Regulation Of Cell-mediated Immunity Research Articles

SON-1010: an albumin-binding IL-12 fusion protein that improves cytokine half-life, targets tumors, and enhances therapeutic efficacy.

Cytokines have been promising cancer immunotherapeutics for decades, yet only two are licensed to date. Interleukin-12 (IL-12) is a potent regulator of cell-mediated immunity that activates NK cells and interferon-γ (IFNγ) production. It plays a central role in multiple pathways that can enhance cancer cell death and modify the tumor microenvironment (TME). Attempts to dose rIL-12 were initially successful but IFNγ toxicity in Phase 2 complicated further development in the late 1990s. Since then, better dosing strategies have been developed, but none have achieved the level of cancer control seen in preclinical models. We set out to develop a novel strategy to deliver fully functional IL-12 and other biologics to the TME by binding albumin, taking advantage of its ability to be concentrated and retained in the tumor. Single-chain variable fragments (scFv) were identified from a human phage display library that bound human, mouse, and cynomolgus macaque serum albumin, both at physiologic and acidic conditions. These were taken through a series of steps to identify strongly binding molecules that don't interfere with the normal physiology of albumin to bind FcRn, giving it prolonged half-life in serum, along with SPARC/GP60, which allows albumin to target the TME. A final molecule was chosen and a single mutation was made that minimizes the potential for immunogenicity. This fully human albumin-binding (FHAB®) domain was characterized and manufacturing processes were developed to bring the first drug candidate into the clinic. Once identified, the murine form of mIL12-FHAB was studied preclinically to understand its mechanism of action and biodistribution. It was found to be much more efficient at blocking tumor growth compared to murine IL-12, while stimulating significant IFNγ production with minimal toxicity. SON-1010, which uses the human IL-12 sequence, passed through all of the characterization and required toxicology and is currently being studied in the clinic. We identified and developed a platform technology with prolonged half-life that can target IL-12 and other immune modulators to the TME. Safety and efficacy are being studied using SON-1010 as monotherapy and in combination with checkpoint blockade strategies.

B Cell-mediated Immune Regulation and the Quest for Transplantation Tolerance.

Pathophysiologic function of B cells in graft rejection has been well recognized in transplantation. B cells promote alloantigen-specific T-cell response and secrete antibodies that can cause antibody-mediated graft failures and rejections. Therefore, strategies targeting B cells, for example, B-cell depletion, have been used for the prevention of both acute and chronic rejections. Interestingly, however, recent mounting evidence indicates that subsets of B cells yet to be further identified can display potent immune regulatory functions, and they contribute to transplantation tolerance and operational tolerance in both experimental and clinical settings, respectively. In this review, we integrate currently available information on B-cell subsets, including T-cell Ig domain and mucin domain 1-positive transitional and T-cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif domain-positive memory B cells, displaying immune regulatory functions, with a focus on transplantation tolerance, by analyzing their mechanisms of action. In addition, we will discuss potential T-cell Ig domain and mucin domain 1-positive and T-cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif domain-positive B cell-based strategies for the enhancement of operational tolerance in transplantation patients.

Functions of mucosal associated invariant T cells in eye diseases.

Mucosal-associated invariant T (MAIT) cells are a unique subset of T cells that recognizes metabolites derived from the vitamin B2 biosynthetic pathway. Since the identification of cognate antigens for MAIT cells, knowledge of the functions of MAIT cells in cancer, autoimmunity, and infectious diseases has been rapidly expanding. Recently, MAIT cells have been found to contribute to visual protection against autoimmunity in the eye. The protective functions of MAIT cells are induced by T-cell receptor (TCR)-mediated activation. However, the underlying mechanisms remain unclear. Thus, this mini-review aims to discuss our findings and the complexity of MAIT cell-mediated immune regulation in the eye.

Selective IL-27 production by intestinal regulatory T cells permits gut-specific regulation of TH17 cell immunity.

