Cytotoxic Lymphocyte Activity Research Articles

Breast Cancer Immune Landscape: Interplay Between Systemic and Local Immunity.

Breast cancer (BC) is one of the most common malignancies in women worldwide. Numerous studies in immuno-oncology and successful trials of immunotherapy have demonstrated the causal role of the immune system in cancer pathogenesis. The interaction between the tumor and the immune system is known to have a dual nature. Despite cytotoxic lymphocyte activity against transformed cells, a tumor can escape immune surveillance and leverage chronic inflammation to maintain its own development. Research on antitumor immunity primarily focuses on the role of the tumor microenvironment, whereas the systemic immune response beyond the tumor site is described less thoroughly. Here, a comprehensive review of the formation of the immune profile in breast cancer patients is offered. The interplay between systemic and local immune reactions as self-sustaining mechanism of tumor progression is described and the functional activity of the main cell populations related to innate and adaptive immunity is discussed. Additionally, the interaction between different functional levels of the immune system and their contribution to the development of the pro- or anti-tumor immune response in BC is highlighted. The presented data can potentially inform the development of new immunotherapy strategies in the treatment of patients with BC.

Effect of Granzyme K, FasL and Interferon-γ Expression in Placentas with Preeclampsia.

This study aimed to investigate the cytotoxic activity of decidual lymphocytes and the mRNA/protein expression of cytotoxic proteins in various cell types in the context of preeclampsia (PE) compared to those of healthy pregnancies. We analyzed fresh decidua basalis tissue and tissue embedded in paraffin (FFPE) from PE pregnancies (n = 15) and compared them with those of healthy pregnancies (n = 15) of the corresponding gestational age. Using double immunofluorescence staining, we observed differences in the intensity and distribution of staining for granzyme K (GZMK) and FasL in extravillous trophoblasts. RT-qPCR analysis of FFPE placental tissue showed that GZMK mRNA expression was statistically higher (p < 0.0001) in PE compared to that of healthy controls. On the contrary, there was a low expression (p < 0.001) of FasL mRNA in PE compared to controls, while there was no statistically significant difference for IFN-γ mRNA between PE and controls. Although the level of cytotoxic activity changed depending on the ratio of effector and target cells, there was no significant difference observed between PE and controls in this in vitro study. In conclusion, in PE, extravillous trophoblasts exhibited increased expression of GZMK and decreased expression of FasL. These changes may contribute to impaired trophoblast invasion. However, these alterations did not appear to affect the cytotoxic properties of decidual lymphocytes. Additionally, the possibility of cell sorter separation of decidual lymphocytes would greatly contribute to a better understanding of single cells' genetic profiles.

Abstract 19: Patient derived tumor-on-a-chip platform potentiates adaptive immune response against primary tumor cells

Abstract Background: Adoptive cellular therapy (ACT) is a promising antitumor immunotherapy showing success in select trials/patients. However, major challenges limit ACT in all tumors including insufficient cell numbers and failure to target heterogeneous tumors. To improve ACT, we developed a novel biomimetic device where patient-derived tumor organoids, enriched with antigen presenting cells, are cocultured with autologous peripheral blood mononuclear cells (PBMCs) in a microfluidic tumor-on-a-chip system (TOC). As PBMCs circulate through the device, they are exposed to dispersed tumor polyclonal neoantigens, leading to formation of patient- and tumor-specific organoid interacting lymphocytes (OILs). Objectives: Demonstrate that a 3D tumor-on-a-chip (TOC) platform generates organoid interacting lymphocytes (OILs) with enhanced antitumor reactivity. Methods: Specimens were collected from 15 surgical patients: melanoma (2), mesothelioma (6), and metastatic appendiceal adenocarcinoma (9). Tumor cells were combined with autologous antigen presenting cells (healthy spleen/lymph node) in a 3D matrix to generate immune-enhanced tumor organoids in the TOC system. Autologous PBMCs circulated through the system to produce OILs (7 days). Uncirculated PBMCs and tumor infiltrating lymphocytes (TILs) were cultured as controls. Resultant CD8+ controls and OILs were examined by single cell proteomics to compare cytokine secretion polyfunctionality. To assess anti-tumor reactivity, OILs and controls were reintroduced to tumor cells via coculture. Secreted bulk cytokine profiles were compared by Isoplexis CodePlex and confirmed by ELISA. NanoString Geomx spatial proteomic analysis of coculture PTOs identified key lymphocyte activity and phenotypes during anti-tumor reactivity. Coculture cytotoxic lymphocyte activation and tumor cell death was confirmed by flow cytometry. Data were analyzed using student t-test and differential expression from linear mixed-effect model. Results: Compared to controls, CD8+ OILs showed increased polyfunctionality within single cells immediately after training, particularly in effector T-cell associated cytokine profiles (INF-γ, Granzyme B, Perforin). Upon re-introduction to PTOs, OILs excreted significantly increased inflammatory-associated proteins IFN-γ, MCP-1, and Granzyme A (p&amp;lt;0.05). Furthermore, OIL re-exposure to tumor led to cytotoxic lymphocyte activation (CD8+CD69+) and significantly increased tumor cell death compared to control (p&amp;lt;0.05). Conclusion: Compared to controls, OILs produced in the TOC system show enhanced cytotoxic lymphocyte polyfunctionality and induce tumor cytotoxicity, independent of tumor type. With further validation of the OILs’ increased tumor-targeting efficacy, we propose a new potential ACT modality to target a broad array of tumors previously untreatable by current therapies. Citation Format: Cecilia R. Schaaf, Tiefu Liu, Damian Hutchins, Mitra Kooshki, Calvin Wagner, Nicholas Edenhoffer, Steven Forsythe, Robyn Greissinger, Lance Miller, Pierre Triozzi, Edward Levine, Shay Soker, Adam R. Hall, Konstantinos I. Votanopoulos. Patient derived tumor-on-a-chip platform potentiates adaptive immune response against primary tumor cells [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 19.

