Natural Killer Cell Populations Research Articles

Enhanced CD95 and interleukin 18 signalling accompany T cell receptor Vβ21.3+ activation in multi-inflammatory syndrome in children

Multisystem inflammatory syndrome in children is a post-infectious presentation SARS-CoV-2 associated with expansion of the T cell receptor Vβ21.3+ T-cell subgroup. Here we apply muti-single cell omics to compare the inflammatory process in children with acute respiratory COVID-19 and those presenting with non SARS-CoV-2 infections in children. Here we show that in Multi-Inflammatory Syndrome in Children (MIS-C), the natural killer cell and monocyte population demonstrate heightened CD95 (Fas) and Interleuking 18 receptor expression. Additionally, TCR Vβ21.3+ CD4+ T-cells exhibit skewed differentiation towards T helper 1, 17 and regulatory T cells, with increased expression of the co-stimulation receptors ICOS, CD28 and interleukin 18 receptor. We observe no functional evidence for NLRP3 inflammasome pathway overactivation, though MIS-C monocytes show elevated active caspase 8. This, coupled with raised IL18 mRNA expression in CD16- NK cells on single cell RNA sequencing analysis, suggests interleukin 18 and CD95 signalling may trigger activation of TCR Vβ21.3+ T-cells in MIS-C, driven by increased IL-18 production from activated monocytes and CD16- Natural Killer cells.

Eomesodermin spatiotemporally orchestrates the early and late stages of NK cell development by targeting KLF2 and T-bet, respectively.

Eomesodermin (Eomes) is a critical factor in the development of natural killer (NK) cells, but its precise role in temporal and spatial coordination during this process remains unclear. Our study revealed that Eomes plays distinct roles during the early and late stages of NK cell development. Specifically, the early deletion of Eomes via the CD122-Cre transgene resulted in significant blockade at the progenitor stage due to the downregulation of KLF2, another important transcription factor. ChIP-seq revealed direct binding of Eomes to the conserved noncoding sequence (CNS) of Klf2. Utilizing the CHimeric IMmune Editing (CHIME) technique, we found that deletion of the CNS region of Klf2 via CRISPRi led to a reduction in the NK cell population and developmental arrest. Moreover, constitutive activation of this specific CNS region through CRISPRa significantly reversed the severe defects in NK cell development caused by Eomes deficiency. Conversely, Ncr1-Cre-mediated terminal deletion of Eomes expedited the transition of NK cell subsets from the CD27+CD11b+ phenotype to the CD27-CD11b+ phenotype. Late-stage deficiency of Eomes led to a significant increase in T-bet expression, which subsequently increased the expression of the transcription factor Zeb2. Genetic deletion of one allele of Tbx21, encoding T-bet, effectively reversed the aberrant differentiation of Eomes-deficient NK cells. In summary, we utilized two innovative genetic models to elucidate the intricate mechanisms underlying Eomes-mediated NK cell commitment and differentiation.

Abstract PO4-09-09: Deep Immunoprofiling Demonstrates Racial Differences in the Peripheral Immune System in Women at Risk for Breast Cancer

Abstract Background: Disparities in breast cancer outcomes between Non-Hispanic Black (NHB) and Non-Hispanic White (NHW) women have been attributed to NHB women presenting with more aggressive subtypes and later stage disease, differences in treatment receipt and adherence, access to care and tumor biology. However, previous work has shown that these known factors do not fully account for the observed disparities in outcomes. Systemic immune fitness is a factor not previously studied that may play a role in dictating disease progression and therapeutic responses. Numerous factors, including age and race, impact immune system function and understanding these changes is critical to address disparities in breast cancer outcomes. Here, we developed a pipeline for deep immunoprofiling of peripheral blood mononuclear cells (PBMC) at single cell resolution by full-spectrum flow cytometry. Using this pipeline, we sought to investigate whether there are differences in immune fitness between NHB and NHW women at high risk for developing breast cancer. Methods: PBMC were collected and processed from age-matched NHB and NHW women (n=20/cohort) identified as being at high risk for developing breast cancer through our institution’s B-PREP (Breast Cancer Personalized Risk Assessment Education and Prevention) clinic. Two custom antibody panels and protocols for comprehensive immunoprofiling of T and B cell subsets (T/B Panel), as well as monocytic, NK, and dendritic cell subsets (M/N/D Panel), along with functional and exhaustion markers were designed, optimized, and implemented. Traditional gating strategies were used to quantify proportions of canonical cell populations, and unbiased high dimensionality reduction analysis was performed using the OMIQ analysis platform to assess phenotypes and functional status of various immune cell subsets. Results: Significant differences between cohorts in abundance as well as phenotype of various cell populations were observed. Specifically, CD4+ follicular helper T cells, TEMRA CD8, and CD45RA Terminal Effector CD8+ cells were enriched in NHB women while PD-1+/CD3+ cells and PD-1+/CD8+ cells were enriched in the NHW women (Table). Additionally, several immunosuppressive and systemic inflammatory immune cell types were enriched in NHB women (Table). Conclusions: Deep immunoprofiling using single-cell spectral flow cytometry revealed differences in peripheral immune cells between NHB and NHW women at high risk for breast cancer. The significant enrichment of immunosuppressive monocytes and several activated NK cell populations in PBMC samples from NHB women relative to those from NHW women indicate an altered immune environment that may influence breast cancer development. Additional analysis, including a larger sample size and patients with invasive cancer, are ongoing to further understand if differences in immune fitness between NHB and NHW women are associated with disparities in breast cancer outcomes. Table: PBMC populations (% of CD45+) that are significantly enriched in NHB relative to NHW patients Citation Format: Esther Ogayo, Milos Spasic, Tasnim Rahman, Olga Kantor, Tari King, Peter van Galen, Sandra McAllister, Elizabeth Mittendorf. Deep Immunoprofiling Demonstrates Racial Differences in the Peripheral Immune System in Women at Risk for Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-09-09.

