of natural killer cells in patients with non-small cell lung cancer. Neopla sia. 2013 Feb;15(2):133-42. 2. Bruno A, Ferlazzo G, Albini A, Noonan DM. A think tank of TINK/TANKs: tumor infiltrating/tumor-associated natural killer cells in tumor progression and angiogenesis. J Natl Cancer Inst. 2014 Aug;106(8):dju200 Citation Format: Antonino Bruno, Barbara Bassani, Davide Giuseppe D’Urso, Silvia Zanellato, Elisabetta Gini, Elisa Cassinotti, Luigi Boni, Lorenzo Mortara, Adriana Albini, Douglas M. Noonan. Tumor infiltrating (TINKs) and tumor-associated (TANKs) natural killer cells (TINKs): A new paradigm in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3244.

Identification and Ex Vivo Expansion of a Circulating NK Cell Progenitor Population That Leads to Sustained Production of CD56+ NK Cells

Natural Killer (NK) cells are a component of the innate immune system that can lyse virally infected cells and tumor cells. Although IL-2 activated and ex vivo expanded NK cells are being developed for adoptive cell therapy of cancer, recent data suggests significant heterogeneity exists within these NK cell populations as defined phenotypically, functionally and developmentally. In order to maximize the efficacy of adoptive NK cell therapy, further insights into the ontogeny and behavior of various NK subsets are needed.NK cells have been historically defined as lymphocytes expressing CD56 but lacking CD3. However, we now report the identification of a population of human peripheral blood mononuclear cells that lack expression of the canonical NK markers CD56 and CD16, but otherwise share phenotypic and functional properties with NK cells, which we term "double negative (DN)" killer cells. Besides CD56 and CD16, these cells lack other standard lymphocyte markers including CD3, CD14, CD19, CD20, and CD33, although the majority of these DN cells express surface markers considered to be NK-specific, including CD8α, CD94, CD224, NKp30, CD127, NKp46, NKG2D, and NKG2A.We sorted CD3- CD19- CD14- CD20- human PBMNCs into CD16- CD56Bright, CD16+ CD56dim, CD16+ CD56- and CD16-CD56-(DN) cellular subsets by flow cytometry, then expanded these subsets in vitro by co-culture with irradiated EBV-transformed LCL feeder cells and IL-2 (500IU/mL). DN cells expanded 40-60 fold after 14 days in culture, with 55% of cells retaining the DN phenotype while 40% of cells acquired expression of CD56 but not CD16 (termed CD56SP). Generation of CD56SP cells from DN cells cultured in vitro suggests that circulating DN cells contain NK cell precursors. When DN cells were expanded for 14 days in culture and the residual remaining DN cells were then resorted and re-expanded for an additional 14 days, exponential cellular growth continued. After this addition 14 days of culture, 10-40% of cells became CD56SP NK cells, while the remainder remained DN. Repeated cycles of resorting residual DN cells demonstrated that the DN subset could continually regenerate additional DN cells as well as produce CD56SP NK cells for at least 45 days, suggesting unique proliferative and differentiative properties of this cell population.CD56SP NK cells generated in a 14 day cell culture from DN cells had up-regulation of a variety of cell surface receptors including CD30 (+57%), CD127 (+0.7%), CD161 (+69%), CD57 (+5.7%), CD8α (+40%), CD244 (+99%), NKp30 (+79%), NKp44 (+53%), CD25 (+3.5%), CD69 (+95%), and had higher intracellular IFN-γ (+35%), and perforin (+60%), suggesting both NK differentiation and education occurred in these progeny (figure). In contrast, expression of inhibitory receptors in the DN population, such as CD94 (88%), CD158e (12%) and NKG2A (24%) showed minimal change in expression levels in their progeny DN cells or CD 56SP NK cells at culture-day 14, consistent with prior studies showing inhibitory receptors are less environmentally influenced.CD56SP cells generated from the DN cells also had higher levels of bone marrow and lymph node homing receptors CXCR4 and CCR7 and possessed superior degranulation capacity against K562 cells compared to pre or post expanded DN or pre-expanded CD56SP cells (figure). Further, gene expression analysis comparing DN vs. CD56SP cells at day 0 and DN and CD56SP cells generated from DN cells at day 14, showed DN cells had a gene expression profile with enhanced expression of genes involved in processes such as cell division and replication, while CD56SP cells generated from DN cells had a more cytotoxic or "active phenotype".Conclusion: CD56- 16- DN cells share many characteristics with canonical NK cells, have long-term in vitro differentiation and proliferation capacity, and may represent a cell population that is a self-renewing progenitor for highly activated and cytotoxic CD56SP NK cells. Isolation and expansion of a progenitor population with long-term NK cell repopulating potential in adoptive NK cell immunotherapy trials could offer advantages over trials transferring more differentiated NK cells with limited in vivo proliferative capacity. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Use of Quantitative Real Time Polymerase Chain Reaction to Assess Gene Transcripts Associated With Antibody-Mediated Rejection of Kidney Transplants.

Microarray studies have shown elevated transcript levels of endothelial and natural killer (NK) cell-associated genes during antibody-mediated rejection (AMR) of the renal allograft. This study aimed to assess the use of quantitative real-time polymerase chain reaction as an alternative to microarray analysis on a subset of these elevated genes. Thirty-nine renal transplant biopsies from patients with de novo donor-specific antibodies and eighteen 1-year surveillance biopsies with no histological evidence of rejection were analyzed for expression of 11 genes previously identified as elevated in AMR. Expression levels of natural killer markers were correlated to microcirculation inflammation and graft outcomes to a greater extent than endothelial markers. Creating a predictive model reduced the number of gene transcripts to be assessed to 2, SH2D1b and MYBL1, resulting in 66.7% sensitivity and 89.7% specificity for graft loss. This work demonstrates that elevated gene expression levels, proposed to be associated with AMR, can be detected by established quantitative real-time polymerase chain reaction technology, making transition to the clinical setting feasible. Transcript analysis provides additional diagnostic information to the classification schema for AMR diagnosis but it remains to be determined whether significant numbers of centres will validate transcript analysis in their laboratories and put such analysis into clinical use.

OMIP-027: Functional analysis of human natural killer cells.

OMIP-027: Functional analysis of human natural killer cells.