Abstract 1424: Epigenetic activation of tumoral IFNγresponse and pyroptosis overcomes immunotherapy resistance in hepatocellular carcinoma

Abstract Background: Immune checkpoint blockade (ICB) therapies by antibodies have revolutionized the treatment paradigm for a variety of cancers. Although subsets of people exhibit durable responses, resistance and relapse are common in hepatocellular carcinoma (HCC). We therefore aim to delineate the mechanism underlying the failure of ICB therapy in HCC, and more importantly to identify ICB-sensitizing therapeutic approaches. Methods: ICB-resistant orthotopic-grafted murine models were established via a serial selection of HCC cells in ICB-treated mice. Integrated multi-omics analysis (single-cell RNA-seq, single-cell ATAC-seq and ChIP-seq) were applied to decode the mechanisms underlying the ICB resistance and an epigenetic-mediated ICB response. Results: We successfully established the ICB-resistant murine models of HCC, in which tumor intrinsic interferon-gamma (IFNγ) response was most significantly suppressed and characterized by a “cold” tumor microenvironment with decreased lymphocyte activation and intratumoral infiltration. On the other hand, single cell RNA-seq analysis of human HCC biopsies revealed that patients with HDAC1/2/3-high HCC exhibit poor survival upon ICB therapy. Notably, we found that a novel specific HDAC1/2/3 inhibitor CXD101 synergized with PD-(L)1 antibody to induce immunogenic cell death, eradicate tumor and prolong survival in our ICB-resistant mouse models, which was accompanied by enhanced intratumoral infiltration, activation and anti-tumor memory formation of cytotoxic lymphocytes. Mechanistically, CXD101 combined with PD-(L)1 inhibition synergistically reactivated tumor IFNγ pathway via enhancing chromatin accessibility of downstream genes associated with antigen presentation and lymphocytes recruitment. Moreover, the activated immune system induced tumor cell pyroptosis, further igniting antitumor immunity and ICB response. Conclusions: Our findings suggest that corruption of tumor intrinsic IFNγ signaling may confer ICB resistance upon ICB therapy, which can be rectified by class I HDAC inhibitor mediated IFNγ response and tumor cell pyroptosis. Based on these findings, we have commenced a Phase II clinical study of a new epigenetic immunotherapy (CXD101 plus anti-PD-1) for HCC patients resisting anti-PD-(L)1 alone. This project is supported by the CRF (C4045-18W), GRF (14115820), TRS (T11-706/18-N), Li Ka Shing Foundation, CUHK Strategic Seed Funding for Collaborative Research Scheme. We also acknowledge support (funding and study medications) by Celleron Therapeutics. Citation Format: Yalin Tu, Haoran Wu, Chengpeng Zhong, Zhewen Xiong, Jing Wang, Patrick Pak-Chun Wong, Weiqin Yang, Jiahuan Lu, Zhixian Liang, Shufen Chen, Lingyun Zhang, Siyun Chen, Jingying Zhou, Ka-Fai To, Joseph Jao-Yiu Sung, Stephen Lam Chan, David Kerr, Nick La Thangue, Alfred Sze-Lok Cheng. Epigenetic activation of tumoral IFNγresponse and pyroptosis overcomes immunotherapy resistance in hepatocellular carcinoma [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 1424.

Abstract 4189: BALB/c-hIL12RB1/hIL12RB2: A robust model for preclinical research in human IL-12 therapy

