Effector Functions Research Articles

Metabolic Plasticity of Regulatory T Cells in Health and Autoimmunity.

Immunometabolism has been demonstrated to control immune tolerance and the pathogenic events leading to autoimmunity. Compelling experimental evidence also suggests that intracellular metabolic programs influence differentiation, phenotype, proliferation, and effector functions of anti-inflammatory CD4+CD25+Foxp3+ regulatory T (Treg) cells. Indeed, alterations in intracellular metabolism associate with quantitative and qualitative impairments of Treg cells in several pathological conditions. In this review, we summarize the most recent advances linking how metabolic pathways control Treg cell homeostasis and their alterations occurring in autoimmunity. Also, we analyze how metabolic manipulations could be employed to restore Treg cell frequency and function with the aim to create novel therapeutic opportunities to halt immune-mediated disorders.

Immunological characteristics of CD103+CD161+ T lymphocytes on chronic rhinosinusitis with nasal polyps

Chronic rhinosinusitis with nasal polyps (CRSwNPs) is a heterogeneous disease characterized by local inflammation of the upper airway and sinus mucosa. T cell-mediated immune responses play irreplaceable roles in the pathogenesis of nasal polyps. CD161+ T cells have been implicated in the pathology of several diseases through cytokine production and cytotoxic activity. However, the immunological characteristics of CD161+ T cells in nasal mucosa are still not well understood, particularly in CRSwNPs. Our research revealed a notable enrichment of CD161+ T cells in nasal tissues compared to peripheral blood, with a significantly more infiltration of CD161+ T cells in CRSwNPs compared to control nasal samples. Phenotypical analysis found that CD161+ T cells predominantly co-expressed tissue-resident memory surface markers CD103, CD69, and CD45RO. CD161+CD103+ T cells demonstrated complicated effector functions, marked by elevated levels of PD-1, CTLA-4, IL-17, and IFN-γ and diminished expression of FoxP3 and CD25. Interestingly, despite CD161+ T cells was more abundant in polyp tissues compared to normal control tissues, and then further categorizing polyp samples into distinct groups based on clinical characteristics, only the recurrent CRSwNP group showed a significant reduction in CD161+CD8+ T cells compared to the primary CRSwNP group. This finding suggested the necessity for further research to comprehensively understand the underlying mechanisms and the broader significance of CD161+ T cells in the advancement and relapse of CRSwNPs.

ABIN1 is a negative regulator of effector functions in cytotoxic T cells

T cells are pivotal in the adaptive immune defense, necessitating a delicate balance between robust response against infections and self-tolerance. Their activation involves intricate cross-talk among signaling pathways triggered by the T-cell antigen receptors (TCR) and co-stimulatory or inhibitory receptors. The molecular regulation of these complex signaling networks is still incompletely understood. Here, we identify the adaptor protein ABIN1 as a component of the signaling complexes of GITR and OX40 co-stimulation receptors. T cells lacking ABIN1 are hyper-responsive ex vivo, exhibit enhanced responses to cognate infections, and superior ability to induce experimental autoimmune diabetes in mice. ABIN1 negatively regulates p38 kinase activation and late NF-κB target genes. P38 is at least partially responsible for the upregulation of the key effector proteins IFNG and GZMB in ABIN1-deficient T cells after TCR stimulation. Our findings reveal the intricate role of ABIN1 in T-cell regulation.

Distribution of Three Verticillium dahliae Races in Coastal California and Evaluation of Resistance in Lettuce.