Regulatory T cells (Treg cells) are instrumental in establishing immunological tolerance. However, the precise effector mechanisms by which Treg cells control a specific type of immune response in a given tissue remains unresolved. By simultaneously studying Treg cells from different tissue origins under systemic autoimmunity, in the present study we show that interleukin (IL)-27 is specifically produced by intestinal Treg cells to regulate helper T17 cell (TH17 cell) immunity. Selectively increased intestinal TH17 cell responses in mice with Treg cell-specific IL-27 ablation led to exacerbated intestinal inflammation and colitis-associated cancer, but also helped protect against enteric bacterial infection. Furthermore, single-cell transcriptomic analysis has identified a CD83+CD62Llo Treg cell subset that is distinct from previously characterized intestinal Treg cell populations as the main IL-27 producers. Collectively, our study uncovers a new Treg cell suppression mechanism crucial for controlling a specific type of immune response in a particular tissue and provides further mechanistic insights into tissue-specific Treg cell-mediated immune regulation.

Prognostic and immunological roles of IL18RAP in human cancers.

Across several cancers, IL18 receptor accessory protein (IL18RAP) is abnormally expressed, and this abnormality is related to tumor immunity and heterogeneous clinical outcomes. In this study, based on bioinformatics analysis, we discovered that IL18RAP is related to the human tumor microenvironment and promotes various immune cells infiltration. Additionally, the multiple immunofluorescence staining revealed that with the increased expression of IL18RAP, the number of infiltrated M1 macrophages increased. This finding was confirmed by coculture migration analysis using three human cancer cell lines (MDA-MB-231, U251, and HepG2) with IL18RAP knockdown. We discovered a positive link between IL18RAP and the majority of immunostimulators, immunoinhibitors, major histocompatibility complex (MHC) molecules, chemokines, and chemokine receptor genes using Spearman correlation analysis. Additionally, functional IL18RAP's gene set enrichment analysis (GSEA) revealed that it is related to a variety of immunological processes, such as positive regulation of interferon gamma production and positive regulation of NK cell-mediated immunity. Moreover, we used single-cell RNA sequencing analysis to detect that IL18RAP was mainly expressed in immune cells, and HALLMARK analysis confirmed that the INF-γ gene set expression was upregulated in CD8Tex cells. In addition, in human and mouse cancer cohorts, we found that the level of IL18RAP can predict the immunotherapy response. In short, our study showed that IL18RAP is a new tumor biomarker and may become a potential immunotherapeutic target in cancer.

Mast cell-mediated immune regulation in health and disease.

Mast cells are important components of the immune system, and they perform pro-inflammatory as well as anti-inflammatory roles in the complex process of immune regulation in health and disease. Because of their strategic perivascular localization, sensitivity and adaptability to the microenvironment, and ability to release a variety of preformed and newly synthesized effector molecules, mast cells perform unique functions in almost all organs. Additionally, Mast cells express a wide range of surface and cytoplasmic receptors which enable them to respond to a variety of cytokines, chemicals, and pathogens. The mast cell's role as a cellular interface between external and internal environments as well as between vasculature and tissues is critical for protection and repair. Mast cell interactions with different immune and nonimmune cells through secreted inflammatory mediators may also turn in favor of disease promoting agents. First and forefront, mast cells are well recognized for their multifaceted functions in allergic diseases. Reciprocal communication between mast cells and endothelial cells in the presence of bacterial toxins in chronic/sub-clinical infections induce persistent vascular inflammation. We have shown that mast cell proteases and histamine induce endothelial inflammatory responses that are synergistically amplified by bacterial toxins. Mast cells have been shown to exacerbate vascular changes in normal states as well as in chronic or subclinical infections, particularly among cigarette smokers. Furthermore, a potential role of mast cells in SARS-CoV-2-induced dysfunction of the capillary-alveolar interface adds to the growing understanding of mast cells in viral infections. The interaction between mast cells and microglial cells in the brain further highlights their significance in neuroinflammation. This review highlights the significant role of mast cells as the interface that acts as sensor and early responder through interactions with cells in systemic organs and the nervous system.

