Frequency Of NK Cells Research Articles

Peripheral immune profiling of soft tissue sarcoma: perspectives for disease monitoring.

Studying the tumor microenvironment and surrounding lymph nodes is the main focus of current immunological research on soft tissue sarcomas (STS). However, due to the restricted opportunity to examine tumor samples, alternative approaches are required to evaluate immune responses in non-surgical patients. Therefore, the purpose of this study was to evaluate the peripheral immune profile of STS patients, characterize patients accordingly and explore the impact of peripheral immunotypes on patient survival. Blood samples were collected from 55 STS patients and age-matched healthy donors (HD) controls. Deep immunophenotyping and gene expression analysis of whole blood was analyzed using multiparametric flow cytometry and real-time RT-qPCR, respectively. Using xMAP technology, proteomic analysis was also carried out on plasma samples. Unsupervised clustering analysis was used to classify patients based on their immune profiles to further analyze the impact of peripheral immunotypes on patient survival. Significant differences were found between STS patients and HD controls. It was found a contraction of B cells and CD4 T cells compartment, along with decreased expression levels of ICOSLG and CD40LG; a major contribution of suppressor factors, as increased frequency of M-MDSC and memory Tregs, increased expression levels of ARG1, and increased plasma levels of IL-10, soluble VISTA and soluble TIMD-4; and a compromised cytotoxic potential associated with NK and CD8 T cells, namely decreased frequency of CD56dim NK cells, and decreased levels of PRF1, GZMB, and KLRK1. In addition, the patients were classified into three peripheral immunotype groups: "immune-high," "immune-intermediate," and "immune-low." Furthermore, it was found a correlation between these immunotypes and patient survival. Patients classified as "immune-high" exhibited higher levels of immune-related factors linked to cytotoxic/effector activity and longer survival times, whereas patients classified as "immune-low" displayed higher levels of immune factors associated with immunosuppression and shorter survival times. In conclusion, it can be suggested that STS patients have a compromised systemic immunity, and the correlation between immunotypes and survival emphasizes the importance of studying peripheral blood samples in STS. Assessing the peripheral immune response holds promise as a useful method for monitoring and forecasting outcomes in STS.

Lifestyle score is associated with cellular immune profiles in healthy Tanzanian adults

Immune system and vaccine responses vary across geographical locations worldwide, not only between high and low-middle income countries (LMICs), but also between rural and urban populations within the same country. Lifestyle factors such as housing conditions, exposure to microorganisms and parasites and diet are associated with rural-and urban-living. However, the relationships between these lifestyle factors and immune profiles have not been mapped in detail. Here, we profiled the immune system of 100 healthy Tanzanians living across four rural/urban areas using mass cytometry. We developed a lifestyle score based on an individual's household assets, housing condition and recent dietary history and studied the association with cellular immune profiles. Seventeen out of 80 immune cell clusters were associated with living location or lifestyle score, with eight identifiable only using lifestyle score. Individuals with low lifestyle score, most of whom live in rural settings, showed higher frequencies of NK cells, plasmablasts, atypical memory B cells, T helper 2 cells, regulatory T cells and activated CD4+ T effector memory cells expressing CD38, HLA-DR and CTLA-4. In contrast, those with high lifestyle score, most of whom live in urban areas, showed a less activated state of the immune system illustrated by higher frequencies of naïve CD8+ T cells. Using an elastic net machine learning model, we identified cellular immune signatures most associated with lifestyle score. Assuming a link between these immune profiles and vaccine responses, these signatures may inform us on the cellular mechanisms underlying poor responses to vaccines, but also reduced autoimmunity and allergies in low- and middle-income countries.

P-436 Uterine NK cells underexpress KIR2DL1/S1 and LILRB1 receptors recognizing HLA-C2 and HLA-G on extravillous trophoblasts in reproductive failure

Abstract Study question Is there a difference in frequency, receptor expression and cytokine production of uterine NK cells (uNK) subpopulations in women with reproductive failure compared to controls? Summary answer Reproductive failure is associated with global underexpression of uNK receptors interacting with extravillous trophoblasts (EVT) that may result in defective implantation and placentation. What is known already A significant proportion of infertility, recurrent miscarriage and recurrent implantation failure are unexplained. This may be caused by immunological dysfunction at the maternal-fetal interface resulting in defective implantation and placentation. uNK is thought to play an important role in early pregnancy by cross-talking with EVT via KIR/HLA-C, LILRB1/HLA-G and CD94/HLA-E interactions to regulate EVT invasion and spiral arteriole remodeling in the decidua. Dysregulation of uNK function may cause reproductive failure; however it is unclear whether NK cell over- or under-activation is the pathology. Previous studies have been conducted on uNK obtained after miscarriage, which may be confounded by inflammatory processes. Study design, size, duration Here, we conducted a cross-sectional study to assess frequency, phenotype and function of matched pNK and uNK during the window of implantation in 16 women with reproductive failure compared to 11 controls. Women with reproductive failure cohort included unexplained subfertility (9), recurrent miscarriage (4) and recurrent implantation failure (3). The women were subsequently followed up after IVF and comparison made between women who did and did not fall pregnant. Participants/materials, setting, methods Matched peripheral blood and endometrial tissue were obtained from participants during the mid-secretory phase. Samples were accurately timed for 7-9 days after ovulation by urine LH test or histological dating. Flow cytometry was used to assess the frequency, phenotype and function. Phenotype was measured by KIR, LILRB1 and CD94 and receptor expression. Function was measured by degranulation (anti-CD107a internalization) and cytokine production (TNF-α, GM-CSF, IFN-γ and IL-8), with and without stimulation by PMA and ionomycin. Main results and the role of chance The expression of KIR2DL1/S1 and LILRB1 were significantly lower in reproductive failure group for both uNK (total uNK, uNK2 and 3) and pNK. The common finding in both uNK and pNK suggests that this is not tissue specific and may have an immunogenetic aetiology. We have previously found that degranulation peaks during secretory phase and in first trimester pregnancy. TNF-α production was also found to peak in secretory phase. Here, we found that degranulation activity of total uNK, as well as TNF-α production all uNK subsets was significantly lower in reproductive failure compared to control group. However, no significant difference was found in women in the reproductive failure cohort who fell pregnant compared to women who did not fall pregnant or miscarried after IVF. Contrary to findings from previous studies, we have not found any difference in uNK or pNK frequencies in any of the groups that were examined. Taken together, our findings suggest that reproductive failure is associated with global underexpression of receptors important for interaction with HLA-C and HLA-G on EVT, resulting in reduced uNK activation. This in turn may lead to dysregulation of implantation and placenta formation in early stage of pregnancy. Limitations, reasons for caution KIR2DL1/S1 antibody used does not distinguish between KIR2DL1 and KIR2DS1 which exerts inhibitory and activating effects respectively. Larger cohort studies on both pNK and uNK that consider KIR2DL1 and KIR2DS1 separately is needed to confirm which of these receptors are specifically reduced in reproductive failure. Wider implications of the findings Overall, our findings suggest that reproductive failure is associated with global underexpression of NK cell receptors leading to impaired implantation and placentation. This is the first study to examine uNK subsets during window of implantation and will serve as a platform to focus on particular uNK subsets in future studies. Trial registration number Not applicable