Verticillium wilt, caused by Verticillium dahliae, is one of the most devastating soilborne diseases of lettuce (Lactuca sativa L.). There are three races of V. dahliae, and each race has been characterized by markers representing race-specific effectors. Race 1 is differentiated by the presence of the functional secretory Ave1 effector. Similarly, races 2 and 3 are differentiated by effectors VdR2e and VdR3e, respectively. Although the presence of race 1 in coastal California was well established, the presence of effector-based races 2 and 3 was uncertain. This study therefore focused on characterizing 727 isolates collected from 142 ranches of symptomatic lettuce and other crops from coastal California. Based on this evaluation, 523 isolates were designated as race 1, 20 isolates as race 2, 23 isolates as race 3, and 17 as race undefined. Isolates representing other Verticillium species totaled 110, and 34 were non-Verticillium fungal species. Because the use of resistant cultivars is a key strategy to manage this disease, we evaluated 48 lettuce germplasm lines and 1 endive (Cichorium endivia L.) line, comprising commercial cultivars and breeding lines, including the race 1-resistant heirloom cultivar La Brillante and the susceptible cultivar Salinas as controls. Resistance against races 1, 2, and 3 along with VdLs17, a virulent isolate of V. dahliae from lettuce that is currently not assigned to a race, was evaluated in replicated greenhouse experiments. Two crisphead lettuce lines, HL28 and HL29, exhibited resistance against race 1 and a partial resistance against race 2, whereas all other lines were highly susceptible to races 1 and 2 and VdLs17. The majority of lines exhibited higher resistance to race 3 relative to the other two races. This study documents the current distribution of the different races in coastal California. In addition, the sources of resistance currently being developed should be effective or partially effective against these races for targeted deployment as soon as they are available.

TRIM21 and Fc-engineered antibodies: decoding its complex antibody binding mode with implications for viral neutralization.

TRIM21 is a pivotal effector in the immune system, orchestrating antibody-mediated responses and modulating immune signaling. In this comprehensive study, we focus on the interaction of TRIM21 with Fc engineered antibodies and subsequent implications for viral neutralization. Through a series of analytical techniques, including biosensor assays, mass photometry, and electron microscopy, along with structure predictions, we unravel the intricate mechanisms governing the interplay between TRIM21 and antibodies. Our investigations reveal that the TRIM21 capacity to recognize, bind, and facilitate the proteasomal degradation of antibody-coated viruses is critically dependent on the affinity and avidity interplay of its interactions with antibody Fc regions. We suggest a novel binding mechanism, where TRIM21 binding to one Fc site results in the detachment of PRYSPRY from the coiled-coil domain, enhancing mobility due to its flexible linker, thereby facilitating the engagement of the second site, resulting in avidity due to bivalent engagement. These findings shed light on the dual role of TRIM21 in antiviral immunity, both in recognizing and directing viruses for intracellular degradation, and demonstrate its potential for therapeutic exploitation. The study advances our understanding of intracellular immune responses and opens new avenues for the development of antiviral strategies and innovation in tailored effector functions designed to leverage TRIM21s unique binding mode.

Converging cytokine and metabolite networks shape asymmetric T cell fate at the term human maternal-fetal interface.

Placentation presents immune conflict between mother and fetus, yet in normal pregnancy maternal immunity against infection is maintained without expense to fetal tolerance. This is believed to result from adaptations at the maternal-fetal interface (MFI) which affect T cell programming, but the identities (i.e., memory subsets and antigenic specificities) of T cells and the signals that mediate T cell fates and functions at the MFI remain poorly understood. We found intact recruitment programs as well as pro-inflammatory cytokine networks that can act on maternal T cells in an antigen-independent manner. These inflammatory signals elicit T cell expression of co-stimulatory receptors necessary for tissue retention, which can be engaged by local macrophages. Although pro-inflammatory molecules elicit T cell effector functions, we show that additional cytokine (TGF-β1) and metabolite (kynurenine) networks may converge to tune T cell function to those of sentinels. Together, we demonstrate an additional facet of fetal tolerance, wherein T cells are broadly recruited and restrained in an antigen-independent, cytokine/metabolite-dependent manner. These mechanisms provide insight into antigen-nonspecific T cell regulation, especially in tissue microenvironments where they are enriched.

Allergen-specific IgA and IgG antibodies as inhibitors of mast cell function in food allergy.

Food allergy, a group of adverse immune responses to normally innocuous food protein antigens, is an increasingly prevalent public health issue. The most common form is IgE-mediated food allergy in which food antigen-induced crosslinking of the high-affinity IgE-receptor, FcεRI, on the surface of mast cells triggers the release of inflammatory mediators that contribute to a wide range of clinical manifestations, including systemic anaphylaxis. Mast cells also play a critical function in adaptive immunity to foods, acting as adjuvants for food-antigen driven Th2 cell responses. While the diagnosis and treatment of food allergy has improved in recent years, no curative treatments are currently available. However, there is emerging evidence to suggest that both allergen-specific IgA and IgG antibodies can counter the activating effects of IgE antibodies on mast cells. Most notably, both antigen-specific IgA and IgG antibodies are induced in the course of oral immunotherapy. In this review, we highlight the role of mast cells in food allergy, both as inducers of immediate hypersensitivity reactions and as adjuvants for type 2 adaptive immune responses. Furthermore, we summarize current understanding of the immunomodulatory effects of antigen-specific IgA and IgG antibodies on IgE-induced mast cell activation and effector function. A more comprehensive understanding of the regulatory role of IgA and IgG in food allergy may provide insights into physiologic regulation of immune responses to ingested antigens and could seed novel strategies to treat allergic disease.