Fundamental Characterization of Antibody Fusion-Single-Chain TNF Recombinant Proteins Directed against Costimulatory TNF Receptors Expressed by T-Lymphocytes.

The costimulatory signal regulated by the members of the tumor necrosis factor receptor (TNFR) superfamily expressed by T cells plays essential roles for T cell responses and has emerged as a promising target for cancer immunotherapy. However, it is unclear how the difference in TNFR costimulation contributes to T cell responses. In this study, to clarify the functional significance of four different TNFRs, OX40, 4-1BB, CD27 and GITR, we prepared corresponding single-chain TNF ligand proteins (scTNFLs) connected to IgG Fc domain with beneficial characteristics, i.e., Fc-scOX40L, Fc-sc4-1BBL, Fc-scCD27L (CD70) and Fc-scGITRL. Without intentional cross-linking, these soluble Fc-scTNFL proteins bound to corresponding TNFRs induced NF-kB signaling and promoted proliferative and cytokine responses in CD4+ and CD8+ T cells with different dose-dependencies in vitro. Mice injected with one of the Fc-scTNFL proteins displayed significantly augmented delayed-type hypersensitivity responses, showing in vivo activity. The results demonstrate that each individual Fc-scTNFL protein provides a critical costimulatory signal and exhibits quantitatively distinct activity toward T cells. Our findings provide important insights into the TNFR costimulation that would be valuable for investigators conducting basic research in cancer immunology and also have implications for T cell-mediated immune regulation by designer TNFL proteins.

Super-killer CTLs are generated by single gene deletion of Bach2.

Bach2 codes for a transcriptional regulator exerting major influences on T cell-mediated immune regulation. Effector CTLs derived from in vitro activation of murine CD8+ T cells showed increased proliferative and cytolytic capacity in the absence of BACH2. Before activation, BACH2-deficient splenic CD8+ T cells had a higher abundance of memory and reduced abundance of naïve cells compared to wild-type. CTLs derived from central memory T cells were more potently cytotoxic than those derived from naïve T cells, but even within separated subsets, BACH2-deficiency conferred a cytotoxic advantage. Immunofluorescence and electron microscopy revealed larger granules in BACH2-deficient compared to wild-type CTLs, and proteomic analysis showed an increase in granule content, including perforin and granzymes. Thus, the enhanced cytotoxicity observed in effector CTLs lacking BACH2 arises not only from differences in their initial differentiation state but also inherent production of enlarged cytolytic granules. These results demonstrate how a single gene deletion can produce a CTL super-killer.

The Multifaceted Role of STAT3 in NK-Cell Tumor Surveillance.

Signal transducer and activator of transcription 3 (STAT3) is a member of the Janus kinase (JAK)-STAT pathway, which is one of the key pathways contributing to cancer. STAT3 regulates transcription downstream of many cytokines including interleukin (IL)-6 and IL-10. In cancer, STAT3 is mainly described as a tumor promoter driving tumor cell proliferation, resistance to apoptosis, angiogenesis and metastasis and aberrant activation of STAT3 is associated with poor prognosis. STAT3 is also an important driver of immune evasion. Among many other immunosuppressive mechanisms, STAT3 aids tumor cells to escape natural killer (NK) cell-mediated immune surveillance. NK cells are innate lymphocytes, which can directly kill malignant cells but also regulate adaptive immune responses and contribute to the composition of the tumor microenvironment. The inborn ability to lyse transformed cells renders NK cells an attractive tool for cancer immunotherapy. Here, we provide an overview of the role of STAT3 in the dynamic interplay between NK cells and tumor cells. On the one hand, we summarize the current knowledge on how tumor cell-intrinsic STAT3 drives the evasion from NK cells. On the other hand, we describe the multiple functions of STAT3 in regulating NK-cell cytotoxicity, cytokine production and their anti-tumor responses in vivo. In light of the ongoing research on STAT3 inhibitors, we also discuss how targeting STAT3 would affect the two arms of STAT3-dependent regulation of NK cell-mediated anti-tumor immunity. Understanding the complexity of this interplay in the tumor microenvironment is crucial for future implementation of NK cell-based immunotherapies.