Unraveling the non-fitness status of NK cells: Examining the NKp30 receptor and its isoforms distribution in HIV/HCV coinfected patients

BackgroundHIV/HCV coinfection is associated with a rapid progression to liver damage. Specifically, NK cell population dysregulation is of particular interest, as these cells have been shown to block HCV replication effectively and have an anti-fibrogenic activity. The NKp30 receptor is linked to tumor cell lysis and has a crucial role during viral infections. In the present study, we determined the subpopulations of NK cells based on CD56 and CD16 expression, NKp30 receptor expression, its isoforms A, B, and C, along with the cytotoxicity molecules in patients with HIV/HCV. Results: evidenced by the APRI and FIB-4 indices, the HCV-infected patients presented greater liver damage than the HIV and HIV/HCV groups. The HCV group presented a decreased expression of NKp30 isoform A, and NK cell frequency was not different between groups; however, CD56brigth subpopulation, NKp30 receptor, and CD247 adaptor chain were decreased in HIV/HCV patients; further, we described increased levels of soluble IL-8, IL-10, IL-12, and IL-23 in the serum of HIV/HCV patients. Conclusions: HCV and HIV/HCV patients have multiple parameters of non-fitness status in NK cells; awareness of these dysfunctional immunological parameters in HIV/HCV and HCV patients can elucidate possible novel therapeutics directed towards the improvement of NK cell fitness status, in order to improve their function against liver damage.

Immune system evaluation in a cohort of Brazilian patients with head and neck squamous cell carcinoma who are non-smokers and alcoholic beverages abstainers.

e18030 Background: Smoking and alcohol abuse are the main risk factors for head and neck squamous cell carcinoma. However, some patients do not present clear risk factors. We are studying a cohort of Brazilian patients that are non-smokers and alcoholic beverage abstainers. Brazilian people have peculiar ethnic characteristics, reflecting a miscegenation of Europeans, Africans and Amerindians. There is evidence that ethnic background may influence germline and somatic mutations, as well as tumor behavior. One of the aims is to evaluate the immune system of this cohort of miscegenated head and neck squamous cell carcinoma patients who are non-smokers and alcoholic beverages abstainers. Methods: In this prospective study, until now we have included 16 patients: 10 women and 6 men, who were non-smokers and alcoholic abstainers. All patients answered a questionnaire and collected blood, saliva and tumor samples before treatment for analysis. Self-declared skin color was mainly brown (n=8,) followed by white (n=6), black (n=1) and yellow (n=1). The median age was 56.5 years. Patients were diagnosed with oral (n=10), glotic (n=1), oropharyngeal (N=1,) lip and gingiva (n=4) carcinoma; clinical stages I (n=3), II (n=6); III (n=1) and IVA (n=5). Fourteen patients were already operated on. Results: Peripheral blood analysis revealed that within the mononuclear cell population CD16+CD66b-HLA-DR- cells, possibly NK, were significantly more frequent in patients with cancer stage III and IVA, than in patients with cancer stages I and II. There was also a significant negative correlation between the frequency of CD16+ cells in the mononuclear population and age of the patients. Finally, we observed that in the granulocyte population, patients with lesion grade I and II have 20-fold more CD16+CD66b+PD-L1+ cells than patients with lesions grade III and IV. It is possible that an IFN gamma driven response may be, in part, responsible for the induction of PD-L1 expression in granulocytes. This hypothesis is corroborated by the positive correlation between PD-L1 expression level in the granulocyte population and the frequency of CD16+CD66b- population (p=0.01, Pearson correlation 0.62). No other parameter, which included lymphocytes T and B, and monocytes, displayed correlation with clinical data. Conclusions: Our results suggest NK and myeloid cell frequency may vary in our cohort according to lesion grade as a consequence of immune responses. However, these are preliminary results, and other parameters acquisition, as well as a larger sample, will be needed for definitive conclusions.