Clonal associations between lymphocyte subsets and functional states in rheumatoid arthritis synovium

Rheumatoid arthritis (RA) is an autoimmune disease involving antigen-specific T and B cells. Here, we perform single-cell RNA and repertoire sequencing on paired synovial tissue and blood samples from 12 seropositive RA patients. We identify clonally expanded CD4 + T cells, including CCL5+ cells and T peripheral helper (Tph) cells, which show a prominent transcriptomic signature of recent activation and effector function. CD8 + T cells show higher oligoclonality than CD4 + T cells, with the largest synovial clones enriched in GZMK+ cells. CD8 + T cells with possibly virus-reactive TCRs are distributed across transcriptomic clusters. In the B cell compartment, NR4A1+ activated B cells, and plasma cells are enriched in the synovium and demonstrate substantial clonal expansion. We identify synovial plasma cells that share BCRs with synovial ABC, memory, and activated B cells. Receptor-ligand analysis predicted IFNG and TNFRSF members as mediators of synovial Tph-B cell interactions. Together, these results reveal clonal relationships between functionally distinct lymphocyte populations that infiltrate the synovium of patients with RA.

Decoding changes in tumor-infiltrating leukocytes through dynamic experimental models and single-cell technologies.

The ability to characterize immune cells and explore the molecular interactions that govern their functions has never been greater, fueled in recent years by the revolutionary advance of single-cell analysis platforms. However, precisely how immune cells respond to different stimuli and where differentiation processes and effector functions operate remain incompletely understood. Inferring cellular fate within single-cell transcriptomic analyses is now omnipresent, despite the assumptions typically required in such analyses. Recently developed experimental models support dynamic analyses of the immune response, providing insights into the temporal changes that occur within cells and the tissues in which such transitions occur. Here we will review these approaches and discuss how these can be combined with single-cell technologies to develop a deeper understanding of the immune responses that should support the development of better therapeutic options for patients.

T-cell immunity induced and reshaped by an anti-HPV immuno-oncotherapeutic lentiviral vector

We recently developed an immuno-oncotherapy against human papillomavirus (HPV)-induced tumors based on a lentiviral vector encoding the Early E6 and E7 oncoproteins of HPV16 and HPV18 genotypes, namely “Lenti-HPV-07”. The robust and long-lasting anti-tumor efficacy of Lenti-HPV-07 is dependent on CD8+ T-cell induction and remodeling of the tumor microenvironment. Here, we first established that anti-vector immunity induced by Lenti-HPV-07 prime has no impact on the efficacy of a homologous boost to amplify anti-HPV T-cell immunity. To longitudinally monitor the evolution of the T-cell repertoire generated after the prime, homologous or heterologous boost with Lenti-HPV-07, we tracked T-cell clonotypes by deep sequencing of T-Cell Receptor (TCR) variable β and α chain mRNA, applied to whole peripheral blood cells (PBL) and a T cell population specific of an immunodominant E7HPV16 epitope. We observed a hyper-expansion of clonotypes post prime, accompanied by increased frequencies of HPV-07-specific T cells. Additionally, there was a notable diversification of clonotypes post boost in whole PBL, but not in the E7HPV16-specific T cells. We then demonstrated that the effector functions of such Lenti-HPV-07-induced T cells synergize with anti-checkpoint inhibitory treatments by systemic administration of anti-TIM3 or anti-NKG2A monoclonal antibodies. While Lenti-HPV-07 is about to enter a Phase I/IIa clinical trial, these results will help better elucidate its mode of action in immunotherapy against established HPV-mediated malignancies.

SACI-IO HR+: A randomized phase II trial of sacituzumab govitecan with or without pembrolizumab in patients with metastatic hormone receptor-positive/HER2-negative breast cancer.