Abstract 2107: CHMP2A regulates NK cell-mediated anti-tumor activity in a syngeneic HNSCC model

Abstract Natural killer (NK) cells are a key effector in antitumor immunity. However, tumors often acquire resistance programs to escape NK cell-mediated immunosurveillance. Identifying targetable vulnerabilities that reinvigorate NK cell-driven antitumor immunity can enable new therapeutic strategies to improve NK cell-mediated anti-tumor activity. Previous studies from our group used a whole genome CRISPR-Cas9 screen that identified CHMP2A as a gene that mediates tumor-intrinsic resistance to NK cell cytotoxicity. CHMP2A is a member of the ESCRTIII complex and regulates secretion of tumor-derived chemokines and extracellular vehicles (EVs) that express NK cell-activating ligands MICA/B and TRAIL, which induce NK apoptosis. Previously, we demonstrated that the deletion of CHMP2A in glioblastoma and head and neck squamous cell carcinoma (HNSCC) increases allogeneic NK cell-mediated killing both in vitro and in vivo. Here, we extend these studies to explore whether CHMP2A may serve as a targetable regulator of NK cell-mediated immunity. Employing our recently characterized syngeneic, tobacco-signature murine HNSCC model, 4MOSC (PMID: 31804466), we deleted CHMP2A in both the immune-responsive 4MOSC1 and immune-insensitive 4MOSC2 cell line. In vitro NK cell cytotoxicity assays reveal that 4MOSC1-CHMP2A-KO cells were more potently killed by NK cells compared to 4MOSC1-WT cells (53% increased cytotoxicity, p<0.001). In contrast, there was no significant difference in NK cell-mediated killing of 4MOSC2-WT versus CHMP2A-KO cells. Following orthotopic transplantation into immunocompetent hosts, we find that 4MOSC1-KO, but not 4MOSC2-WT, tumors spontaneously regress in vivo compared to wild type tumors (4MOSC1 KO with 80% regression, p=0.0476). Moreover, we find that NK cell depletion, achieved with systemic delivery of the blocking antibody PK136, was sufficient to reverse the complete response of 4MOSC1 tumors to anti CTLA-4 immune checkpoint inhibition (p<0.01), implying promise for combination therapeutic strategies in immunotherapy insensitive tumors. Ongoing studies are using IHC and mass cytometry (Cytof) to characterize the immune infiltrates to better define immune cell populations regulated by CHMP2A-mediated resistance of tumor cells to NK cell and possibly T-cell-mediated responses. Together, these studies demonstrate that CHMP2A provides a targetable, tumor-derived inhibitor of NK cell-driven antitumor immunity. Moreover, our preclinical model features HNSCC cell lines with variable sensitivity to CHMP2A-deletion to enable future studies to target key pathways to overcome resistance programs and mediate improved anti-tumor activity. Citation Format: Jiyoung Yun, Robert Saddawi-Konefka, Benjamin Goldenson, Riyam Al-msari, J Silvio Gutkind, Dan S. Kaufman. CHMP2A regulates NK cell-mediated anti-tumor activity in a syngeneic HNSCC model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2107.