Abstract Interleukin-12 (IL-12) is a multifaceted cytokine primarily produced by antigen-activated dendritic cells, macrophages, and neutrophils. Its receptor is a composite of IL12RB1 and IL12RB2, and the union of these two subunits forms a high-affinity receptor complex proficient in mediating IL-12 signaling. As a pivotal regulator of innate and adaptive immunity, IL-12 plays a crucial role in steering the differentiation of naïve T cells into Th1 cells. It is renowned as a T cell stimulatory factor, fostering T cell proliferation. Furthermore, IL-12 has the capacity to augment the activation of cytotoxic lymphocytes and natural killer cells (NK), thereby enhancing interferon (IFN-γ) production. Empirical evidence consistently demonstrates that IL-12 synergistically exerts anti-tumor effects in conjunction with various immune cells, such as T cells and NK cells. Its effectiveness in preclinical studies has solidified IL-12 as an appealing drug target, leading to the development of activating antibodies, therapeutic IL-12 proteins, and tumor vaccines as potential treatments. To delve deeper into the functionality and therapeutic potential of IL-12, we have engineered a humanized mouse model, known as BALB/c-hIL12RB1/hIL12RB2. Notably, this model fully preserves the intracellular domains of mouse IL12RB1 and IL12RB2 proteins, ensuring the normal propagation of intracellular signaling. It also effectively expresses human IL12RB1 and IL12RB2. In vitro functional validation experiments have verified that BALB/c-hIL12RB1/hIL12RB2 mice can proficiently generate IFN-γ when induced by human IL-12. Furthermore, in vivo efficacy experiments have yielded compelling results. Intratumoral administration of human IL-12 mRNA in BALB/c-hIL12RB1/hIL12RB2 mice led to a significant suppression in the growth of CT26 tumors, culminating in complete tumor regression. Subsequent rechallenge experiments conducted after the discontinuation of treatment revealed no tumor recurrence, signifying the enduring inhibitory effect of hIL12 mRNA on mouse tumors. In conclusion, the development of BALB/c-hIL12RB1/hIL12RB2 mice stands as a robust model for advancing preclinical research in the realm of human IL-12 therapy. Citation Format: Yuan Fang, Huiyi Wang, Jun Xing, Lu Yang, Jing Zhao, Xiang Gao, Cunxiang Ju. BALB/c-hIL12RB1/hIL12RB2: A robust model for preclinical research in human IL-12 therapy [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 4189.

Essential role of CD155 glycosylation in functional binding to DNAM-1 on natural killer cells.

The cluster of differentiation 155 (CD155) is highly expressed on tumor cells and augments or inhibits the cytotoxic activities of natural killer (NK) cells and T cells through its receptor ligands DNAX accessory molecule 1 (DNAM-1) and T-cell immunoglobulin (Ig) and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), respectively. Although CD155 is heavily glycosylated, the role of glycosylation of CD155 in the cytotoxic activity of effector lymphocytes remains unknown. Here, we show that the N-linked glycosylation at residue 105 (N105 glycosylation) in the first Ig-like domain of CD155 is involved in the binding of CD155 to both DNAM-1 and TIGIT. The N105 glycosylation also plays an essential role to induce signaling in both DNAM-1 and TIGIT reporter cells. Moreover, we show that the N105 glycosylation of CD155 contributes preferentially to the DNAM-1-mediated activating signal over the TIGIT-mediated inhibitory signal in NK cells. Our results demonstrated the important role of the N105 glycosylation of CD155 in DNAM-1 and TIGIT functions and shed new light on the understanding of tumor immune responses.

Activation of cytotoxic lymphocytes through CD6 enhances killing of cancer cells

Immune checkpoint inhibitors (ICIs) have demonstrated efficacy and improved survival in a growing number of cancers. Despite their success, ICIs are associated with immune-related adverse events that can interfere with their use. Therefore, safer approaches are needed. CD6, expressed by T-lymphocytes and human NK cells, engages in cell–cell interactions by binding to its ligands CD166 (ALCAM) and CD318 (CDCP1). CD6 is a target protein for regulating immune responses and is required for the development of several mouse models of autoimmunity. Interestingly, CD6 is exclusively expressed on immune cells while CD318 is strongly expressed on most cancers. Here we demonstrate that disrupting the CD6-CD318 axis with UMCD6, an anti-CD6 monoclonal antibody, prolongs survival of mice in xenograft mouse models of human breast and prostate cancer, treated with infusions of human lymphocytes. Analysis of tumor-infiltrating immune cells showed that augmentation of lymphocyte cytotoxicity by UMCD6 is due to effects of this antibody on NK, NKT and CD8 + T cells. In particular, tumor-infiltrating cytotoxic lymphocytes from UMCD6-treated mice expressed higher levels of perforin and were found in higher proportions than those from IgG-treated mice. Moreover, RNA-seq analysis of human NK-92 cells treated with UMCD6 revealed that UMCD6 up-regulates the NKG2D-DAP10 receptor complex, important in NK cell activation, as well as its downstream target PI3K. Our results now describe the phenotypic changes that occur on immune cells upon treatment with UMCD6 and further confirm that the CD6-CD318 axis can regulate the activation state of cytotoxic lymphocytes and their positioning within the tumor microenvironment.

EFFECT OF B. SUBTILIS ІМV B-7724 LECTIN ON THE ACTIVITY OF EFFECTORS OF CELLULAR ANTITUMOR IMMUNITY OF MICE WITH LEWIS LUNG CARCINOMA.