Functional Heterogeneity of Umbilical Cord Blood Monocyte-Derived Dendritic Cells.

Human umbilical cord blood (UCB) represents a unique resource for hematopoietic stem cell transplantation for children and patients lacking suitable donors. UCB harbors a diverse set of leukocytes such as professional APCs, including monocytes, that could act as a novel source for cellular therapies. However, the immunological properties of UCB monocytes and monocyte-derived dendritic cells (MoDCs) are not fully characterized. In this study, we characterized the phenotype and functions of UCB-MoDCs to gauge their potential for future applications. UCB exhibited higher frequencies of platelets and lymphocytes as well as lower frequencies of neutrophils in comparison with adult whole blood. Leukocyte subset evaluation revealed significantly lower frequencies of granulocytes, NK cells, and CD14+CD16- monocytes. Surface marker evaluation revealed significantly lower rates of costimulatory molecules CD80 and CD83 while chemokine receptors CCR7 and CXCR4, as well as markers for Ag presentation, were similarly expressed. UCB-MoDCs were sensitive to TLR1-9 stimulation and presented quantitative differences in the release of proinflammatory cytokines. UCB-MoDCs presented functional CCR7-, CXCR4-, and CCR5-associated migratory behavior as well as adequate receptor- and micropinocytosis-mediated Ag uptake. When cocultured with allogeneic T lymphocytes, UCB-MoDCs induced weak CD4+ T lymphocyte proliferation, CD71 expression, and release of IFN-γ and IL-2. Taken together, UCB-MoDCs present potentially advantageous properties for future medical applications.

Plasma immune signatures can predict rejection-free survival in the first year after pediatric liver transplantation

After pediatric liver transplantation (pLT), children undergo life-long immunosuppression since reliable biomarkers for the assessment of rejection probability are scarce. In the multicenter (n= 7) prospective clinical cohort "ChilSFree" study, we aimed to characterize longitudinal dynamics of soluble and cellular immune mediators during the first year after pLT and identify early biomarkers associated with outcome. Using a Luminex-based multiplex technique paired with flow cytometry, we characterized longitudinal dynamics of soluble immune mediators (SIMs, n= 50) and immune cells in the blood of 244 patients at eight visits over 1 year: before, and 7/14/21/28 days and 3/6/12 months after pLT. The unsupervised clustering of patients based on SIM profiles revealed six unique SIM signatures associated with clinical outcome. From three signatures linked to improved outcome, one was associated with 1-year-long rejection-free survival and stable graft function and was characterized by low levels of pro-inflammatory SIMs (CXCL8/9/10/12, CCL7, SCGF-β, sICAM-1), and high levels of regenerative (SCF, TNF-β) and pro-apoptotic (TRAIL) SIMs (all, p <0.001, fold change >100). Of note, this SIM signature appeared 2 weeks after pLT and remained stable over the entire year, pointing towards its potential as a novel early biomarker for minimizing or weaning immunosuppression. In the blood of these patients, a higher frequency of CD56bright natural killer cells (p <0.01), a known hallmark also associated with operationally tolerant pLT patients, was detected. The concordance of the model for prediction of rejection based on identified SIM signatures was 0.715, and 0.795, in combination with living-related transplantation as a covariate, respectively. SIM blood signatures may enable the non-invasive and early assessment of rejection risks in the first year after pLT, paving the way for improved clinical management. ChilSFree represents the largest pediatric liver transplant (pLT) cohort with paired longitudinal data on soluble immune mediators (SIMs) and immune phenotyping in the first year after pLT. SIM signatures allow for the selection of rejection-free patients 2 weeks after pLT independently of patient diagnosis, sex, or age. The SIM signatures may enable the non-invasive and early assessment of rejection risks, paving the way for minimization or withdrawal of immunosuppression after pLT.

Lower peripheral blood CD4+ lymphocyte ratio is associated with severe bronchopulmonary dysplasia.

To elucidate the characteristics of lymphocyte subsets in bronchopulmonary dysplasia (BPD) diagnosis following Jensen's criterion to understand the spectrum of lymphocytes in different degrees of BPD. This single-center retrospective cohort study included 120 neonates admitted to the neonatal intensive care unit between 1 July 2014 and 30 June 2021, who had undergone peripheral blood lymphocyte subpopulation detection. Thirty-one neonates were included in the control group, whereas 33 infants with BPD were included in the case group. In addition, we selected 56 infants with a gestational age (GA) <37 weeks without BPD who were receiving oxygen therapy. Among the three groups, the B cell and NK cell frequencies were significantly higher and the frequencies of T cells and CD4+ cells were significantly lower in the BPD group. In newborns without BPD, the distribution of T lymphocyte subsets was similar at different GAs. Comparing different degrees of BPD, the patients in the grades 2-3 BPD group had significantly lower percentages of T lymphocytes and CD4+ T cells than those in the other groups. Remarkably, the frequencies of NK cells were significantly higher in patients with grades 2-3 BPD, and the Treg cells slightly increased with BPD severity, although the differences were not significant. Healthy neonates had similar ratios of lymphocyte subsets among different GAs; although as the GAs increased, the percentage of lymphocytes increased slightly. Severe BPD was associated with lower CD4+ T cells and higher NK cells. However, whether such changes were the cause or the consequence of BPD has not been determined.