Effect of propofol and isoflurane on NK Cells: A pilot study on perioperative breast cancer patients from Eastern India

Introduction: In onco-anesthesiology, the selection between intravenous and volatile anesthetics for maintaining NK cell response during cancer surgery has been an ambiguous area. In the absence of any such comparative Indian report, this pilot study aimed to investigate the effect of intravenous anesthetic, Propofol, and volatile anesthetic, Isoflurane on the NK cell-mediated immune-inflammatory response of perioperative breast cancer patients. Materials and Methods: Treatment naive breast cancer patients (N, 50) selected for surgery were randomly subjected to either Isoflurane (N, 25) or Propofol (N, 25) anesthetic group. The peripheral blood samples were collected 1 day before surgery (Pre), 1 h after incision (Intra) and 48 h after surgery (Post). The blood samples were subjected to immunophenotyping, activation, degranulation, and cytokine analysis. Results: The frequency of NK cell populations did not differ between the Isoflurane and Propofol groups irrespective of cancer stage II/III. NK cell degranulation marker CD107a and activation marker CD335 expression were significantly reduced in the postoperative period compared to the preoperative group in the Isoflurane group but Propofol did not inflict such alterations. Both Isoflurane/Propofol elicited similar trends in the cytokine profiles of interleukin (IL)-6, IL-15, interferon (IFN)-g and transforming growth factor (TGF)-β levels. Isoflurane significantly increased IL-8 during the intraoperative period and matrix metalloproteinase (MMP)-9 during the postoperative period whereas no such changes were observed with Propofol. Conclusion: This pilot study indicated that Propofol partially proved better over Isoflurane in maintaining the activation (CD335) and degranulation of NK cells (CD107a) and alleviating inflammation (IL-8 and MMP-9) during the perioperative period in the breast cancer patients.

Peripheral-central network analysis of cancer cachexia status accompanied by the polarization of hypothalamic microglia with low expression of inhibitory immune checkpoint receptors

While the excessive inflammation in cancer cachexia is well-known to be induced by the overproduction of inflammatory mediators in the periphery, microflora disruption and brain dysfunction are also considered to contribute to the induction of cancer cachexia. Hypothalamic microglia play a crucial role in brain inflammation and central-peripheral immune circuits via the production of inflammatory mediators. In the present study, we evaluated possible changes in excessive secretion of gut microbiota-derived endotoxin and the expression timeline of several inflammation-regulatory mediators and their inhibiting modulators in hypothalamic microglia of a mouse model of cancer cachexia following transplantation of pancreatic cancer cells. We demonstrated that the plasma level of lipopolysaccharide (LPS) was significantly increased with an increase in anaerobic bacteria, especially Firmicutes, in the gut at the late stage of tumor-bearing mice that exhibited dramatic appetite loss, sarcopenia and severe peripheral immune suppression. At the early stage, in which tumor-bearing mice had not yet displayed “cachexia symptoms”, the mRNA expression of pro-inflammatory cytokines, but not of the neurodegenerative and severe inflammatory modulator lipocalin-2 (LCN2), was significantly increased, whereas at the late “cachexia stage”, the level of LCN2 mRNA was significantly increased along with significant decreases in levels of inhibitory immune checkpoint receptors programmed death receptor-1 (PD-1) and CD112R in hypothalamic microglia. In addition, a high density of activated neurons in the paraventricular nucleus (PVN) of the hypothalamus region and a significant increase in corticosterone secretion were found in cachexia model mice. Related to the cachexia state, released corticosterone was clearly increased in normal mice with specific activation of PVN neurons. A marked decrease in the natural killer cell population was also observed in the spleen of mice with robust activation of PVN neurons as well as mice with cancer cachexia. On the other hand, in vivo administration of LPS in normal mice induced hypothalamic microglia with low expression of inhibitory immune checkpoint receptors. These findings suggest that the induction of cancer cachexia may parallel exacerbation of the hypothalamic inflammatory status with polarization to microglia expressed with low levels of inhibitory immune checkpoint receptors following LPS release from the gut microflora.