To evaluate the effect of B. subtilis IMV B-7724 lectin on the functional activity of macrophages (Mph), natural killer (NK) cells and cytotoxic lymphocytes (CTL) of mice bearing Lewis lung carcinoma (LLC). The studies were performed on C57Bl/6J mice; LLC was used as an experimental transplantable tumor. The lectin from B. subtilis IMV B-7724 was administered to LLC-bearing mice subcutaneously at a dose of 1 mg/kg of body weight for 10 days. The immunological testing was performed on days 14, 21, and 28 after tumor grafting. The cytotoxic activity of Mph, NK, and CTL was estimated in MTT-assay; the content of the stable metabolites of nitric oxide (NO) was measured by a standard Griess reaction; the arginase activity (Arg) was determined based on the measurement of urea. The administration of the B. subtilis IMV B-7724 lectin to LLC-bearing mice exerted its antitumor and antimetastatic effects partially via a significant (p < 0.05) increase of Mph and NK activities after the completion of the treatment. In the group of animals injected with lectin, the NO/Arg ratio increased significantly, indicating the prevalence of Mph with proinflammatory and antitumor properties. The cytotoxic activity of Mph exceeded the indices of untreated mice and intact control by 1.8 times and 5.3 times respectively; of NK - by 2.8 and 1.3 times respectively. The effect of treatment on the CTL activity was less pronounced. Antitumor and antimetastatic activity of the lectin from B. subtilis IMV B-7724 ensured the preservation of the cytotoxic activity of the main effectors of antitumor immunity (Mph, NK, and CTL) throughout LLC growth.

Abstract A011: Targeting the FES tyrosine kinase in antigen presenting cells to enhance anti-tumor cytotoxic lymphocyte responses

Abstract For cytotoxic lymphocytes (CTLs) to effectively kill cancer cells, inflammatory signals from antigen presenting cells (APCs) are required. Targeting the signaling pathways that regulate production of these inflammatory cytokines represents an exciting strategy to bolster CTL activation in the context of cancer immunotherapy. Achieving optimal levels of CTL activation is critical to the success of immunotherapy treatments. Compelling advancements in CTL therapies including CAR-T cells and immune checkpoint inhibitors are showing great promise for improving patient outcomes. However, enhancing the innate immune system’s ability to strengthen anti-tumor CTL immune responses has been explored to a lesser extent. We intend to fill this gap by studying the APC-intrinsic role of the non-receptor tyrosine kinase FES in regulating the production of inflammatory cytokines. The first evidence of FES’s potential immune regulating role came from observations of increased lipopolysaccharide (LPS) sensitivity in fes-null mice. A transgenic mouse model of breast cancer expressing activated HER2/Neu in the mammary glands showed delayed tumor onset in mice targeted with a fes mutation that catalytically inactivated FES; and this delay correlated with increased immune infiltration and inflammation in pre-malignant mammary tissue. Previous studies have implicated FES in the activation of the SHP-2 phosphatase in macrophages, which can suppress toll-like receptor (TLR) pathways. TLR pathways govern strong innate inflammatory responses, including the expression of the so-called signal 3 cytokines required for full CTL activation (e.g. IL-12, IFNα/β). We therefore hypothesized that disrupting FES in APCs will relieve suppression of inflammatory cytokine production pathways, leading to increased CTL activation and cancer cell cytotoxicity. To investigate the impact of FES disruption on inflammatory signaling cascades, immunoblotting analysis of proteins in these signaling pathways was conducted on LPS-stimulated fes−/− and WT mouse bone marrow derived macrophages or dendritic cells. qRT-PCR was used to measure inflammatory cytokine transcript levels, including IFNα/β, IL-12, IL-1β and TNFα. To compare the ability of WT and fes−/− macrophages to present antigen and activate CTLs, OT-1 CTLs were co-cultured with OVA-presenting WT or fes−/− macrophages; and then evaluated by flow cytometry for IFNγ production. Evidence of increased activation of downstream mediators of TLR signaling including NFκB and TBK1 were seen in fes−/− macrophages and dendritic cells post LPS stimulation. fes−/− macrophages displayed increased inflammatory cytokine mRNA production in response to LPS stimulation. Finally, CTLs showed increased IFNγ expression when co-cultured with LPS-stimulated fes−/− macrophages compared to WT macrophages, indicating a higher degree of activation by fes−/− macrophages. The immunosuppressive function of FES makes it an actionable target for improving the efficacy of cancer immunotherapies and anti-cancer adaptive immune responses. Citation Format: Natasha Dmytryk, Brian Laight, Peter Greer. Targeting the FES tyrosine kinase in antigen presenting cells to enhance anti-tumor cytotoxic lymphocyte responses [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A011.

Novel NKG2D-directed bispecific antibodies enhance antibody-mediated killing of malignant B cells by NK cells and T cells.