Abstract LB119: The immune-modulation of HM16390, firing up the poor tumor microenvironment to induce a potent anti-tumor efficacy

Abstract Immunotherapy, encompassing immune checkpoint blockades (ICBs) and immune stimulators, has become a widespread approach in cancer treatment. However, the effectiveness of these strategies relies significantly on the characteristics of the current tumor microenvironment (TME). This reliance underscores the urgent need for a potent immune modulator capable of inducing a favorable TME, particularly in non-immunogenic cold tumors. Here, we demonstrate that HM16390, a long acting IL-2 analog, has the potential to modify the immunogenicity of the TME by activating, expanding, and recruiting cytotoxic effector cells in cold tumor. In the murine B16F10 melanoma model, following the early upregulated proliferative potential, the proportions of CD8+ T as well as NK cells increased in both blood and tumor. While NK cell frequency initially increased and then recovered, CD8 T cells exhibited a dose-dependent upregulation in the late phase in both blood and tumor. Given the IL-2Rα (CD25) affinity of HM16390, there was an increase in Treg proportions in blood; however, intriguingly, Treg frequency decreased in the tumor. This pattern emphasizes the crucial role of HM16390 in balancing the hyper-activation of peripheral effector cells while simultaneously inducing a profoundly immune-favorable TME. Effector functions were also upregulated in both blood and tumor. Early upregulation of IFN-γ and TNF occurred in both blood and tumor, but the upregulation of granzyme B displayed a sustained expression pattern in the tumor. These immune-modulated features correlated with the exposures of HM16390, particularly noting a negative correlation between Treg down-regulation and Its exposure in blood. The immune modulations of HM16390 were validated in terms of efficacy in the Pancreatic Ductal Adenocarcinoma (PDAC) model. HM16390 was administered subcutaneously to panc02 tumor syngeneic mice via once-a-week administration for 6 weeks, with or without PD-1 combination treatment. HM16390 treatment group was well-tolerated and resulted in a dose-dependent anti-tumor effect. Notably, all mice treated with the highest dose exhibited a complete response. Furthermore, in panc02 syngeneic mice, known for a poor response to ICBs, the combination treatment of HM16390 with mAnti-PD-1 demonstrated synergistic anti-tumor effects. In conclusion, HM16390 demonstrates improved expansion and functions of effector tumor-infiltrating lymphocytes (TILs), correlating with exposure, and exhibits a safe Treg modulation pattern. These modulations culminate in a significant anti-tumor effect and synergies with PD-1 blockade in the B16F10 and PDAC model. Given that both B16F10 melanoma and Panc02 PDAC are recognized as poor immunogenic murine models with low TIL frequency, HM16390 shows promise in modifying the immunogenicity of the TME, thereby activating proper immune responses in preclinical models. Citation Format: Seongju Jeong, Yunjae Kim, Jooyun Byun, Jinyoung Kim, Jaehyuk Choi, Sungmin Bae, Daejin Kim, In Young Choi. The immune-modulation of HM16390, firing up the poor tumor microenvironment to induce a potent anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB119.

Abstract 4074: Discovery of WTX-518, an IL-18 pro-drug that is conditionally activated within the tumor microenvironment and induces regressions in mouse tumor models

Abstract Systemic administration of proinflammatory cytokines is a promising approach to treat cancer. IL-18 has been shown to promote activation of both innate and adaptive anti-tumor responses in pre-clinical models. Specifically, IL-18 has been shown to impact macrophage suppressive function as well as drive CD4+ T cells towards a TH1 phenotype. However, IL-18 therapies have been hampered by a lack of efficacy due to the suppressive activity of IL-18 binding protein (IL-18BP), which binds to IL-18 and prevents its interaction with its receptor. Several groups have taken the approach of modifying IL-18 to render it resistant to IL-18BP as a potential strategy to enhance anti-tumor activity. To explore the therapeutic benefit of delivering a conditionally-activated IL-18 to the tumor microenvironment, we have developed inducible polypeptides (INDUKINE™ molecules) consisting of mouse IL-18 or IL-18BP resistant IL-18 (BPR IL-18) tethered via protease-sensitive linkers to a high affinity antibody blockade domain, and a half-life extension (HLE) domain which improves exposure in the tumor. IL-18 INDUKINE™ molecules are inactive until reaching the tumor microenvironment, where the linkers are cleaved by intra-tumoral proteases, releasing active IL-18. Intraperitoneal (i.p.) administration of an IL-18BP resistant IL-18 (BPR IL-18) INDUKINE™ molecule led to complete tumor regression in the MC38 tumor model. In contrast, equimolar doses of wild-type IL-18 INDUKINE™ protein were not as efficacious. Moreover, BPR IL-18 INDUKINE treatment led to increased activation and frequencies of NK cells and tumor specific CD8 T cells in MC38 tumors. WTX-518 is a novel INDUKINE™ molecule that is designed to selectively deliver active human BPR IL-18 to the tumor microenvironment. WTX-518 is inducible in vitro, either when tested using a reporter assay or primary immune cells. Importantly, the active BPR IL-18 payload is resistant to IL-18BP in reporter or primary immune cell assays. WTX-518 is also selectively inducible, as incubation ex vivo with various primary human tumors led to the release of active BPR IL-18, while WTX-518 was stable in human serum and after exposure to primary cells from healthy tissues. Pharmacological and efficacy data support continued preclinical development of this innovative and differentiated engineered IL-18 therapy. Citation Format: Kristin R. Morris, Heather Brodkin, Kyriakos Economides, Daniel J. Hicklin, Julie LePrevost, Celesztina Nagy-Domonkos, Christopher Nirschl, Andres Salmeron, Cynthia Seidel-Dugan, Cierra Spencer, Zoe Steuert, William M. Winston. Discovery of WTX-518, an IL-18 pro-drug that is conditionally activated within the tumor microenvironment and induces regressions in mouse tumor models [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 4074.