LBA1004 Background: Sacituzumab govitecan (SG) is a TROP2 antibody drug conjugate (ADC) with a topoisomerase I inhibitor payload (SN-38) approved for previously treated triple negative and hormone receptor-positive/HER2-negative (HR+/HER2-) metastatic breast cancer (MBC). Double-strand DNA breaks induced by SN-38 activate cGAS-STING, stimulating type I IFN production and T cell recruitment. SN-38 can upregulate MHC class-I and PD-L1 expression, enhance cytotoxic T cell effector functions and deplete regulatory T cells. To evaluate if SG synergizes with pembrolizumab (PD-1 inhibitor) we conducted a randomized, open-label phase 2 study comparing SG with or without pembrolizumab in HR+/HER2- MBC (NCT04448886). Methods: Eligible patients (pts) had unresectable locally advanced or metastatic HR+ (ER≥1% and/or PR≥1%), HER2- breast cancer treated with ≥1 prior endocrine therapy and 0-1 chemotherapy (CT) for MBC. Pts with brain metastases were eligible if locoregional therapy was completed and steroids discontinued ≥7 days before study therapy. Pts who received prior topoisomerase I inhibitor ADC, irinotecan or PD-1/L1 inhibitors were excluded. Pts were randomized 1:1 to Arm A [SG 10 mg/kg IV (D1, D8) + pembrolizumab 200 mg IV (D1), 21-day cycle] or Arm B (SG alone). The primary endpoint was progression-free survival (PFS); secondary endpoints included PFS in PD-L1+ pts (22C3 CPS ≥1), overall survival (OS), objective response rate (ORR) and toxicity (NCI CTCAE v5.0). Baseline and on-treatment biopsies were performed for correlative analyses. For this preliminary analysis, data were locked 1/12/24. Results: Between 03/2021-01/2024, 110 pts enrolled; 104 pts (52 Arm A; 52 Arm B) started study therapy and were included in the analysis. Median age was 57 yrs (range: 27-81); 102 pts (98.1%) were female. 80 pts (76.9%) received prior CDK4/6 inhibitor for MBC; 58 (55.8%) had no prior CT, 46 (44.2%) had 1 prior line of CT for MBC. At a median follow-up of 9.2 months (mo), median PFS was 8.4 mo in Arm A vs 6.2 mo in Arm B (HR 0.76, 95% CI 0.47-1.23, log-rank p=0.26); ORR 21.2% and 17.3%, respectively. OS data are immature with only 26 events to date; OS was 16.9 mo vs 17.1 mo (HR 0.65, 95% CI 0.30-1.41, log-rank p=0.28), respectively. The most frequent treatment-related toxicities (≥G2) in Arm A were neutropenia (67.3%), fatigue (36.5%), alopecia (36.5%), anemia (32.7%), leukopenia (26.9%), diarrhea (21.2%) and nausea (21.2%); in Arm B, neutropenia (59.6%), alopecia (38.5%), diarrhea (34.6%), nausea (32.7%), fatigue (32.7%) and anemia (21.2%). Conclusions: Addition of pembrolizumab to SG showed a non-significant trend toward improved PFS in unselected HR+/HER2- MBC at this preliminary time point. Final PFS and updated OS with further follow-up will be presented at the meeting. Exploratory outcome analyses by TROP2 and PD-L1 expression will be reported. Clinical trial information: NCT04448886 .

Expression of a single inhibitory Ly49 receptor is sufficient to license NK cells for effector functions.

Natural killer (NK) cells recognize target cells through germline-encoded activation and inhibitory receptors enabling effective immunity against viruses and cancer. The Ly49 receptor family in the mouse and killer immunoglobin-like receptor family in humans play a central role in NK cell immunity through recognition of MHC class I and related molecules. Functionally, these receptor families are involved in licensing and rejection of MHC-I-deficient cells through missing-self. The Ly49 family is highly polymorphic, making it challenging to detail the contributions of individual Ly49 receptors to NK cell function. Herein, we showed mice lacking expression of all Ly49s were unable to reject missing-self target cells in vivo, were defective in NK cell licensing, and displayed lower KLRG1 on the surface of NK cells. Expression of Ly49A alone on a H-2Dd background restored missing-self target cell rejection, NK cell licensing, and NK cell KLRG1 expression. Thus, a single inhibitory Ly49 receptor is sufficient to license NK cells and mediate missing-self in vivo.