The activating receptor natural killer group 2, member D (NKG2D) represents an attractive target for immunotherapy as it exerts a crucial role in cancer immunosurveillance by regulating the activity of cytotoxic lymphocytes. In this study, a panel of novel NKG2D-specific single-chain fragments variable (scFv) were isolated from naïve human antibody gene libraries and fused to the fragment antigen binding (Fab) of rituximab to obtain [CD20×NKG2D] bibodies with the aim to recruit cytotoxic lymphocytes to lymphoma cells. All bispecific antibodies bound both antigens simultaneously. Two bibody constructs, [CD20×NKG2D#3] and [CD20×NKG2D#32], efficiently activated natural killer (NK) cells in co-cultures with CD20+ lymphoma cells. Both bibodies triggered NK cell-mediated lysis of lymphoma cells and especially enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) by CD38 or CD19 specific monoclonal antibodies suggesting a synergistic effect between NKG2D and FcγRIIIA signaling pathways in NK cell activation. The [CD20×NKG2D] bibodies were not effective in redirecting CD8+ T cells as single agents, but enhanced cytotoxicity when combined with a bispecific [CD19×CD3] T cell engager, indicating that NKG2D signaling also supports CD3-mediated T cell activation. In conclusion, engagement of NKG2D with bispecific antibodies is attractive to directly activate cytotoxic lymphocytes or to support their activation by monoclonal antibodies or bispecific T cell engagers. As a perspective, co-targeting of two tumor antigens may allow fine-tuning of antibody cancer therapies. Our proposed combinatorial approach is potentially applicable for many existing immunotherapies but further testing in different preclinical models is necessary to explore the full potential.

The state of the humoral and cellular links of immunity of recipient rabbits following allogeneic transfusion of erythrocyte mass

The study of activation of the humoral link of immunity of recipient rabbits following allogeneic transfusion of erythrocyte mass is important for a fundamental understanding of the formation of immunity in model species of animals. We measured the contents of B-lymphocytes, immunoglobulins of M, G, A classes, circulating immune complexes in blood of the recipient rabbits and antibody-dependent cytotoxic activity of lymphocytes. Number of B-lymphocytes in the blood of the rabbits was determined according to Bianco. Immunoglobulin content in blood serum of the rabbits was measured based on the Mancini method. The spectrophotometry method was used to measure circulating immune complexes. The antibody-dependent cytotoxic activity of lymphocytes was identified by the colorimetric method. Modeling of erythrocyte mass transfusion was performed on five clinically healthy rabbits by intravenous injection of allogeneic erythrocyte mass in the dose of 5.5 mL/kg of body weight. The materials for the study were the blood samples, gathered on the 3rd, 7th, and 23rd days after transfusion. We found that the content of B-lymphocytes in blood of the recipient rabbits increased throughout the research: on the 3rd day the content of B lymphocytes, compared with the initial state (11.4%), increased by 1.94 times and was 22.2%; on the 7th day their content increased by 2.22 times and was 25.4%; and on the 23rd day of the experiment the content of lymphocytes increased by 2.46 times, compared with the initial state, equaling 28.6%. The content of IgM on the 3rd day after transfusion of erythrocyte mass increased by 54.9% and was 2.20 ± 0.70 g/L. On the 7th day it increased by 19.0%, equaling 1.69 ± 0.44 g/L, and on the 23rd day the content of immunoglobulins decreased by 54.2% compared with the initial state (1.42 ± 0.18 g/L, intact rabbits), accounting for 0.77 ± 0.25 g/L. The content of immunoglobulins of class G on the 3rd, 7th, and 23rd days of the experiment was nearly the same as in the initial state. The content of class A immunoglobulins decreased 4.16-fold on the 3rd day of the experiment, approaching the control values afterwards. After the transfusion of erythrocyte mass to the recipient rabbits, the content of circulating immune complexes was higher compared with the initial state throughout the research: on the 3rd day the CICs content increased by 2.13 times, measuring 6.40 conventional units, and on the 7th and 23rd days it approached the initial values of the control group. On the 3rd day after transfusion, the antibody-dependent cytotoxic activity of blood lymphocytes of erythrocyte mass of the rabbits increased compared with the initial state (15.9%), measuring 17.3%; on the 7th day it was 19.4%; and on the 23rd day it increased to 27.9%. The results revealed presence of characteristic immunological changes after allogeneic transfusion of erythrocyte mass to the recipient rabbits, which were a consequence of progressive increase in the activity of the humoral link of immunity.

Perforin and granzyme A release as novel tool to measure NK cell activation in chickens

Natural killer (NK) cells are cytotoxic lymphocytes that are present in the circulation but also in many organs including spleen and gut, where they play an important role in the defense against infections. Interaction of NK cells with target cells leads to degranulation, which results in the release of perforin and granzymes in the direct vicinity of the target cell. Chicken NK cells have many characteristics similar to their mammalian counterparts and based on similarities with studies on human NK cells, surface expression of CD107 was always presumed to correlate with granule release. However, proof of this degranulation or in fact the actual presence of perforin (PFN) and granzyme A (GrA) in chicken NK cells and their release upon activation is lacking. Therefore, the purpose of the present study was to determine the presence of perforin and granzyme A in primary chicken NK cells and to measure their release upon degranulation, as an additional tool to study the function of chicken NK cells. Using human specific antibodies against PFN and GrA in fluorescent and confocal microscopy resulted in staining in chicken NK cells. The presence of PFN and GrA was also confirmed by Western blot analyses and its gene expression by PCR. Stimulation of NK cells with the pectin SPE6 followed by flow cytometry resulted in reduced levels of intracellular PFN and GrA, suggesting release of PFN and GrA. Expression of PFN and GrA reversely correlated with increased surface expression of the lysosomal marker CD107. Finally it was shown that the supernatant of activated NK cells, containing the NK cell granule content including PFN and GrA, was able to kill Escherichia coli. This study correlates PFN and GrA release to activation of chicken NK cells and establishes an additional tool to study activity of cytotoxic lymphocytes in chickens.