Abstract 1335: Generating functional CAR-NK cells for cancer immunotherapy

Abstract Chimeric Antigen Receptor-Natural Killer (CAR-NK) cells are rapidly emerging as a promising cellular therapeutic overcoming the potential life-threatening adverse events that may result from CAR-T therapy, including cytokine release syndrome (CRS), neurotoxicity and graft-versus-host disease (GVHD). Due to their intrinsic characteristics, NK cells can kill tumor cells without the requirement for recognition of antigen in the context of MHC. This removes the constraint of HLA matching and supports antigen independent cytotoxicity through NK cell intrinsic mechanisms. As such CAR-NK cells kill via both CAR-dependent and -independent mechanisms. For these reasons, they offer the advantage of being an “off-the-shelf” product; satisfying the requirement for large-scale production of cancer immunotherapies, with the potential to reach more patients at a reduced cost. The frequency of NK cells in the blood is low (~ 2-5%), therefore improving their in vitro expansion is key to generating sufficient material to offer a viable CAR-NK cell therapy. Here we outline an in-vitro method to generate, expand, and test primary human CAR-NK cells using engineered feeder cells, and go on to demonstrate the efficacy of the CAR-NK against target cancer cells. Primary human NK cells were cultured and expanded in the presence of irradiated K562 cells (iK562) modified to express 4-1BBL and membrane bound IL-21. Following initial expansion, NK cells were transduced with a clinically tested CD19-targeting CAR lentiviral vector (LV) or GFP expressing LV; as an empty vector transduced control. Following transduction, CD19 CAR-NK and GFP-NK were enriched by FACS sorting to reach &amp;gt;80% purity and further expanded in the presence of 4-1BBL/mbIL21 iK562 feeder cells. Following sufficient expansion, cytotoxicity of fresh or cryopreserved CD19 CAR-NK cells was tested in vitro in a Tumor Killing Assay (TKA) against CD19+ or CD19- target cells. As expected, CD19 CAR-NK cells showed increased on-target cytotoxicity, increased degranulation and IFNꝩ secretion compared to controls. This effect was not observed with CD19 negative cancer cells, strongly suggesting CD19 CAR engagement of the target antigen to mediate killing. Taken together these data show the generation, expansion, and enrichment of CD19 CAR-NK cells with enhanced cytotoxic activity towards CD19+ target cells. This approach provides a platform for in vitro characterization of novel CAR-constructs, allied with in-vitro safety assessment and in vivo pharmacology testing. Together, this offers the potential to further develop new and more widely available cellular therapeutics to treat cancer. Citation Format: Karina Di Gregoli, Henry Leonard, Dan Rocca, Sabrina de Munnik, Gemma Moiset, Robert Nunan, Lauren Schewitz-Bowers, Louise Brackenbury. Generating functional CAR-NK cells for cancer immunotherapy [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 1335.

Abstract 3915: BMS-986442 (AGEN1777), a novel TIGIT/CD96 bispecific antibody, demonstrates superior monotherapy and combination activity versus conventional anti-TIGIT antibodies in preclinical models

Abstract TIGIT immune checkpoint blockade can enhance anti-tumor immunity, however conventional anti-TIGIT antibodies have shown suboptimal activity in the clinic. Here, we present the first preclinical disclosure of BMS-986442 (AGEN1777), a novel and fully human Fc-enhanced bispecific antibody against TIGIT and CD96 co-receptor, that demonstrates superior pharmacological activity compared to conventional TIGIT monospecific antibodies (mAbs). Gene expression of TIGIT, CD96, and the co-stimulatory counter-receptor CD226 in tumor samples from patients with cancer was identified using bulk and single cell RNA-sequencing. To target this interaction, Fc-engineered knob-in-hole bispecific antibodies against TIGIT and CD96 were developed and affinity optimized to achieve potent and complete blockade of TIGIT and CD96 ligand interactions. BMS-986442 and corresponding monospecific anti-TIGIT and anti-CD96 monoclonal antibodies were evaluated with or without anti-PD-1 in a variety of human T and NK cell stimulation assays. Mouse surrogate TIGIT/CD96 bispecific antibodies were developed and evaluated as monotherapy or together with anti-PD-1 in colorectal CT26 and breast orthotopic 4T1 tumor bearing mice. In CT26 and 4T1 tumor-bearing mouse models, the Fc-enhanced TIGIT/CD96 bispecific antibody demonstrated superior tumor control compared to either anti-TIGIT, anti-CD96, or the combination of anti-TIGIT and anti-CD96 mAbs. Tumor control was dependent on co-engaging activating Fcγ receptors, as an Fc-silent bispecific antibody lacked anti-tumor activity. In the tumor microenvironment, efficacy was associated with increased frequency of CD226+ effector and TCF-1+ GrzB- memory precursor CD8 T cells, increased frequency and activation of cytotoxic NK cells, and increased activation of professional APCs, including B cells, MHC Class II monocytes, and dendritic cells as determined by expression of CD40, CD83, and CD86. In primary human in vitro assays, BMS-986442 potently enhanced T cell responsiveness and activation compared with conventional anti-TIGIT mAbs alone and in combination with anti-PD-1 (nivolumab). Lastly, BMS-986442 promoted superior NK cell activation in tumor co-culture assays. In conclusion, BMS-986442 demonstrated a differentiated FcγR-dependent mechanism-of-action to enhance innate and adaptive immune responses compared with anti-TIGIT or anti-CD96 mAbs. BMS-986442 is currently advancing in phase I clinical studies in patients with advanced solid cancers (NCT05543629). Citation Format: Rebecca Ward, Nicola Ramsay, Spencer Campbell, Hema Patel, Mark Bushell, Benjamin M. Morin, Beth Wensley, David A. Savitsky, Pilar Garcia-Broncano, Bishnu Joshi, Emmanuel Briend, Olga Ignatovich, Zahra Jawad, Nils-Petter Rudqvist, Nicholas Wilson, Dhan Chand. BMS-986442 (AGEN1777), a novel TIGIT/CD96 bispecific antibody, demonstrates superior monotherapy and combination activity versus conventional anti-TIGIT antibodies in preclinical models [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 3915.