Multifactorial determinants of NK cell repertoire organization: insights into age, sex, KIR genotype, HLA typing, and CMV influence.

Polymorphisms in the KIR and HLA genes contribute to the diversity of the NK cell repertoire. Extrinsic factors also play a role in modifying this repertoire. The best example is cytomegalovirus, which promotes the expansion of memory-like NK cells. However, the mechanisms governing this phenotypic structure are poorly understood. Furthermore, the influence of age and sex has been understudied. In this study, we examined these parameters in a cohort of 200 healthy volunteer blood donors, focusing on the major inhibitory KIR receptors and CD94/NKG2A, as well as the differentiation marker CD57 and the memory-like population marker NKG2C. Flow cytometry and two joint analyses, unsupervised and semi-supervised, helped define the impact of various intrinsic and extrinsic markers on the phenotypic structure of the NK cell repertoire. In the KIR NK cell compartment, the KIR3DL1 gene is crucial, as unexpressed alleles lead to a repertoire dominated by KIR2D interacting only with HLA-C ligands, whereas an expressed KIR3DL1 gene allows for a greater diversity of NK cell subpopulations interacting with all HLA class I ligands. KIR2DL2 subsequently favors the KIR2D NK cell repertoire specific to C1/C2 ligands, whereas its absence promotes the expression of KIR2DL1 specific to the C2 ligand. The C2C2Bw4+ environment, marked by strong -21T motifs, favors the expansion of the NK cell population expressing only CD57, whereas the absence of HLA-A3/A11 ligands favors the population expressing only NKG2A, a population highly represented within the repertoire. The AA KIR genotype favors NK cell populations without KIR and NKG2A receptors, whereas the KIR B+ genotypes favor populations expressing KIR and NKG2A. Interestingly, we showed that women have a repertoire enriched in CD57- NK cell populations, while men have more CD57+ NK cell subpopulations. Overall, our data demonstrate that the phenotypic structure of the NK cell repertoire follows well-defined genetic rules and that immunological history, sex, and age contribute to shaping this NK cell diversity. These elements can contribute to the better selection of hematopoietic stem cell donors and the definition of allogeneic NK cells for cell engineering in NK cell-based immunotherapy approaches.cters are displayed correctly.

Generation of an Inhibitory NK Cell Subset by TGF-β1/IL-15 Polarization.

NK cells have been shown to exhibit inflammatory and immunoregulatory functions in a variety of healthy and diseased settings. In the context of chronic viral infection and cancer, distinct NK cell populations that inhibit adaptive immune responses have been observed. To understand how these cells arise and further characterize their immunosuppressive role, we examined invitro conditions that could polarize human NK cells into an inhibitory subset. TGF-β1 has been shown to induce regulatory T cells invitro and invivo; we therefore investigated if TGF-β1 could also induce immunosuppressive NK-like cells. First, we found that TGF-β1/IL-15, but not IL-15 alone, induced CD103+CD49a+ NK-like cells from peripheral blood NK cells, which expressed markers previously associated with inhibitory CD56+ innate lymphoid cells, including high expression of GITR and CD101. Moreover, supernatant from ascites collected from patients with ovarian carcinoma also induced CD103+CD49a+ NK-like cells invitro in a TGF-β-dependent manner. Interestingly, TGF-β1/IL-15-induced CD103+CD56+ NK-like cells suppressed autologous CD4+ T cells invitro by reducing absolute number, proliferation, and expression of activation marker CD25. Collectively, these findings provide new insight into how NK cells may acquire an inhibitory phenotype in TGF-β1-rich environments.

Fuyuan decoction prevents nasopharyngeal carcinoma metastasis by inhibiting circulating tumor cells/ endothelial cells interplay and enhancing anti-cancer immune response.

Distant metastasis is a major cause of treatment failure in cancer patients and a key challenge to improving cancer care today. We hypothesized that enhancing anti-cancer immune response and inhibiting circulating tumor cells (CTCs) adhesion and transendothelial migration through synergistic multi-target approaches may effectively prevent cancer metastasis. "Fuyuan Decoction" (FYD) is a traditional Chinese medicine compound that is widely used to prevent postoperative metastasis in cancer patients, but its underlying mechanism remains unclear. In this work, we systematically elucidated the underlying molecular mechanism by which FYD prevents cancer metastasis through multi-compound and multi-target synergies in vitro and in vivo. FYD significantly prevented cancer metastasis at non-cytotoxic concentrations by suppressing the adhesion of CTCs to endothelial cells and their subsequent transendothelial migration, as well as enhancing anti-cancer immune response. Mechanistically, FYD interrupts adhesion of CTCs to vascular endothelium by inhibiting TNF-α-induced CAMs expression via regulation of the NF-κB signaling pathway in endothelial cells. FYD inhibits invasion and migration of CTCs by suppressing EMT, PI3K/AKT and FAK signaling pathways. Moreover, FYD enhances the anti-cancer immune response by significantly increasing the population of Tc and NK cells in the peripheral immune system. In addition, the chemical composition of FYD was determined by UPLC-HRMS, and the results indicated that multiple compounds in FYD prevents cancer metastasis through multi-target synergistic treatment. This study provides a modern medical basis for the application of FYD in the prevention of cancer metastasis, and suggesting that multi-drug and multi-target synergistic therapy may be one of the most effective ways to prevent cancer metastasis.