CXCR4 blockade reduces the severity of murine heart allograft rejection by plasmacytoid dendritic cell-mediated immune regulation

Allograft-specific regulatory T cells (Treg cells) are crucial for long-term graft acceptance after transplantation. Although adoptive Treg cell transfer has been proposed, major challenges include graft-specificity and stability. Thus, there is an unmet need for the direct induction of graft-specific Treg cells. We hypothesized a synergism of the immunotolerogenic effects of rapamycin (mTOR inhibition) and plerixafor (CXCR4 antagonist) for Treg cell induction. Thus, we performed fully-mismatched heart transplantations and found combination treatment to result in prolonged allograft survival. Moreover, fibrosis and myocyte lesions were reduced. Although less CD3+ T cell infiltrated, higher Treg cell numbers were observed. Noteworthy, this was accompanied by a plerixafor-dependent plasmacytoid dendritic cells-(pDCs)-mobilization. Furthermore, in vivo pDC-depletion abrogated the plerixafor-mediated Treg cell number increase and reduced allograft survival. Our pharmacological approach allowed to increase Treg cell numbers due to pDC-mediated immune regulation. Therefore pDCs can be an attractive immunotherapeutic target in addition to plerixafor treatment.

Epigenetic Regulation of NK Cell-Mediated Antitumor Immunity.

Natural killer (NK) cells are critical innate lymphocytes that can directly kill target cells without prior immunization. NK cell activation is controlled by the balance of multiple germline-encoded activating and inhibitory receptors. NK cells are a heterogeneous and plastic population displaying a broad spectrum of functional states (resting, activating, memory, repressed, and exhausted). In this review, we present an overview of the epigenetic regulation of NK cell-mediated antitumor immunity, including DNA methylation, histone modification, transcription factor changes, and microRNA expression. NK cell-based immunotherapy has been recognized as a promising strategy to treat cancer. Since epigenetic alterations are reversible and druggable, these studies will help identify new ways to enhance NK cell-mediated antitumor cytotoxicity by targeting intrinsic epigenetic regulators alone or in combination with other strategies.

Immunoprofiling of Drosophila Hemocytes by Single-cell Mass Cytometry

Single-cell mass cytometry (SCMC) combines features of traditional flow cytometry (i.e., fluorescence-activated cell sorting) with mass spectrometry, making it possible to measure several parameters at the single-cell level for a complex analysis of biological regulatory mechanisms. In this study, weoptimizedSCMC to analyze hemocytes of the Drosophila innate immune system. We used metal-conjugated antibodies (against cell surface antigens H2, H3, H18, L1, L4, and P1, and intracellular antigens 3A5 and L2) and anti-IgM (against cell surface antigen L6) to detect the levels of antigens, while anti-GFP was used to detect crystal cells in the immune-induced samples. We investigated the antigen expression profile of single cells and hemocyte populations in naive states, in immune-induced states, in tumorous mutants bearing a driver mutation in the Drosophila homologue of Janus kinase (hopTum) and carrying a deficiency of the tumor suppressor gene lethal(3)malignant blood neoplasm-1 [l(3)mbn1], as well as in stem cell maintenance-defective hdcΔ84 mutant larvae. Multidimensional analysis enabled the discrimination of the functionally different major hemocyte subsets for lamellocytes, plasmatocytes, and crystal cells, anddelineated the unique immunophenotype of Drosophila mutants. We have identified subpopulations of L2+/P1+ and L2+/L4+/P1+ transitional phenotype cells in the tumorous strains l(3)mbn1 and hopTum, respectively, and a subpopulation of L4+/P1+ cells upon immune induction. Our results demonstrated for the first time that SCMC, combined with multidimensional bioinformatic analysis, represents a versatile and powerful tool to deeply analyze the regulation of cell-mediated immunity of Drosophila.