Epitope Mapping of an Anti-Mouse CCR8 Monoclonal Antibody C8Mab-2 Using Flow Cytometry.

The C-C motif chemokine receptor 8 (CCR8) is highly and selectively expressed in regulatory T (Treg) cells and is associated with tumor progression. The massive accumulation of Treg cells into tumors suppresses the effector function of CD8+ cells against tumor cells. Therefore, selective depletion of Treg cells using anti-CCR8 monoclonal antibodies (mAbs) reinvigorates antitumor immune responses and improves responses to cancer immunotherapy. Previously, we developed an anti-mouse CCR8 (mCCR8) mAb, C8Mab-2, using the Cell-Based Immunization and Screening method. In this study, the binding epitope of C8Mab-2 was investigated using flow cytometry. The mCCR8 extracellular domain-substituted mutant analysis showed that C8Mab-2 recognizes the N-terminal region (1-33 amino acids) of mCCR8. Next, 1×alanine (or glycine) scanning and 2×alanine (or glycine) scanning were conducted in the N-terminal region. The results revealed that the 17-DFFTAP-22 sequence is important for the recognition by C8Mab-2, and Thr20 is a central amino acid of the epitope. These results revealed the involvement of the N-terminus of mCCR8 in the recognition by C8Mab-2.

Complement-dependent virion lysis mediated by dengue-Zika virus cross-reactive antibodies correlates with protection from severe dengue disease.

Primary infection with one of four dengue virus serotypes (DENV1-4) may generate antibodies that protect or enhance subsequent secondary heterotypic infections. However, the characteristics of heterotypic cross-reactive antibodies associated with protection from symptomatic infection and severe disease are not well-defined. We selected plasma samples collected before a secondary DENV heterotypic infection that was classified either as dengue fever (DF, n = 31) or dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS, n = 33) from our longstanding pediatric cohort in Nicaragua. We screened various antibody properties to determine the features correlated with protection from DHF/DSS. Protection was associated with high levels of binding of various antibody isotypes, IgG subclasses and effector functions, including antibody-dependent complement deposition, ADCD. Although the samples were derived from DENV-exposed, Zika virus (ZIKV)-naïve individuals, the protective ADCD association was stronger when assays were conducted with recombinant ZIKV antigens. Further, we showed that a complement-mediated virion lysis (virolysis) assay conducted with ZIKV virions was strongly associated with protection, a finding reproduced in an independent sample set collected prior to secondary heterotypic inapparent versus symptomatic DENV infection. Virolysis was the main antibody feature correlated with protection from DHF/DSS and severe symptoms, such as thrombocytopenia, hemorrhagic manifestations, and plasma leakage. Hence, anti-DENV antibodies that cross-react with ZIKV, target virion-associated epitopes, and mediate complement-dependent virolysis are correlated with protection from secondary symptomatic DENV infection and DHF/DSS. These findings may support the rational design and evaluation of dengue vaccines and development of therapeutics.

Enhanced abscopal anti-tumor response via a triple combination of thermal ablation, IL-21, and PD-1 inhibition therapy

Despite the success of immune checkpoint inhibitors (ICIs) in treating solid tumors, lots of patients remain unresponsive to this therapy. Microwave ablation (MWA) stimulates systemic adaptive immunity against tumor cells by releasing tumor antigens. Additionally, IL-21 has demonstrated importance in stimulating T-cell effector function. The combination of these three therapies—MWA, IL-21, and anti-PD-1 monoclonal antibodies (mAbs)—has yet to be explored in the context of cancer treatment.In this study, we explored the impact of thermal ablation on IL-21R expression in tumor-infiltrating lymphocytes (TILs). Subsequently, we assessed alterations in the tumor microenvironment (TME) and peripheral lymphoid organs. Additionally, we conducted a thorough examination of tumor-infiltrating CD45+ immune cells across various treatment groups using single-cell RNA sequencing (scRNA-seq). Moreover, we determined the potential anti-tumor effects of the triple combination involving MWA, IL-21, and anti-PD-1 mAbs.Our findings revealed that MWA upregulated the expression of IL-21R on various immune cells in the untreated tumors. The combination of MWA with IL-21 exhibited a robust abscopal anti-tumor effect, enhancing the effector function of CD8+ T cells and facilitating dendritic cells' maturation and antigen presentation in the untreated tumor. Notably, the observed abscopal anti-tumor effect resulting from the combination is contingent upon T-cell recirculation, indicating the reliance of systemic adaptive immunity for this treatment regimen. Additionally, the combination of MWA, IL-21, and PD-1 mAbs demonstrated profound abscopal anti-tumor efficacy. Our findings provide support for further clinical investigation into a triple combination therapy involving MWA, IL-21, and ICIs for the treatment of metastatic cancer.