Immune landscape and prognostic immune-related signature in KRAS-mutated lung adenocarcinoma.

The heterogeneity of lung adenocarcinoma (LUAD) indicated that target therapies and immunotherapies may not be effective in all patients. The exploration of the feature of the immune landscape of different gene mutations may provide novel perspectives. In this study, we obtained LUAD samples from The Cancer Genome Atlas. By applying ESTIMATE and ssGSEA, KRAS-mutated group was discovered to be associated with lower immune infiltration, lower expression of immune checkpoints, especially, a lower abundance of B cell, CD8+ T cell, dendritic cell, natural killer cell, and macrophage, higher abundance of neutrophil and endothelial cell. Through ssGSEA, we found that the process of antigen-presenting cell co-inhibition and co-stimulation were inhibited, cytolytic activity and human leukocyte antigen molecules were downregulated in the KRAS-mutated group. And KRAS mutation is negatively related to antigen presentation and procession, cytotoxic lymphocyte activity, cytolytic activities, and cytokine interaction signaling pathway via gene function enrichment analysis. Finally, 24 immune-related genes were identified to establish an immune-related gene signature with excellent prognostic prediction capacity, whose 1-, 3- and 5-year AUCs were 0.893, 0.986, and 0.999. Our findings elucidate the features of the immune landscape of KRAS-mutated groups and successfully established a prognostic signature on the basis of immune-related genes in LUAD.

Изменения иммунологических показателей у пострадавших с политравмой при применении в комплексном лечении электромагнитных волн терагерцевого диапазона

Introduction Improving the results of treatment and rehabilitation is essential for polytrauma patients. The aim of the study was to prove the effectiveness of terahertz electromagnetic waves (TEW) in polytrauma patients early after traumatic event to improve immune parameters. Material and methods The study included 20 polytrauma patients who were divided into two groups. Patients of the treatment group (n = 10) received TEW early after traumatic event in addition to standard treatment using the damage control approach. Control patients (n = 10) received no TEW therapy in the complex of therapeutic measures. Immunological studies were performed during treatment. The effectiveness of rehabilitation was assessed at a long term using an evaluation scale. Results The mean rehabilitation score was significantly higher in the treatment group compared to the controls. The treatment group showed significantly higher production of TNFα and IL-2, spontaneous production of IFNγ by lymphocytes, phagocyte activity (NBT-test) and absorption activity of monocytes after the use of TEW in comparison with controls. An increased level of IgG and IgM was also noted in patients of the treatment group. Discussion The findings showed that the use of TEW in polytrauma patients early after traumatic event resulted in the increase in cytotoxic activity of lymphocytes and phagocytic cells. The immunostimulating effect of TEW was also achieved through boosting humoral immunity. Conclusion The TEW used early after traumatic event in polytrauma patients resulted in strengthening of the immune system which contributed to more effective rehabilitation.

Elucidating the role of heat shock protein receptor CD91 on antigen presenting cells during cancer immunosurveillance

Abstract The premise of immunosurveillance is that the immune system, particularly by T cells, allows detection and elimination of emerging tumor cells. However, the mechanisms that allow efficient T cell priming against nascent tumors despite limited antigen availability remain unclear. We have shown that immunogenic heat shock proteins (HSPs) such as gp96 and their receptor CD91 are one such mechanism. HSPs efficiently chaperone minute levels of peptides from tumors to elicit antigen-specific immunity. Cross presentation of HSP-chaperoned peptides by CD91 +dendritic cells is a key mechanism for priming T cell responses. Diverse DC subsets vary in their capacity to modulate such anti-tumor immune responses. In this project, we aim to understand which CD91 +DC subset(s) is/are important for HSP-mediated cancer immunosurveillance. We generated mouse strains with deletion of CD91 on specific DC subsets. cDC1s are thought to be superior cross-presenters that excel in activation of cytotoxic lymphocytes. CD91 deletion in cDC1 render mice highly susceptible to chemically induced tumors. To better understand the CD91-HSP pathway, we tracked the dispersal of tumor-derived gp96 among myeloid cells using tumor cells that express EGFP-tagged gp96. We show that migratory DC subpopulations, such as CD103 +(cDC1) dendritic cells acquire gp96 from the tumor microenvironment, while a different DC subset (CD11b +CD11c +) transfers protective antitumor immunity. The hand-off of tumor-derived gp96 between migratory and lymph node resident DCs is currently under investigation. These results advance our understanding of the HSP-CD91 pathway and its role in DC function which can have significant implications for cancer prognosis and therapy. 5R01CA233803-04

Correction of Immunological and Behavioral Parameters of Rats with Experimental Traumatic Brain Injury with a Preparation of Monoclonal Antibodies to the C3 Component of Complement