Mesenchymal Stromal Cell-Mediated Immune Regulation: A Promising Remedy in the Therapy of Type 2 Diabetes Mellitus

Type 2 diabetes mellitus (T2DM) is a major threat to global public health, with increasing prevalence as well as high morbidity and mortality, to which immune dysfunction has been recognized as a crucial contributor. Mesenchymal stromal cells (MSCs), obtained from various sources and possessing potent immunomodulatory abilities, have displayed great therapeutic potential for T2DM. Interestingly, the immunomodulatory capabilities of MSCs are endowed and plastic. Among the multiple mechanisms involved in MSC-mediated immune regulation, the paracrine effects of MSCs have attracted much attention. Of note, extracellular vesicles (EVs), an important component of MSC secretome, have emerged as pivotal mediators of their immunoregulatory effects. Particularly, the necrobiology of MSCs, especially apoptosis, has recently been revealed to affect their immunomodulatory functions in vivo. In specific, a variety of preclinical studies have demonstrated the beneficial effects of MSCs on improving islet function and ameliorating insulin resistance. More importantly, clinical trials have further uncovered the therapeutic potential of MSCs for T2DM. In this review, we outline current knowledge regarding the plasticity and underlying mechanisms of MSC-mediated immune modulation, focusing on the paracrine effects. We also summarize the applications of MSC-based therapies for T2DM in both preclinical studies and clinical trials, with particular emphasis on the modulation of immune system.

Integrated analysis of the molecular mechanisms in idiopathic pulmonary fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) is one of the most aggressive forms of idiopathic interstitial pneumonia. Some miRNAs may be associated with IPF and may affect the occurrence and development of IPF in various pathways. Many miRNAs and genes that may be involved in the development of IPF have been discovered using chip and high throughput technologies.Methods: We analyzed one miRNA and four mRNA databases. We identified hub genes and pathways related to IPF using GO, KEGG enrichment analysis, gene set variation analysis (GSVA), PPI network construction, and hub gene analysis. A comprehensive analysis of differentially expressed miRNAs (DEMs), predicted miRNA target genes, and differentially expressed genes (DEGs) led to the creation of a miRNA-mRNA regulatory network in IPF.Results: We found 203 DEGs and 165 DEMs that were associated with IPF. The findings of enrichment analyses showed that these DEGs were mainly involved in antimicrobial humoral response, antimicrobial humoral immune response mediated by antimicrobial peptide, extracellular matrix organization, cell killing, and organ or tissue specific immune response. The VEGFA, CDH5, and WNT3A genes overlapped between hub genes and the miRNA-mRNA regulatory network. The miRNAs including miR-199b-5p, miR-140-5p, miR-199a-5p, miR-125A-5p, and miR-107 that we predicted would regulate the VEGFA, CDH5, and WNT3A genes, which were also associated with IPF or other fibrosis-related diseases. GSVA indicated that metabolic processes of UTP and IMP, immune response, regulation of Th2 cell cytokine production, and positive regulation of NK cell-mediated immunity are associated with the pathogenesis and treatment of IPF. These pathways also interact with VEGFA, CDH5, and WNT3A.Conclusion: These findings provide a new research direction for the diagnosis and treatment of IPF.

Effects of Single-Dose Interleukin-12 Exposure on Interleukin-12–Associated Toxicity and Interferon-γ Production

AbstractInterleukin-12 (IL-12) is a key regulator of cell-mediated immunity that has therapeutic potential in cancer and infectious disease. In a previous Phase 1 dose escalation study of a single test dose of recombinant human IL-12 (rhIL-12) followed 14 days later by cycles of five consecutive daily intravenous injections every 3 weeks, we showed that a dose level up to 500 ng/kg could be administered with acceptable levels of safety. Based on these results, a Phase 2 study was conducted. In the Phase 2 study, however, administration of rhIL-12 at this same dose level resulted in severe toxicities with some patients unable to tolerate more than two successive doses. Of the 17 patients receiving rhIL-12 in the Phase 2 study, 12 patients were hospitalized and two patients died. A thorough scientific investigation to determine the cause of this unexpected toxicity failed to identify any difference in the drug products used or the patient populations enrolled in the Phase 1 and Phase 2 studies that could have accounted for the profound difference in toxicity. The focus of the investigation therefore shifted to the schedule of rhIL-12 administration. We determined that a single injection of rhIL-12 2 weeks before consecutive dosing included in the Phase 1 study, but not in the schedule of administration in the Phase 2 study, has a profound abrogating effect on IL-12–induced interferon-γ (IFN-γ) production and toxicity. This observation of schedule-dependent toxicity of IL-12 has been verified in mice, as well as nonhuman primates. In this regard, a single injection of IL-12 before consecutive daily dosing protected mice and cynomolgus monkeys from acute toxicity including mortality and was associated with an attenuated IFN-γ response. Because of this unique biologic response, careful attention to the schedule of administration is required to assure safe and effective clinical development of this highly promising cytokine.