Dysfunctional natural killer cells can be reprogrammed to regain anti-tumor activity.

Natural killer (NK) cells are critical to the innate immune system, as they recognize antigens without prior sensitization, and contribute to the control and clearance of viral infections and cancer. However, a significant proportion of NK cells in mice and humans do not express classical inhibitory receptors during their education process and are rendered naturally "anergic", i.e., exhibiting reduced effector functions. The molecular events leading to NK cell anergy as well as their relation to those underlying NK cell exhaustion that arises from overstimulation in chronic conditions, remain unknown. Here, we characterize the "anergic" phenotype and demonstrate functional, transcriptional, and phenotypic similarities to the "exhausted" state in tumor-infiltrating NK cells. Furthermore, we identify zinc finger transcription factor Egr2 and diacylglycerol kinase DGKα as common negative regulators controlling NK cell dysfunction. Finally, experiments in a 3D organotypic spheroid culture model and an in vivo tumor model suggest that a nanoparticle-based delivery platform can reprogram these dysfunctional natural killer cell populations in their native microenvironment. This approach may become clinically relevant for the development of novel anti-tumor immunotherapeutic strategies.

Repression of YEATS2 induces cellular senescence in hepatocellular carcinoma and inhibits tumor growth

ABSTRACT Hepatocellular carcinoma (HCC) stands as the third leading cause of cancer-related fatalities globally. In this study, we observed a significant increase in the expression level of the YEATS2 gene in HCC patients, and it is negatively correlated with the patients’ survival rate. While we have previously identified the association between YEATS2 and the survival of pancreatic cancer cells, the regulatory mechanisms and significance in HCC are still to be fully elucidated. Our study shows that knockdown (KD) of YEATS2 expression leads to DNA damage, which in turn results in an upregulation of γ-H2A.X expression and activation of the canonical senescence-related pathway p53/p21Cip1. Moreover, our transcriptomic analysis reveals that YEATS2 KD cells can enhance the expression of p21Cip1 via the c-Myc/miR-93-5p pathway, consequently fostering the senescence of HCC cells. The initiation of cellular senescence through dual-channel activation suggests that YEATS2 plays a pivotal regulatory role in the process of cell proliferation. Ultimately, our in vivo research utilizing a nude mouse tumor model revealed a notable decrease in both tumor volume and weight after the suppression of YEATS2 expression. This phenomenon is likely attributable to the attenuation of proliferative cell activity, coupled with a concurrent augmentation in the population of natural killer (NK) cells. In summary, our research results have supplemented the understanding of the regulatory mechanisms of HCC cell proliferation and indicated that targeting YEATS2 may potentially inhibit liver tumor growth.

Lingering Effects of Early Institutional Rearing and Cytomegalovirus Infection on the Natural Killer Cell Repertoire of Adopted Adolescents.

Adversity during infancy can affect neurobehavioral development and perturb the maturation of physiological systems. Dysregulated immune and inflammatory responses contribute to many of the later effects on health. Whether normalization can occur following a transition to more nurturing, benevolent conditions is unclear. To assess the potential for recovery, blood samples were obtained from 45 adolescents adopted by supportive families after impoverished infancies in institutional settings (post-institutionalized, PI). Their immune profiles were compared to 39 age-matched controls raised by their biological parents (non-adopted, NA). Leukocytes were immunophenotyped, and this analysis focuses on natural killer (NK) cell populations in circulation. Cytomegalovirus (CMV) seropositivity was evaluated to determine if early infection contributed to the impact of an atypical rearing. Associations with tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ), two cytokines released by activated NK cells, were examined. Compared to the NA controls, PI adolescents had a lower percent of CD56bright NK cells in circulation, higher TNF-α levels, and were more likely to be infected with CMV. PI adolescents who were latent carriers of CMV expressed NKG2C and CD57 surface markers on more NK cells, including CD56dim lineages. The NK cell repertoire revealed lingering immune effects of early rearing while still maintaining an overall integrity and resilience.

Preclinical Evaluation of Off-The-Shelf PD-L1+ Human Natural Killer Cells Secreting IL15 to Treat Non-Small Cell Lung Cancer.