Abstract 1543: 40-color spectral flow cytometry for comprehensive immunophenotyping of murine cancers

Abstract Introduction: High-dimensional immunoprofiling is essential for studying host response to cancer immunotherapy. Synergistic agents often induce systemic changes in leukocyte abundance/function due to targeting, dosage, or delivery, necessitating multi-tissue analyses to decipher immune response mechanisms. We set out to dissect leukocyte composition in lymphoid tissues and tumors by using spectral cytometry, a multiplexed, high-throughput technology. Methods: We engineered a 40-color spectral flow cytometry panel to immunophenotype all major leukocytes and their functional subsets. Naïve and tumor-burdened C57BL/6 mice treated with combinations of agonist CD40 (aCD40) and checkpoint inhibitors were sacrificed, and tissues were processed. Stained cells were acquired on a 5-laser CyTEK Aurora. Unmixing accuracy and resolution was validated via 780 NxN plots, and high-dimensional analysis was performed. Results: Spectral cytometry affords resolution of 43+ parameters compared to 26 or less in conventional systems. We first designed a 25-marker backbone panel to identify all immune lineages. Additional channels were assigned to functional markers, allowing for profiling of migration, maturation, and activation in tumor-infiltrating leukocytes (TILs). In naïve secondary lymphoid tissues (SLTs), 35 distinct CD45+ populations were resolved. Germinal center B cells (17% vs. ~0.3% of CD45+ in other SLTs) and T follicular cells (31% vs. ~1-5% of CD4+ T cells in other SLTs) were mostly found in the Peyer’s patches, representing a hypothesized model tissue for studying tertiary immune structures in tumors. Peripheral blood contained the highest frequency of NK cells (4%), monocytes (6%), basophils (0.3%), eosinophils (4%), and neutrophils (7%) as a percentage of CD45+ cells. The spleen held the largest reservoir of CD4/8 central/effector memory cells (5% of CD45+) while ~80% of all T cells in the lymph nodes were naïve. In primary lymphoid tissues (PLTs), 12 distinct thymocyte and 7 B cell populations were resolved. Wanderlust trajectory analysis revealed CD24 and CD127 as key developmental regulators in lymphocyte differentiation. Autofluorescence extraction was essential for myeloid phenotyping. With this approach, we found monocytes comprised ~60% of TILs following aCD40 treatment (~8% in non-treatment and checkpoint only controls) and had altered macrophage differentiation fates. aCD40-treated tumors also contained higher frequencies of activated CD40/80/86+ DCs (~80/86/60% vs. ~60/51/50% of DCs in NTCs). Conclusion: This work describes a novel tool that efficiently resolves leukocytes on the axes of time, tissue, and treatment. Applications range from serial monitoring of circulating blood, endpoint analysis, validation of multiplexed-IHC, and cell sorting. PLT and SLT data has been made publicly available to serve as a training tool and reference proteomic atlas in future work. Citation Format: Aris John Kare, Lisa Nichols, Ricardo Zermeno, Marina N. Raie, Spencer K. Tumbale, Katherine W. Ferrara. 40-color spectral flow cytometry for comprehensive immunophenotyping of murine cancers [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 1543.

Abstract 5932: Immunomodulatory and anticancer effects of cyclical gemcitabine/nab-paclitaxel with continuous dosing of tinodasertib (AUM001) in preclinical KPC model

Abstract Background: PDAC has a poor 5-year survival outcome, and its aggressiveness limits the treatment efficacy. This is often driven by dysregulated mRNA translation of pro-tumorigenic genes that contribute to the proliferative and metastatic nature of PDAC. Synergizing chemotherapy with inhibition of oncogenic mRNA translation via MNK-coupled phosphorylation of eIF4E could potentially be viable. To support this hypothesis, we combined AUM001, a MNK inhibitor, with gemcitabine (GEM)-nab-paclitaxel (nPTX) with the aim of modulating tumor growth kinetics and maintaining it in a stable state. Methods: PDAC KPC cells were subcutaneously inoculated into BL6 mice and randomized to five treatment groups. Chemotherapy involved administering GEM-nPTX intraperitoneally on days 1, 4, and 8. AUM001 was given orally daily for 8 days for both mono- and combination therapies and was further administered for an additional 3 weeks in continuous dosing group. IHC staining for Ki67, MVD, and stromal markers was conducted on FFPE sections as well as Western blot analysis of tumor tissues. Apoptosis was assessed using Annexin V. Immune profiling were evaluated by flow cytometric assay and GeoMx spatial transcriptomics analysis. Results: The administration of GEM-nPTX and AUM001 (GPA) or GEM-nPTX alone (GP) led to an average 38% reduction in net tumor growth with no observed toxicity. Notably, continuous dosing of AUM001 (GPAc) following the cessation of chemotherapy exhibited a 45% decrease in net tumor growth (P=0.056) when compared to GPA. EIF4E phosphorylation and Myc expression were markedly inhibited in GPAc with lower recovery rate for MVD as compared to the GPA group at EOO (P &amp;lt; 0.05). A higher fraction of apoptotic cells was detected in the GPAc group at EOO than GPA group in reference to GP group (P=NS). GPA also resulted in a lower frequency of NK cells which was partially lifted in GPAc during observation phase. Macrophages which are known to have a pro-tumor role initially increased in frequency upon addition of AUM during chemotherapy, but subsequently decreased during observation. These modulations in infiltrating immune cells correlated with the differential expression of several immune related genes in the tumor associated CD45+ immune cells, revealed by the GeoMx analysis. Conclusions: This study showed that sustained administration of AUM001 alongside cyclical GEM-nPTX is well-tolerated with good anti-tumor outcomes. Additionally, the maintenance of AUM001 may enhance the decrease in pro-oncogenic protein expression and remodel the tumor immune microenvironment. In conclusion, continuous dosing of AUM001 following the cessation of chemotherapy could stabilize the tumor state, potentiating its application in clinical settings as maintenance therapy for PDAC. Citation Format: Wai Fook Leong, Sze Sing Lee, Jyue Yuan Lim, Siwen Sylvialin Chen, Fara Faye Desacola Regis, Romana Vidergar, Subhra K. Biswas, Balram Chowbay. Immunomodulatory and anticancer effects of cyclical gemcitabine/nab-paclitaxel with continuous dosing of tinodasertib (AUM001) in preclinical KPC 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 5932.

A single bout of vigorous intensity exercise enhances the efficacy of rituximab against human chronic lymphocytic leukaemia B-cells ex vivo

Chronic lymphocytic leukaemia (CLL) is characterised by the clonal proliferation and accumulation of mature B-cells and is often treated with rituximab, an anti-CD20 monoclonal antibody immunotherapy. Rituximab often fails to induce stringent disease eradication, due in part to failure of antibody-dependent cellular cytotoxicity (ADCC) which relies on natural killer (NK)-cells binding to rituximab-bound CD20 on B-cells. CLL cells are diffusely spread across lymphoid and other bodily tissues, and ADCC resistance in survival niches may be due to several factors including low NK-cell frequency and a suppressive stromal environment that promotes CLL cell survival. It is well established that exercise bouts induce a transient relocation of NK-cells and B-cells into peripheral blood, which could be harnessed to enhance the efficacy of rituximab in CLL by relocating both target and effector cells together with rituximab in blood. In this pilot study, n = 20 patients with treatment-naïve CLL completed a bout of cycling 15 % above anaerobic threshold for ∼ 30-minutes, with blood samples collected pre-, immediately post-, and 1-hour post-exercise. Flow cytometry revealed that exercise evoked a 254 % increase in effector (CD3−CD56+CD16+) NK-cells in blood, and a 67 % increase in CD5+CD19+CD20+ CLL cells in blood (all p < 0.005). NK-cells were isolated from blood samples pre-, and immediately post-exercise and incubated with primary isolated CLL cells with or without the presence of rituximab to determine specific lysis using a calcein-release assay. Rituximab-mediated cell lysis increased by 129 % following exercise (p < 0.001). Direct NK-cell lysis of CLL cells – independent of rituximab – was unchanged following exercise (p = 0.25). We conclude that exercise improved the efficacy of rituximab-mediated ADCC against autologous CLL cells ex vivo and propose that exercise should be explored as a means of enhancing clinical responses in patients receiving anti-CD20 immunotherapy.

Immunohistochemical Evaluation of NKP46 Receptor Expression and the Number of NK Cells in the Endometrium of Patients with Endometriosis.

Endometriosis is a medical condition that can cause infertility in women. Women with endometriosis experience a decrease in NK cell cytotoxic activity against endometrial cells, ultimately contributing to the spread of these cells. To assess the frequency of NK cells and the expression of the NKP46 receptor in endometrial tissue from patients with endometriosis using immunohistochemistry. 30 endometrial tissue specimens were collected from three groups of cases with mild (n=11), moderate (n=10), and severe endometriosis (n=9), respectively. Additionally, 20 normal endometrial tissue specimens were collected as the control group. Immunohistochemical staining was carried out using specific human monoclonal antibodies against CD56 and NKP46 molecules. Cases with severe endometriosis had a significantly higher number of CD56+ uterine NK cells (26.19±2.50) compared to fertile women (15.02±0.622) and women with mild to moderate endometriosis (p<0.001). However, there was no significant difference between the mild to moderate patients compared with the healthy women (p>0.05). Endometrial NKp46 expression was lower in women with severe endometriosis (0.447±0.0829) compared to fertile women (0.987±0.115, p=0.03). The NKp46+/CD56+ cell ratio was also lower in women with severe endometriosis (0.019±0.003) compared to fertile women (0.072±0.011, p=0.01). Women with severe endometriosis demonstrated an increased rate of infiltrated uterine NK cells and a significant decrease in NKP46 expression compared to fertile women. Therefore, NK cells and the NKp46 receptor may be involved in the development of endometriosis.

Low Percentage of Perforin-Expressing NK Cells during Severe SARS-CoV-2 Infection: Consumption Rather than Primary Deficiency.

Genetic defects in the ability to deliver effective perforin have been reported in patients with hemophagocytic lymphohistiocytosis. We tested the hypothesis that a primary perforin deficiency might also be causal in severe SARS-CoV-2 infection. We recruited 54 volunteers confirmed as being SARS-CoV-2-infected by RT-PCR and admitted to intensive care units or non-intensive care units and age- and sex-matched healthy controls. Compared with healthy controls, the percentage of perforin-expressing CD3-CD56+ NK cells quantified by flow cytometry was low in COVID-19 patients (69.9 ± 17.7 versus 78.6 ± 14.6%, p = 0.026). There was no correlation between the proportions of perforin-positive NK cells and T8 lymphocytes. Moreover, the frequency of NK cells producing perforin was neither linked to disease severity nor predictive of death. Although IL-6 is known to downregulate perforin production in NK cells, we did not find any link between perforin expression and IL-6 plasma level. However, we unveiled a negative correlation between the degranulation marker CD107a and perforin expression in NK cells (r = -0.488, p = 10-4). PRF1 gene expression and the frequency of NK cells harboring perforin were normal in patients 1 y after acute SARS-CoV-2 infection. A primary perforin defect does not seem to be a driver of COVID-19 because NK perforin expression is 1) linked neither to T8 perforin expression nor to disease severity, 2) inversely correlated with NK degranulation, and 3) normalized at distance from acute infection. Thus, the cause of low frequency of perforin-positive NK cells appears, rather, to be consumption.

Elevated levels of cell-free NKG2D-ligands modulate NKG2D surface expression and compromise NK cell function in severe COVID-19 disease.

It is now clear that coronavirus disease 19 (COVID-19) severity is associated with a dysregulated immune response, but the relative contributions of different immune cells is still not fully understood. SARS CoV-2 infection triggers marked changes in NK cell populations, but there are contradictory reports as to whether these effector lymphocytes play a protective or pathogenic role in immunity to SARS-CoV-2. To address this question we have analysed differences in the phenotype and function of NK cells in SARS-CoV-2 infected individuals who developed either very mild, or life-threatening COVID-19 disease. Although NK cells from patients with severe disease appeared more activated and the frequency of adaptive NK cells was increased, they were less potent mediators of ADCC than NK cells from patients with mild disease. Further analysis of peripheral blood NK cells in these patients revealed that a population of NK cells that had lost expression of the activating receptor NKG2D were a feature of patients with severe disease and this correlated with elevated levels of cell free NKG2D ligands, especially ULBP2 and ULBP3 in the plasma of critically ill patients. In vitro, culture in NKG2DL containing patient sera reduced the ADCC function of healthy donor NK cells and this could be blocked by NKG2DL-specific antibodies. These observations of reduced NK function in severe disease are consistent with the hypothesis that defects in immune surveillance by NK cells permit higher levels of viral replication, rather than that aberrant NK cell function contributes to immune system dysregulation and immunopathogenicity.

Unveiling the role of NK cells, NKT-like cells, and γδ cells in pathogenesis of type 1 reactions in leprosy

Leprosy is a disease with spectral clinical manifestations along with two types of reactions, type 1 reaction (T1R) and type 2 reaction (T2R). T1R especially occurs because of the defensive upgradation of cell-mediated immunity (CMI) to M. leprae antigens. T1R is the main cause of disability in leprosy. The role of conventional adaptive T cells has been well studied to understand T1R. A comprehensive understanding of the role of unconventional T cells in the manifestation of inflammation during T1R is crucial and has not been studied. In our study, we found significantly higher plasma levels of TNFα, IL1β, IL17, and IP10 in T1R when compared to non-reaction (NR). Gene expression for cytokines in blood circulation by qPCR showed significantly higher expression of IFNγ, IP10, TNFα, IL6, IL17A and chemokines CCL3, CCR1, CCR5, and CXCR3 in T1R as compared to NR. Frequencies of NKT-like cells (48.7 %) and NK cells (22.3 %) were found significantly higher in T1R in comparison to NR (36.9 %, 18.3 %, respectively) (p = 0.0001). Significantly lower levels of γδT cells (3.32 %) were observed in T1R in comparison to NR (5.16 %). The present study has provided evidence for the first time on the role of plausible unconventional T cells in the immunopathogenesis of T1R in leprosy.

Non-fatal outcomes of COVID-19 disease in pediatric organ transplantation associates with down-regulation of senescence pathways

This is a cross-sectional study examining kinetics and durability of immune response in children with solid organ transplants (SOTs) who had COVID-19 disease between November 2020 through June 2022, who were followed for 60-days at a single transplant center. Blood was collected between 1–14 (acute infection), and 15–60 days of a positive PCR (convalescence). SOT children with peripheral blood mononuclear cells (PBMC) cryopreserved before 2019 were non-infected controls (ctrls). PBMCs stimulated with 15-mer peptides from spike protein and anti-CD49d/anti-CD28. Testing done included mass cytometry, mi-RNA sequencing with confirmatory qPCR. 38 children formed the study cohort, 10 in the acute phase and 8 in the convalescence phase. 20 subjects were non-infected controls. Two subjects had severe disease. Subjects in the acute and convalescent phases were different subjects. The median age and tacrolimus level at blood draw was not significantly different. There was no death, and no subject was lost to follow-up. During acute infection CD57 expression was low in NKT, Th17 effector memory, memory Treg, CD4−CD8−, and γδT cells (p = 0.01, p = 0.04, p = 0.03, p = 0.03, p = 0.004 respectively). The frequencies of NK and Th2 effector memory cells increased (p = 0.01, p = 0.02) during acute infection. Non-switched memory B and CD8 central memory cell frequencies were decreased during acute infection (p = 0.02; p = 0.02), but the decrease in CD8 central memory cells did not persist. CD4−CD8− and CD14 monocyte frequencies increased during recovery (p = 0.03; p = 0.007). Our observations suggest down regulation of CD57 with absence of NK cell contraction protect against death from COVID-19 disease in children with SOTs.