IL-35 is critical in suppressing superantigenic Staphylococcus aureus-driven inflammatory Th17 responses in human nasopharynx-associated lymphoid tissue

The human nasopharynx is frequently exposed to microbial pathogens, including superantigen-producing Staphylococcus aureus (SAg-Sau), which activates potent pro-inflammatory T cell responses. However, cellular mechanisms that control SAg-Sau-driven T cell activation are poorly understood. Using human nasopharynx-associated lymphoid tissue (NALT), we show that SAg-Sau drove a strong Th17 activation, which was associated with an impaired CD4+ T cell-mediated immune regulation. This impairment of immune control correlated with a significant downregulation of interleukin-35 (IL-35) expression in tonsillar CD4+ T cells by SAg-Sau. Supplementing recombinant IL-35 suppressed SAg-Sau-activated Th17 responses, and this IL-35-mediated suppression positively correlated with the level of Th17 activation. Interestingly, SAg-Sau stimulation induced Foxp3+ Treg expansion and interleukin-10 (IL-10) production, which effectively suppressed the Th1 response, but failed to control the activation of Th17 cells. Overall, our results reveal an aberrant T cell regulation on SAg-Sau-driven Th17 activation and identify IL-35 as a critical cytokine to control superantigenic S.aureus-activated Th17 responses.

PSVI-6 Effect of cow neutrophil activation on expression of T cell immunoglobulin and Mucin-Domain-Containing-Protein 3 (TIM-3) and its ligand Galectin 9 gene

Abstract The objective of this study was to assess the effect of neutrophil activation on the expression of T cell immunoglobulin and mucin domain-containing protein-3 (TIM-3) and its ligand Galectin 9. T cell immunoglobulin and Mucin-Domain-Containing-Protein 3 is a type I transmembrane protein involved in the regulation of cell-mediated immunity. Galectin-9 is a β-galactoside-binding lectin, that can function as an alarmin in response to diverse pathogens. Binding of Gal-9 to TIM-3 and stimulation with phorbol 12-myristate 13-acetate (PMA) induced activation of NADPH oxidase on human neutrophils. Increased understanding of Gal-9 to TIM-3 gene expression and modulation in cow neutrophils is needed. Blood was collected from the jugular vein of clinically healthy Holstein-Friesian cows (n = 3) from North Carolina A&T dairy unit. Neutrophils were isolated by differential centrifugation and incubated with either 10ng/ml of PMA or maintained in Phosphate buffer saline for 30 minutes at 370 C. Total RNA was isolated from neutrophils reverse transcribed to cDNA. Specific primers for TIM-3 and LGALS -9 were designed using the NCBI Primer 3 tool. Pooled cDNA samples were used for real-time PCR. Housekeeping genes RPLP0 and UCHL5 served as internal controls. Fold change in transcript abundance was calculated using the Livak method (Fold change >2 were considered as significant). Both LGAS9 and TIM-3 were expressed in resting neutrophils. Treatment with PMA upregulated transcription of TIM-3 (5.55 fold). Whereas, LGALS -9 transcription remained unchanged. Activation of cow neutrophils using PMA changes in the expression of TIM-3. Further studies are needed to define the signaling pathways involved and functional implications for animal health and production.

Peripheral immune response in the African green monkey model following Nipah-Malaysia virus exposure by intermediate-size particle aerosol.