We described previously a human natural killer (NK) cell population that upregulates PD-L1 expression upon recognizing and reacting to tumor cells or exposure to a combination of IL12, IL18 and IL15. Here, to investigate the safety and efficacy of tumor-reactive and cytokine-activated (TRACK) NK cells, human NK cells from umbilical cord blood were expanded, transduced with a retroviral vector encoding soluble (s) IL15, and further cytokine activated to induce PD-L1 expression. Our results show cryopreserved and thawed sIL15_TRACK NK cells had significantly improved cytotoxicity against non-small cell lung cancer (NSCLC) in vitro when compared to non-transduced (NT) NK cells, PD-L1+ NK cells lacking sIL15 expression (NT_TRACK NK), or NK cells expressing sIL15 without further cytokine activation (sIL15 NK cells). Intravenous injection of sIL15_TRACK NK cells into immunodeficient mice with NSCLC significantly slowed tumor growth and improved survival when compared to NT NK and sIL15 NK cells. The addition of the anti-PD-L1 atezolizumab further improved control of NSCLC growth by sIL15_TRACK NK cells in vivo. Moreover, a dose-dependent efficacy was assessed for sIL15_TRACK NK cells without observed toxicity. These experiments indicate that the administration of frozen, off-the-shelf allogeneic sIL15_TRACK NK cells is safe in preclinical models of human NSCLC and has potent antitumor activity without and with the administration of atezolizumab. A Phase I clinical trial modeled after this preclinical study using sIL15_TRACK NK cells alone or with atezolizumab for relapsed or refractory NSCLC is currently underway (NCT05334329).

306 Endothelial Infiltrating Regulatory B Cells and NK T Cells Precipitate Inflammatory Cascade That Leads to Aneurysmal Rupture

INTRODUCTION: Current literature examining the mechanisms delineating intracranial aneurysm development and subsequent rupture is sparse. Studies from decades prior defined the aneurysm model as being one largely driven by flow changes. However, the scope of these models is limited and does not fully explain why only a subset of patients with existing intracranial aneurysms subsequently go on to rupture. METHODS: We obtained aneurysm endothelial lining from 20 patients with ruptured, unruptured, sentinel hemorrhage, and recurrent aneurysms during endovascular coil embolization, which were processed and analyzed using mass spectrometry. RESULTS: Compared to unruptured aneurysms, ruptured aneurysms had significantly increased infiltrating populations of NK cells (5.5% vs 1.4%, p < 0.01), regulatory B cells (27.6% vs 16.0%, p < 0.05), and cytotoxic CD8 T cells (23.7% vs 13.5%, p < 0.05). To understand whether these cells were recruited at the time of rupture or whether they were present prior to the ictus event, we examined aneurysms that were treated after presenting with sentinel hemorrhages – again these patients had that same population of early-NK cells (7.5%, p = 0.2), suggesting that these cells were present at the nexus point immediately prior to rupture. Further, the regulatory B cells were even more prominent in the sentinel population, (45.6%, p < 0.01). CONCLUSIONS: These findings all point to a complex, coordinated immune response that is driving the remodeling of the aneurysm vessel wall. We interpret the results to suggest that regulatory B cell driven destabilization of the aneurysmal wall leads to a series of events; namely the recruitment of NK cells, subsequent inflammatory cascade, and ultimate rupture. While there remains much to be understood about this multi-faceted picture, these findings provide the basis for further studies looking at targets for adjunctive immune therapies in both our unruptured and ruptured patient populations.

Vaccination with a combination of STING agonist-loaded lipid nanoparticles and CpG-ODNs protects against lung metastasis via the induction of CD11bhighCD27low memory-like NK cells

BackgroundNatural killer (NK) cells are effective in attacking tumor cells that escape T cell attack. Memory NK cells are believed to function as potent effector cells in cancer immunotherapy. However, knowledge of their induction, identification, and potential in vivo is limited. Herein, we report on the induction and identification of memory-like NK cells via the action of a combination of a stimulator of interferon genes (STING) agonist loaded into lipid nanoparticles (STING-LNPs) and cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG-ODNs), and the potential of the inducted memory-like NK cells to prevent melanoma lung metastasis.MethodsThe antitumor effects of either the STING-LNPs, CpG-ODNs, or the combination therapy were evaluated using a B16-F10 lung metastasis model. The effect of the combined treatment was evaluated by measuring cytokine production. The induction of memory-like NK cells was demonstrated via flow cytometry and confirmed through their preventative effect.ResultsThe combination of STING-LNPs and CpG-ODNs tended to enhance the production of interleukin 12 (IL-12) and IL-18, and exerted a therapeutic effect against B16-F10 lung metastasis. The combination therapy increased the population of CD11bhighCD27low NK cells. Although monotherapies failed to show preventative effects, the combination therapy induced a surprisingly strong preventative effect, which indicates that CD11bhighCD27low cells could be a phenotype of memory-like NK cells.ConclusionAs far as could be ascertained, this is the first report of the in vivo induction, identification, and confirmation of a phenotype of the memory-like NK cells through a prophylactic effect via the use of an immunotherapeutic drug. Our findings provide novel insights into the in vivo induction of CD11bhighCD27low memory-like NK cells thus paving the way for the development of efficient immunotherapies.Graphical

Abstract 112: The NSG-Flt3KO; Tg(hFLT3LG); hIL15 mouse strain improves engraftment of innate immune cells post-CD34+ humanization