Fibrotic pathways and fibroblast-like synoviocyte phenotypes in osteoarthritis.

Osteoarthritis (OA) is the most common form of arthritis, characterized by osteophyte formation, cartilage degradation, and structural and cellular alterations of the synovial membrane. Activated fibroblast-like synoviocytes (FLS) of the synovial membrane have been identified as key drivers, secreting humoral mediators that maintain inflammatory processes, proteases that cause cartilage and bone destruction, and factors that drive fibrotic processes. In normal tissue repair, fibrotic processes are terminated after the damage has been repaired. In fibrosis, tissue remodeling and wound healing are exaggerated and prolonged. Various stressors, including aging, joint instability, and inflammation, lead to structural damage of the joint and micro lesions within the synovial tissue. One result is the reduced production of synovial fluid (lubricants), which reduces the lubricity of the cartilage areas, leading to cartilage damage. In the synovial tissue, a wound-healing cascade is initiated by activating macrophages, Th2 cells, and FLS. The latter can be divided into two major populations. The destructive thymocyte differentiation antigen (THY)1─ phenotype is restricted to the synovial lining layer. In contrast, the THY1+ phenotype of the sublining layer is classified as an invasive one with immune effector function driving synovitis. The exact mechanisms involved in the transition of fibroblasts into a myofibroblast-like phenotype that drives fibrosis remain unclear. The review provides an overview of the phenotypes and spatial distribution of FLS in the synovial membrane of OA, describes the mechanisms of fibroblast into myofibroblast activation, and the metabolic alterations of myofibroblast-like cells.

Intestinal cDC1s provide cues required for CD4+ T cell-mediated resistance to Cryptosporidium.

Cryptosporidium is an enteric pathogen and a prominent cause of diarrheal disease worldwide. Control of Cryptosporidium requires CD4+ T cells, but how protective CD4+ T cell responses are generated is poorly understood. Here, Cryptosporidium parasites that express MHCII-restricted model antigens were generated to understand the basis for CD4+ T cell priming and effector function. These studies revealed that parasite-specific CD4+ T cells are primed in the draining mesenteric lymph node but differentiate into Th1 cells in the gut to provide local parasite control. Although type 1 conventional dendritic cells (cDC1s) were dispensable for CD4+ T cell priming, they were required for CD4+ T cell gut homing and were a source of IL-12 at the site of infection that promoted local production of IFN-γ. Thus, cDC1s have distinct roles in shaping CD4+ T cell responses to an enteric infection: first, to promote gut homing from the mesLN, and second, to drive effector responses in the intestine.

PECULIARITIES OF NEUTROPHIL EXTRACELLULAR TRAPS FORMATION IN CHINCHILLA RABBITS

Neutrophil extracellular traps (NETs) are decondensed nuclear chromatin, decorated with bactericidal proteins of various cell organelles and performing an eff ector function aimed to combat pathogens at the site of infl ammation. At the same time, NETs play an important role in the pathogenesis of many autoimmune and infl ammatory diseases as well as malignancies. Rabbits are one of the most commonly used species of laboratory animals in medical and biological research. A large number of models of various diseases of the cardiovascular, immune and other human systems have been developed in rabbits. However, there is no information in the scientifi c literature about the ability of rabbit neutrophils to undergo NETosis in response to well-known pharmacological stimuli. The purpose of the present work was to study in in vitro system the ability of neutrophils of Soviet chinchilla rabbit to form NETs in response to mimetic of diacylglycerol phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187. To isolate rabbit neutrophils, the one-step density gradient centrifugation on Ficoll-Hypaque method with modifi cations was used. Oxidative burst was assessed with luminol-amplifi ed chemiluminescence method, and NET formation was assessed with immunofl uorescence analysis. The work shows for the fi rst time that neutrophils of Soviet chinchilla rabbit do not form NETs in response to PMA, but form traps in response to A23187, as well as have a low level of oxidative burst in response to PMA, A23187 and chemoattractant N-formyl-methionylleucyl-phenylalanine.