The ability to appropriately mimic human disease is critical for using animal models as a tool for understanding virus pathogenesis. In the case of Nipah virus (NiV), infection of humans appears to occur either through inhalation, contact with or consumption of infected material. In two of these circumstances, respiratory or sinusoidal exposure represents a likely route of infection. In this study, intermediate-size aerosol particles (~7 μm) of NiV-Malaysia were used to mimic potential routes of exposure by focusing viral deposition in the upper respiratory tract. Our previous report showed this route of exposure extended the disease course and a single animal survived the infection. Here, analysis of the peripheral immune response found minimal evidence of systemic inflammation and depletion of B cells during acute disease. However, the animal that survived infection developed an early IgM response with rapid development of neutralizing antibodies that likely afforded protection. The increase in NiV-specific antibodies correlated with an expansion of the B cell population in the survivor. Cell-mediated immunity was not clearly apparent in animals that succumbed during the acute phase of disease. However, CD4+ and CD8+ effector memory cells increased in the survivor with correlating increases in cytokines and chemokines associated with cell-mediated immunity. Interestingly, kinetic changes of the CD4+ and CD8bright T cell populations over the course of acute disease were opposite from animals that succumbed to infection. In addition, increases in NK cells and basophils during convalescence of the surviving animal were also evident, with viral antigen found in NK cells. These data suggest that a systemic inflammatory response and “cytokine storm” are not major contributors to NiV-Malaysia pathogenesis in the AGM model using this exposure route. Further, these data demonstrate that regulation of cell-mediated immunity, in addition to rapid production of NiV specific antibodies, may be critical for surviving NiV infection.

Phenotyping Gamma Delta T cells in Bladder Cancer

Event Abstract Back to Event Phenotyping Gamma Delta T cells in Bladder Cancer Magdalene Joseph1*, Fidelma Cahill2, Harriet Wylie2, Deborah Enting2, Mieke Van Hemelrijck1 and Adrian Hayday1 1 King's College London, United Kingdom 2 Guy's and St Thomas' NHS Foundation Trust, United Kingdom Background Bladder cancer is known to be immunologically modulated since the 1970s when Bacille-Calmette Guerin (BCG) was shown effective in treating superficial bladder tumours. Moreover, immune checkpoint inhibitors have now demonstrated survival benefit in advanced bladder cancer and are considered standard treatment for chemotherapy-resistant disease. Of special interest in immuno-oncology are unconventional, innate-like T cells bearing the gamma delta T cell receptor. They are found enriched at the epithelial surfaces and are capable of rapid, non-MHC restricted responses to various stressors. These cells are found to be the strongest correlate of positive prognosis in a recent transcriptomic analysis of over 18,000 tumours 1. Studies on mouse models of bladder cancer have shown that survival can be prolonged by injecting gamma delta T cells intravesically2 and that responses to BCG are abolished in gamma delta T cell knockout mice suggesting an anti-tumorigenic role3. This project aims to characterise the presence and phenotype of gamma delta T cells in the blood and tissue in human bladder cancer to understand their contribution to the immune regulation of the disease. Methods Mononuclear cells from peripheral blood of healthy, age-matched controls and patients with bladder cancer will be phenotyped by flow cytometry with a focus on gamma delta T cells to elucidate the differences between health and disease. Tissue infiltrating lymphocytes will also be isolated from bladder tumour tissue and peri-tumoural normal tissue using a cell-foam matrix culture system described previously4 and phenotyped in a similar fashion. Phenotypes from flow cytometry will be confirmed by polymerase chain reaction targeting the markers of interest. The localisation of gamma delta T cells within the tissue will be examined by immunohistochemistry. Results Preliminary results from healthy volunteers show differences in the expression of various markers between different gamma delta T cell populations and differences between fresh and frozen samples. Conclusion The aim is to understand gamma delta T cell mediated immune regulation of bladder cancer. Acknowledgements The King's Health Partners Urological Cancer Biobank and the King's College London Infectious Diseases Biobank for their help in obtaining patient and control samples. References