Abstract Human immune system mouse models play critical roles in numerous preclinical drug development pipelines, including immunology, infectious diseases, and immuno-oncology. These models are constantly evolving, with addition of human cytokines to sustain engraftment of human immune cells. To date, most humanized models have primarily focused on T lymphocyte cell biology, leaving room for improvement in developing models that effectively represent NK cells and the myeloid lineage. Previously, we demonstrated that the introduction of human IL-15 (NSG-IL-15) or FLT3L (NSG-FLT3L) to NOD-scid IL2rγnull (NSG) mice enhanced engraftment of innate immune cells, including human CD56+ NK cells, CD141+ and CD1c+ DC subsets, CD123+ pDCs, and CD14+ monocytes. In this study, we describe a new NSG mouse variant (NSG-Flt3KO; Tg(hFLT3LG); hIL15) that expresses physiological levels of both human IL-15 and FLT3L and detail the immune cell diversity following HSC engraftment. NSG-Flt3KO; Tg(hFLT3LG); hIL15 mice, along with control strains NSG, NSG-IL15, and NSG-FLT3L, were engrafted with human umbilical cord blood derived CD34+ hematopoietic stem cells from three different donors, and blood samples were collected until Week 20 post-transplantation. Blood samples were analyzed via flow cytometry to assess the engraftment of innate and adaptive immune cells. Spleen and bone marrow samples were collected at Week 12 post-transplantation and analyzed for lymphoid and myeloid immune cell populations via flow cytometry. To compare the differences among the strains in terms of immune cell sub-populations across weeks, a Two-Way ANOVA (mixed-model) was conducted, with p<0.05 considered statistically significant. Overall, the results demonstrate that NSG-Flt3KO; Tg(hFLT3LG); hIL15 mice have enhanced CD56+/dim CD16+ NK cells compared to NSG-FTL3L strain (20.5±12.8% vs 7.0±3.5% at Week 12), but comparable to NSG-IL15. Furthermore, myeloid sub-lineage cells in NSG-IL15-FLT3L mice, such as monocytes, macrophages (M1), granulocytes, dendritic cells, and neutrophils, were greater than in NSG or NSG-IL15 strains, but comparable to NSG-FLT3L mice. Overall levels of hCD45+ and T cell sub-populations (CD3+ or CD4+ or CD8+) were comparable across all strains tested. In sum, our NSG-Flt3KO; Tg(hFLT3LG); hIL15 engraftment data suggest an improved model that supports drug development targeting human myeloid and NK cell populations. Citation Format: Srinivas Reddy Boreddy, Shuang Hu, Brian Soper. The NSG-Flt3KO; Tg(hFLT3LG); hIL15 mouse strain improves engraftment of innate immune cells post-CD34+ humanization [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 112.

Abstract 1195: Prospective study for the identification of nivolumab biomarkers via analyses of pre-treatment plasma exosome mRNAs from head and neck cancer patients (BIONEXT)

Abstract Nivolumab paved a new way in the treatment of recurrent or metastatic (RM) head and neck squamous cell carcinoma of (HNSCC). However, the limited rates of long-term survivors (< 20%) demand a robust prognostic biomarker. This nationwide multi-centric prospective study aimed to identify a plasma exosome (PEX) mRNA signature, which serves as a companion diagnostic of nivolumab and as well provides a biological clue to develop effective therapies for a majority of non-survivors. Pre-treatment plasmas (N = 104) were subjected to comprehensive PEX mRNA analyses for prognostic marker discovery and validation. In parallel, paired treatment naïve tumor and plasma samples (N = 20) were assayed to elucidate biological implications of the PEX mRNA signature. A combination of 6 candidate PEX mRNAs plus neutrophil-to-lymphocyte ratio precisely (P = 0.000124) distinguished non-survivors from >2-yr survivor (0% vs 57.7%) with a high hazard ratio (2.878; 95% confidence interval 1.639-5.055; P = 0.0002348). In paired samples, high PEX HLA-E mRNA (a non-survivor-predicting marker) reflected the increased HLA-E protein expression (P = 0.0191) and the dense population of tumor-infiltrating NK cells (P = 0.024) in the corresponding tumor. It is estimated that the effects of PD-1blockade is canceled by the HLA-E-NKG2A immune checkpoint in patients with high PEX HLA-E mRNA. The combination of NKG2A antibody (i.e., monalizumab) and nivolumab may be a promising strategy for non-survivors predicted by a RT-qPCR-based pre-treatment measurement of PEX mRNAs. A candidate companion diagnostic of nivolumab that indicates a possible treatment option is identified. Citation Format: Muneyuki Masuda, Satoshi Toh, Mioko Matsuo, Masashi Sugasawa, Keisuke Yamazaki, Ueki Yushi, Torahiko Nakashima, Hideoki Uryu, Takeharu Ono, Tsutomu Ueda, Hirohito Umeno, Satoshi Kano, Kiyoaki Tsukahara. Prospective study for the identification of nivolumab biomarkers via analyses of pre-treatment plasma exosome mRNAs from head and neck cancer patients (BIONEXT) [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 1195.

Abstract 4032: ATRA induces an interferon response to reprogram the immunosuppressive tumor microenvironment and overcomes resistance to immune checkpoint inhibition in murine models of LKB1-deficient non-small cell lung cancer

Abstract Lung cancer remains the deadliest form of cancer, claiming 1.8 million lives worldwide in 2020. While treatment options have improved with the advent of immune checkpoint inhibitors (ICI), many patients do not respond to immunotherapy or develop resistance following initial response. A significant subset of non-small cell lung cancer (NSCLC) patients harbors somatic co-mutations in Kirsten rat sarcoma virus (KRAS) and Liver kinase 1 [LKB1, also known as serine/threonine kinase 11 (STK11)] genes, whose tumors are characterized by an immunosuppressive tumor microenvironment (TME) that are resistant to ICI. Our studies revealed that all-trans retinoic acid (ATRA), a metabolite derived from vitamin A, sensitized a murine model of NSCLC with increased tumor mutational burden (Kras G12D P53-/-Lkb1-/-; KPL-3M) to PD-1 blockade. The ATRA and anti-PD-1 combination therapy improved local and systemic T cell activation and generated systemic tumor-specific immunity. To understand ATRA-mediated anti-tumor effects, we performed single cell RNA sequencing (scRNA-seq) of KPL-3M murine tumors with or without 6 daily ATRA treatments. scRNA-seq analysis revealed a reduction of neutrophils as well as an enrichment in T cell and natural killer (NK) cell populations in the TME. Furthermore, scRNA-seq analysis revealed elevated expression of interferon (IFN) downstream genes in multiple immune subpopulations in the TME. Preliminary ex vivo studies indicated that ATRA increases NOX2 levels, intracellular reactive oxygen species (ROS), and IFN signaling in the neutrophils in KPL-3M tumors. Our findings suggest that ATRA may reshape the TME by activating IFN signaling in multiple cell subtypes to sensitize resistant tumors to ICI immunotherapy. Citation Format: William P. Crosson, Rui Li, Ramin Salehi-Rad, Raymond J. Lim, Jensen Abascal, Bitta P. Kahangi, Edgar Perez Reyes, Michael Oh, Camelia Dumitras, Nico Edgar, Ryan Chew, Rashel Jocobo, Adam Tfayli, Zhe Jing, Kostyantyn Krysan, Linh Tran, Steven M. Dubinett, Bin Liu. ATRA induces an interferon response to reprogram the immunosuppressive tumor microenvironment and overcomes resistance to immune checkpoint inhibition in murine models of LKB1-deficient non-small cell lung cancer [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 4032.

Abstract 5531: Loss of csf2(gm-csf) with cisplatin treatment decreases metastasis and improves survival in a hpv positive head and neck cancer murine model

Abstract Granulocyte macrophages colony stimulating factor (GM-CSF) also known as CSF2 is a popular cytokine responsible for homeostasis. According to TCGA databank CSF2 expression upregulated in human HPV+ (Human Papilloma Virus) Head and Neck Small Cell Carcinoma (HNSCC) compared to normal tissue. The mechanism of CSF2 in tumor progression and chemo resistance has not been well studied. Previously, our lab generated a murine model of HPV+ HNSCC that is partially responsive to cisplatin and radiation (like human disease). The role of our study is to evaluate the importance of Csf2 with cisplatin treatment (CT) in HPV + HNSCC in the murine model. Using CRISPR-Cas9 technology we knocked out the expression of the Csf2 ligand in our parental mEERL (murine E6 E7 Ras Luciferase) tumor cell line and implanted both the parental mEERL tumor and the Csf2 KO mEERL tumor in the oral cavity of C57BL6/J mice. When tumors reached a palpable size, we administered Cisplatin (20mg/m2, 3 doses total). We observed slow tumor growth in absence of Csf2 without treatment. On the other hand, we observed tumor regression in both tumor models with cisplatin treatment. Post treatment, mice with Csf2 KO tumors had a significantly prolonged rate of survival. To evaluate the role of Csf2 in the Tumor Microenvironment (TME), we collected the tumors 96 hours post CT and characterized the TME using multicolored flow cytometry. The results showed that post treatment mice with Csf2 KO tumors increased the number of the Natural Killer cell population and a surge of M-MDSC (Ly6Chi) population. The synergistic effect of knocking out Csf2 and cisplatin treatment in our tumor model appears to be driven by chemokine and cytokine changes in the TME. Simultaneously, we observed significantly lower levels of lung metastasis in Csf2 KO tumor bearing mice compared to those with parental mEERL tumors. Whereas we have seen increased metastasis in ipsilateral lymph node of Csf2 KO mice. This metastasis data mimics human disease where many patients have regional lymph node involvement, but few develop distant metastasis. These results indicate that GM-CSF (CSF2) in the TME could be an excellent target that would cause less metastasis and improved overall survival. Further investigations are needed to understand the underlying mechanism of immune response in the absence of CSF2 in the TME. Citation Format: Payal Ghosh, Craig Welbon, William C. Spanos. Loss of csf2(gm-csf) with cisplatin treatment decreases metastasis and improves survival in a hpv positive head and neck cancer murine model [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 5531.

Abstract 6397: Immune profiling for improving responsiveness to ipilimumab plus nivolumab checkpoint blockade treatment in patients with metastatic melanoma

Abstract Background:aCTLA-4 and aPD-1 combination therapy has significantly improved clinical outcomes in patients with metastatic melanoma, with 50%-60% of patients responding to treatment, but predictors of response and treatment induced immunological effects are poorly characterized. Identifying circulating immune populations as biomarkers could reveal crucial insights into the mechanisms driving treatment success. Methods:Using unbiased single-cell profiling (with cytometry by Time-Of-Flight, CyTOF), we characterized the systemic immune compartments of over 250 peripheral blood mononuclear samples from 89 patients with advanced melanoma before and during combination therapy. We evaluated the association between circulating immune populations and clinical outcome using nonparametric Mann-Whitney tests, and investigated the prognostic values of these features using survival analysis. Additionally, we assessed treatment-induced immunological effects, providing insights into the dynamic changes within the immune system during therapy. Results:We characterized circulating T cells, NK cells, B cells, and monocytes from peripheral blood. Following combination therapy, there was a shift from naïve and central memory T cells to effector populations in both CD4+ and CD8+ T cells compartments. There was also a broad activation observed in both adaptive and innate populations, characterized by an elevation of multiple co-stimulatory and co-inhibitory molecules. Lower CD8+ NK cell populations prior to combination therapy were predictive of durable clinical benefit (DCB). Pre-treatment measurements of Tregs were not predictive of outcome, but higher Tregs one month after starting therapy, as well as increases of Tregs from pre to post treatment, predicted better progression free survival (PFS) and overall survival (OS). Starting from early on treatment, higher “exhausted-like” CD8+ T cells, characterized by positive expression of at least two co-inhibitory markers, is associated with resistance. Conclusions:aCTLA-4 and aPD-1 combination therapy led to a longitudinal shift from naïve and central memory to effector phenotypes in T cells, along with a broad activation in both adaptive and innate populations. Within innate immune compartment, favorable clinical outcome was associated with lower CD8+ NK cells, capable of regulating proliferation and activation of CD4+ T cells. Interestingly, one month after the start of the therapy, the increase in Tregs and a decrease in 'exhausted-like' CD8+ T populations are associated with a better response. Collectively, these analyses suggest potential treatment-specific correlates of efficacy and may enable biomarker-selected patient populations in future. Citation Format: Joanna Baginska, Jiajia Chen, Giuseppe Tarantino, Anita Giobbie-Hurder, Jason L. Weirather, Mariano Severgnini, Michael Manos, Kelly Burke, Janice D. Russell, Martha Brainard, Elizabeth Gabriel, Ryan Brennick, Matthew Nazzaro, Emma Hathaway, Marta Holovatska, Claire Manuszak, Srinika Ranashinge, David Liu, F. Stephen Hodi. Immune profiling for improving responsiveness to ipilimumab plus nivolumab checkpoint blockade treatment in patients with metastatic melanoma [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 6397.

Overexpression of circulating CD38+ NK cells in colorectal cancer was associated with lymph node metastasis and poor prognosis.

Lymph node metastasis (LNM) is a critical prognostic factor for colorectal cancer (CRC). Due to the potential influence of immune system on CRC progression, investigation into lymphocyte subsets as clinical markers has gained attention. The objective of this study was to assess the capability of lymphocyte subsets in evaluating the lymph node status and prognosis of CRC. Lymphocyte subsets, including T cells (CD3+), natural killer cells (NK, CD3- CD56+), natural killer-like T cells (NK-like T, CD3+ CD56+), CD38+ NK cells (CD3- CD56+ CD38+) and CD38+ NK-like T cells (CD3+ CD56+ CD38+), were detected by flow cytometry. Univariate and multivariate analyses were used to assess the risk factors of LNM. The prognostic role of parameters was evaluated by survival analysis. The proportion of CD38+ NK cells within the NK cell population was significantly higher in LNM-positive patients (p <0.0001). However, no significant differences were observed in the proportions of other lymphocyte subsets. Poorer histologic grade (odds ratio [OR] =4.76, p =0.03), lymphovascular invasion (LVI) (OR =22.38, p <0.01), and CD38+ NK cells (high) (OR =4.54, p <0.01) were identified as independent risk factors for LNM. Furthermore, high proportion of CD38+ NK cells was associated with poor prognosis of CRC patients (HR=2.37, p =0.03). It was demonstrated that the proportion of CD38+ NK cells was a marker overexpressed in LNM-positive patients compared with LNM-negative patients. Moreover, an elevated proportion of CD38+ NK cells is a risk factor for LNM and poor prognosis in CRC.