After traumatic brain injury (TBI), inflammation develops in the CNS, an active participant in which is the complement system. Activated complement fragments initiate inflammation, and subsequently significantly affect the processes of repair and regeneration. The aim of the work is to reduce neuroimmune disorders after experimental TBI by blocking excessive inflammation in the early stages of traumatic disease with monoclonal antibodies to the C3 component of complement. The work was carried out on 65 male Wistar rats using the “falling weight” model. To correct neuroinflammation, a preparation of a recombinant monoclonal antibody 3A8, specific for the C3 neodeterminant of the rat complement component, blocking the activation of the alternative complement pathway was administered (i.v., 100 mg/kg). As a reference drug, a recombinant human interleukin-1 receptor antagonist (rIL-1RA) was used, which was administered s.c. (dose of 50 mg/kg). Both drugs were administered once after 30 min of TBI (mode 1) or 24 hours after TBI (mode 2). We studied the levels of corticosterone in the blood, the cytotoxic and proliferative activity of lymphocytes, and behavioral responses in the “plus maze” test. The obtained data indicate that on the 7th day after TBI in rats treated with 3A8 antibodies in mode 1, post-traumatic weight loss was decreased, the natural cytotoxicity of splenocytes and their proliferative activity were increased, and motor and exploratory activity were increased with a significant decrease in the level of anxiety. The introduction of rIL-1RA in these regimens, as well as the combined use of both drugs, did not have a significant effect on the studied parameters.

Abstract 6367: BSI-507, a first-in-class bispecific antibody targeting PD1 and PVRIG for cancer immunotherapy

Abstract Introduction: Despite the success of anti-PD1/PDL1 therapies, only a small fraction of patients benefit from these checkpoint inhibitors (CPIs). Novel approaches to improve outcomes for patients who are resistant to current CPIs are needed. PVRIG is expressed on CD4+ and CD8+ T cells, NKT and NK cells. PVRIG binds with high affinity with its ligand PVRL2, which is expressed on tumor cells and some myeloid cells. The PVRIG-PVRL2 axis exerts an inhibitory effect on the cytotoxic activity of lymphocytes. Bispecific antibodies that exhibit dual blockade of PD1 and PVRIG provide a promising strategy to enhance anti-tumor immune response. Methods: An anti-PD1 mAb was identified from PD1 KO mice immunized with PD1-ECD-Fc and screened by our proprietary H3 (High-throughput, High-content and High-efficiency) platform. An anti-PVRIG mAb was identified from rats immunized with recombinant PVRIG-ECD-Fc and screened by the H3 platform. Both anti-PD1 and anti-PVRIG antibodies were humanized, and the anti-PD1 scFv was fused to the N-terminus of the heavy chain of the anti-PD1 antibody via a flexible linker. The binding activities and affinities were evaluated by ELISA, FACS and SPR, and the ligand blocking activities were measured by ELISA and FACS. Cell-based reporter assays were used to evaluate the functions of the anti-PD1 and anti-PVRIG mAbs alone and the bispecific antibody. In addition, the activity of the bispecific antibody to reverse PD1 and PVRIG mediated suppression of CMV pp65495-503 antigen specific CD8+ T-cell cytotoxicity was evaluated. Results: BSI-507, an anti-PD1xanti-PVRIG bispecific antibody demonstrated comparable activity to the parental anti-PD1 antibody regarding PD1 binding and PD1/PDL1 blocking. It also exhibited comparable activity to the parental anti-PVRIG antibody in PVRIG binding and PVRIG/PVRL2 blocking. Based on cell-based reporter assays, BSI-507 was able to reverse either PD1 or PVRIG mediated T-cell suppression and exhibited comparable potency to the parental antibodies. BSI-507 was also able to show enhanced reversal of both PD1 and PVRIG mediated T-cell suppression, much better than anti-PD1 or anti-PVRIG alone. In addition, BSI-507 showed the ability to reverse PD1 and PVRIG mediated suppression of CMV pp65495-503 antigen specific CD8+ T-cell cytotoxicity, stronger than either the anti-PD1 or anti-PVRIG monoclonal antibody. Summary: BSI-507 is a first-in-class anti-PD1xanti-PVRIG bispecific antibody for dual blockade of PD-1 and PVRIG pathways to enhance reversal of T cell inhibition. BSI-507 demonstrates favorable biophysical and functional characteristics, supporting the initiation of development activities including manufacturing and IND-enabling studies. Citation Format: Zeyu Peng, Xiaodong F. Liu, Hongyan Li, Wenwen Dai, Jinyu Liu, Xiaoyao Hao, Shukai Xia, Qun Lv, Hugh M. Davis, Mingjiu Chen, Mark Z. Ma. BSI-507, a first-in-class bispecific antibody targeting PD1 and PVRIG for cancer immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6367.