PECULIARITIES OF NEUTROPHIL EXTRACELLULAR TRAPS FORMATION IN CHINCHILLA RABBITS

Neutrophil extracellular traps (NETs) are decondensed nuclear chromatin, decorated with bactericidal proteins of various cell organelles and performing an eff ector function aimed to combat pathogens at the site of infl ammation. At the same time, NETs play an important role in the pathogenesis of many autoimmune and infl ammatory diseases as well as malignancies. Rabbits are one of the most commonly used species of laboratory animals in medical and biological research. A large number of models of various diseases of the cardiovascular, immune and other human systems have been developed in rabbits. However, there is no information in the scientifi c literature about the ability of rabbit neutrophils to undergo NETosis in response to well-known pharmacological stimuli. The purpose of the present work was to study in in vitro system the ability of neutrophils of Soviet chinchilla rabbit to form NETs in response to mimetic of diacylglycerol phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187. To isolate rabbit neutrophils, the one-step density gradient centrifugation on Ficoll-Hypaque method with modifi cations was used. Oxidative burst was assessed with luminol-amplifi ed chemiluminescence method, and NET formation was assessed with immunofl uorescence analysis. The work shows for the fi rst time that neutrophils of Soviet chinchilla rabbit do not form NETs in response to PMA, but form traps in response to A23187, as well as have a low level of oxidative burst in response to PMA, A23187 and chemoattractant N-formyl-methionylleucyl-phenylalanine.

A phase II trial of monalizumab in combination with durvalumab (MEDI4736) plus platinum-based chemotherapy for first-line treatment of extensive stage small cell lung cancer (MOZART): Hoosier Cancer Research Network LUN21-530 study.

TPS8129 Background: Chemoimmunotherapy consisting of platinum, etoposide, and PD-L1 inhibitor remains the standard of care first-line treatment for extensive stage small cell lung cancer (SCLC) yielding median overall survival (OS) of slightly over a year. NKG2A/CD94 is an immune checkpoint that is selectively expressed on natural killer (NK) cells and CD8+ T cells in the tumor microenvironment. When engaging with its ligand, HLA-E, NKG2A transduces inhibitory signals that suppress immune-mediated cytotoxicity. Thus, NKG2A blockade enhances anti-tumor immunity by promoting both, NK and CD8+ T cell, effector functions. Monalizumab, a monoclonal antibody targeting NKG2A, enhances NK cell activity against tumor cells and rescues CD8+ T cell function in combination with PD-(L)1 axis blockade. Pre-clinical data have demonstrated that the absence of NK cells substantially enhances metastatic dissemination of SCLC tumor cells in vivo. NK cell function is modulated in response to cytotoxic chemotherapy, especially platinum. Post-chemotherapy NK cells display an induced expression of NKG2A compared with pre-chemotherapy patients and is associated with a reduced NK cell mediated anti-tumor activity. Hyperactivation of NK cell activity ameliorates SCLC metastases, an effect that is enhanced when combined with anti PD-(L)1 therapy. Altogether, these data suggest that addition of monalizumab to standard of care first-line treatment may be associated with improved efficacy in patients with SCLC. Methods: Patients with extensive-stage SCLC with ECOG performance status of 0-2 and adequate organ function are eligible for enrollment on this single-arm phase II study with an initial safety lead-in cohort. Patients may have received one prior cycle of platinum doublet with or without durvalumab and may have treated or untreated asymptomatic brain metastasis. Patients will receive platinum (cis or carbo), etoposide, durvalumab, and monalizumab every 3 weeks for 4 cycles followed by durvalumab and monalizumab every 4 weeks until disease progression or unacceptable toxicity. Primary endpoints include 1-year progression-free survival, safety, and tolerability. Secondary endpoints include objective response rate, 1-year overall survival, and intracranial progression-free survival. Exploratory objectives include analyzing association of treatment efficacy with minimal residual disease status, blood- and tissue-based genomic and transcriptomic signatures, tumor infiltrating immune cells, and peripheral blood NK cell and CD8+ T cell activity. Five of the planned 30 patients have been enrolled to date. Clinical trial information: NCT05903092 .