Abstract 1831: A long-acting and CD122-enhanced IL-2 analog, HM16390, synergizes with immune checkpoint inhibitor by remodeling an immune cell profile in tumor microenvironment

Abstract Immune checkpoint inhibitors (CPI) are widely used for cancer immunotherapy. However, the response to CPIs is dependent on phenotype of tumor microenvironment (TME). A cold tumor known as immune-excluded or -desert has shown poor response to CPIs due to an absence of effector T cells in TME. IL-2 analog, which is immune stimulator and able to recruit cancer-fighting cells into TME, may be promising therapeutic partner for overcoming a limitation of CPIs. Previously, HM16390, a long-acting CD-122-enhanced IL-2 analog, exerted dose-dependent anti-tumor activity in low immunogenic B16F10 melanoma mice. Here, we further investigated the immune cells composition in TME following HM16390 treatment and synergistic anti-tumor effect after combination with CPI. B16F10 mice were sacrificed at days 1, 3, and 8 following single subcutaneous administration of HM16390 or 5 consecutive daily administrations of aldesleukin. According to the result, HM16390 transiently increased peripheral cytokines (IFNγ and TNFα) more than aldesleukin. In line with this, we observed that the tumor-infiltrating NK/Treg ratio was significantly increased approximately 19 by treatment of HM16390 while showing 4.2 in aldesleukin treated group at Day 3. Furthermore, tumor-infiltrating CD8+/Treg ratio was also upregulated approximately 74 by treatment of HM16390 while only showing 6.1 in aldesleukin treated group at Day 8. Significantly increased pro-inflammatory molecules such as GrzB and IFNγ were also observed in HM16390 treated group compared to aldesleukin (p&amp;lt;0.01 and p&amp;lt;0.001, respectively). Next, we investigated synergistic anti-tumor effects in combination with CPI. B16F10 mice were repeatedly given HM16390 once a week or aldesleukin 5 consecutive days per week with or without mouse anti-PD1. After four weeks treatment, 25% of B16F10 mice showed complete response by treatment of HM16390, and significantly increased up to 88% by combination with anti-PD1. However, none of the mice showed complete response by treatment of aldesleukin/anti-PD1 combo. In conclusion, HM16390 effectively induced tumor growth inhibition through the activation and tumor infiltration of cytotoxic lymphocytes. This favorable immune alteration in tumor elicited a TME remodeling in which CPI could sufficiently respond. Citation Format: Jaehyuk Choi, Jinyoung Kim, Yu Yon Kim, Seongju Jeong, Sungmin Bae, Daejin Kim, In Young Choi. A long-acting and CD122-enhanced IL-2 analog, HM16390, synergizes with immune checkpoint inhibitor by remodeling an immune cell profile in tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1831.

Effect of allogeneic blood transfusion on neutrophil functional activity and lymphocyte cytotoxicity in recipient rabbits

The relevance of this paper is that transfusion of allogeneic blood to recipient animals is always associated with immunological risks. In this regard, the purpose of this study was to assess the state of phagocytic activity of blood neutrophils by indicators of phagocytic index, phagocytic number, and oxygen-dependent bactericidal activity, as well as to establish changes in antibody-dependent cytotoxic activity of lymphocytes in recipient rabbits during allogeneic whole blood transfusion. Modelling of blood transfusions was performed on five clinically healthy rabbits by intravenous administration of whole blood at the rate of 5.5 ml/kg of body weight. Blood samples were taken from animals on Days 3, 7, and 23 after blood transfusion. Neutrophil populations were obtained from blood samples by centrifugation on a double density gradient of 1.077 and 1.093 Ficoll-Verografin. The absorption activity of phagocytes was determined in a microscopic test. To investigate the oxygen-dependent bactericidal activity of neutrophils, a spontaneous test with nitroblue tetrazolium was performed. Antibody-dependent cytotoxic activity of lymphocytes was investigated by colorimetric method. It was found that after the transfusion of whole blood, the phagocytic activity of neutrophils increases with a simultaneous decrease in their absorption capacity. On Days 3 and 7, the results of the spontaneous test with nitroblue tetrazolium decreased. This indicates inactivation of the oxygen-dependent bactericidal activity of neutrophil granulocytes during the first phase of post-transfusion immunological reactions. On Day 23, there was an increase in the values of the indicators of the spontaneous test with nitroblue tetrazolium, which indicates the activation of the bactericidal properties of phagocytes. It was found that on Day 3, the antibody-dependent cytotoxic activity of lymphocytes significantly decreased relative to the initial state, and on Days 7 and 23, it increased. An increase in the antibody-dependent cytotoxic activity of lymphocytes should be associated with the active synthesis of antibodies of the late phase of the immune response. Consequently, transfusion of allogeneic blood causes an immune response in recipient rabbits, without causing immediate and long-term transfusion reactions (changes in heart rate, respiratory rate, body temperature). The obtained results are of practical value both for scientists and practising doctors who use transfusion of whole blood and its components to animals with acute anaemia, impaired functional activity of blood coagulation factors, parasitic, and oncological diseases

A RARE CASE OF ACUTE LIVER FAILURE, HEPATITIS ASSOCIATED APLASTIC ANEMIA, AND HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS

A RARE CASE OF ACUTE LIVER FAILURE, HEPATITIS ASSOCIATED APLASTIC ANEMIA, AND HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS