IFN-gamma Expression Research Articles

Saurian-associated Leishmania tarentolae in dogs: Infectivity and immunogenicity evaluation in the canine model.

In canine leishmaniosis endemic areas, Leishmania infantum may occur in sympatry with the non-pathogenic Leishmania tarentolae, which is associated to reptiles. The potential infectivity of L. tarentolae for mammals raises questions about the interactions between the two Leishmania species, and the potential cross-immune protection in dogs. This study aimed to assess the outcome of experimental L. tarentolae infection in dogs, determining: i) the anti-L. tarentolae antibody production, ii) the duration of the immunity and cytokine expression, and iii) the possible pathogenic effect in the canine host. Twelve purpose-bred beagle dogs were randomly allocated to three groups (intravenous inoculation, G1; intradermal inoculation, G2; negative control, G3). G1 and G2 dogs were inoculated twice (day 0, day 28) with 108 promastigotes of L. tarentolae strain (RTAR/IT/21/RI-325) isolated from a Tarentola mauritanica gecko. The animals were followed until day 206. Blood, serum, conjunctival swabs and lymph node aspirate samples were collected monthly and bone marrow, liver and spleen biopsies on day 91. Hematological and biochemical parameters were assessed monthly, as well as serology (IFAT and ELISA) and molecular identification of L. tarentolae. Mononuclear cells (PBMC) were obtained to assess the cytokine expression through in vitro stimulation or (re-) infection. Data from this study demonstrated that DNA from L. tarentolae is detectable up to 3 months post-infection, with seroconversion after day 28. Moreover, the non-pathogenic nature of L. tarentolae was confirmed, with a neutral Th1/Th2 polarization, and a possible shift to Th1 phenotype after derived macrophages (re-) infection, as demonstrated by the expression of IFN-gamma. Therefore, L. tarentolae demonstrated a great potential as a surrogate pathogen and/or immune-prophylaxis/immune-therapy against Leishmania infections in dogs and humans.

Abstract 5003: Efficacy of the STC-1010 a new allogenic cancer vaccine in different colorectal cancer models

Abstract Colorectal cancer (CRC) is the 2nd cause of cancer-related death worldwide. Recent tremendous progress has installed immunotherapy as a treatment option and opened multiple ways to address therapeutic challenges for CRC patients. Accordingly, Brenus-Pharma developed a therapeutic cancer vaccine, based on stimulating tumor cells (STC) technology with physical (irradiation and heat shock) or chemical (chemotherapy) stress, coupled to haptenization making vaccine more immunogenic. We first validated STC technology using a “one cell line” mouse surrogate vaccine in CT26 mouse model showed an efficacy of the combination of stress plus haptenization. We next prepared a mouse surrogate vaccine using three murine tumoral cell lines the mSTC-1010 (CT26, CMT93 and LTPA), and confirmed the value of using 3 cell lines to increase the antigenicity. In addition, mSTC-1010 associated with immunostimulant (cyclophosphamide and mGM-CSF), combined or not with chemotherapy (FOLFOX or FOLFIRI) led to a significant decrease of tumor volume, a M1-oriented macrophage response (immunohistochemical, iNOS/CD163 >1), and an increase of T lymphocyte infiltration. Following, we confirmed these proofs-of-concept with the human vaccine, STC-1010, composed of 6 drug substances (DS) derived from 3 CRC human cell lines (HCT116, HT29 and Lovo), stressed physically or chemically and then haptenized. Transcriptomic analysis showed the induction of stress-related pathways with a certain specificity for each DS, allowing their quantification in the STC-1010 by deconvolution strategy. Moreover, proteomic analysis showed specific protein expression modification of each DS and confirmed the rationale of using 3 cell lines with 2 stresses to cover the CRC heterogeneity. More than 200 cancer-related proteins from the Atlas proteins’ database were identified in theSTC-1010 including CRC-specific proteins, tumor plasticity and tumor-associated antigens. In ex vivo model, the efficacy of the vaccine was shown, through that CD8+ T cells primed by the STC-1010 treated DCs (versus control), induced massive apoptosis of cancer cells (p<0,001). In chicken embryo Chorio-Allantoid Membrane (CAM) model, STC-1010 significantly increased IL-12, IL-2 and IFN-gamma expression (versus control p<0.02). Results from this immune reactive model showed a significant increase of tumor necrosis (p=0,0267), metastasis regression (-49%), and increased infiltration of CD4+, CD8+ (versus control). The good tolerability and reproducible efficiency of the STC-1010 vaccine in these different clinical models allow to plan a first-in-human phase I/II clinical trial for metastatic CRC(mCRC) patients. A dose-escalation and cohort extension phase I followed by a phase IIa with STC-1010 plus immunostimulant, associated to mFOLFOX6 w/o bevacizumab, will be performed for MSS mCRC patients to evaluate the safety and preliminary effectiveness in human. Citation Format: George Alzeeb, Céline Gongora, Corentin Richard, Romain Boidot, Tanguy Fortin, Yan Wang, Alban Bessede, Benoit Pinteur, Lionel Chalus, Corinne Tortorelli, Paul Bravetti, François Ghiringhelli. Efficacy of the STC-1010 a new allogenic cancer vaccine in different colorectal cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5003.

Effect of Vitamin D3 Levels on Varicella-Zoster Virus Infection and IFN-Gamma Expression in Children: A Cross-Sectional Study in Iraq

Background: This study aimed to explore the correlation between Vitamin D3 levels and IFN-Gamma expression in children residing in the southern and central provinces of Iraq. Vitamin D3 plays a pivotal role in immune function, and IFN-Gamma is a crucial cytokine involved in antiviral defense. Investigating the connection between Vitamin D3 and IFN-Gamma offers valuable insights into immune responses and potential implications for infectious diseases.Methods: A case-control study was conducted, involving children from various schools and kindergartens in the southern and central provinces of Iraq. The study assessed Vitamin D3 levels and measured IFN-Gamma expression. Statistical analyses were performed to determine the relationship between these variables.Results: The outcomes revealed a significant positive correlation between Vitamin D3 levels and IFN-Gamma expression within the study population (p < 0.05). Children with higher Vitamin D3 levels exhibited elevated IFN-Gamma expression, suggesting a possible immunomodulatory impact of Vitamin D3 on IFN-Gamma production.Conclusion:These findings underscore the importance of maintaining adequate Vitamin D3 levels to support immune function, particularly in relation to IFN-Gamma expression. Improving Vitamin D3 status could potentially bolster antiviral defense mechanisms and reduce susceptibility to viral infections among children in the southern and central provinces of Iraq. Further investigation is warranted to delve into the underlying mechanisms and potential clinical ramifications of this significant association. Additionally, exploring the long-term effects of maintaining optimal Vitamin D3 levels on immune function and the outcomes of infectious diseases in this population would offer valuable insights for preventive and therapeutic strategies.

Pedagogical insights from immune responses: analyzing Covid-19's severity through systemic immune-inflammation indexes and gene expressions

Following the COVID-19 pandemic, it's crucial to understand the relationship between immune responses and clinical results, which also underpins interdisciplinary educational discussions. This study goes beyond standard medical evaluation, linking clinical medicine to sociodemography, genetics, and education, emphasizing the importance of an interconnected view for a comprehensive understanding. Severe COVID-19 cases show imbalances in the immune system, highlighting the need for early and precise prognosis, given its wider sociodemographic consequences. This study aims to investigate potential associations between systemic immune-inflammation indexes, gene expression of IFNA1, IFN receptors, IL17A, and plasmatic IFN-gamma in COVID-19 patients. Our observational study recruited 67 COVID-19 patients, both mild (28) or severe (39). Data was gathered on demographics, clinical symptoms, and laboratory results. Blood tests were used to analyze gene expressions and plasma IFNG levels. Most severe cases were males (61.5%) and of the black ethnic group (87.2%), with a clear age difference compared to mild cases. Elevated levels of systemic immune-inflammation measures were seen in severe cases, with gene expressions of IFNA1 and IL17A increasing, whereas IFNAR1 was more common in mild cases. From a pedagogical standpoint, the study emphasizes the link between clinical findings, sociodemographic and genetics data, underlining its profound implications for medical education. It suggests using specific gene expressions and hematological tests as COVID-19 severity indicators and underscores the need for further research to validate and explore broader clinical and pedagogical implications.

Phase 1 Trial of Dendritic Cell/AML Fusion Cell Vaccine after Allogeneic Stem Cell Transplant in Acute Myeloid Leukemia

Introduction We have developed a vaccine in which patient-derived AML cells are fused with donor-derived dendritic cells (DCs). Vaccination after initial remission and consolidation was shown to elicit leukemia-specific immune responses. We hypothesized that donor-derived DC/AML vaccination post-transplant would elicit the durable expansion of leukemia-specific T cells within the donor T cell repertoire to effectively protect against disease relapse. We report updated results of a phase 1 clinical trial (NCT03679650) evaluating the use of DC/AML fusion vaccine following allogeneic transplant. Methods DC/AML fusion vaccine was generated with patient leukemia blasts, cryopreserved at the time of diagnosis, and donor-derived DCs, from leukapheresis of patients who achieved hematopoietic recovery and remained in CR 25-90 days after an allogeneic transplant. DCs were differentiated via culture of adherent mononuclear cells in the presence of GM-CSF, IL-4 and TNFa. Vaccine was administered starting day 70-100 post-transplant as long as the patient did not have active GVHD requiring therapy. Two doses of the vaccine were given subcutaneously at 3 week intervals, in conjunction with 100 mcg GMCSF daily at the vaccine site for 4 days. A booster vaccine was given 30-60 days following the taper of immune suppression, in the absence of GVHD. Longitudinal profiling of the immune landscape by single cell RNA sequencing and CyTOF from peripheral blood samples obtained before and after vaccination is underway. Immunologic response of leukemia-specfic T cells is being evaluated via IFN-gamma expression following stimulation with autologous tumor lysate. OS was calculated using Kaplan Meier Curve. Results 27 participants have been enrolled with median age 59 years (range 23-74). 15 patients were transplanted with a matched unrelated donor, 10 with a matched sibling donor and two with a haplo-identical donor. One patient relapsed prior to leukapheresis and was ineligible. The median yield of leukemia cells was 218x10 6 (range 62-818x10 6)and mean viability was 97%. The median yield of DCs was 106x10 6 (range 19-182x10 6) and mean viability 81%. Fusion vaccine was successfully generated in 25 of 26 patients who underwent leukapheresis; one patient had insufficient DC. Mean fusion efficiency was 47% with viability of 79%. Mean fusion vaccine dose was 3.8 x10 6 cells. 6 patients from whom vaccine was generated did not meet eligibility to initiate vaccination due to GVHD or COVID19 infection. 19 participants initiated vaccine administration and are evaluable for toxicity and immunologic response. The most common side effects were vaccine site reactions (n=10 grade 1, n=2 grade 2). 5 patients developed GVHD that was attributed as possibly related to vaccination, at a median time of 17 days after vaccination (range 5-70 days). Acute GVHD included two events of skin grade 2, one GI grade 2, one liver and skin grade 3. Chronic GVHD included a moderate event affecting 3 organs and severe event affecting 4 organs. There were 10 additional GVHD events with a median time of 108 days post vaccination (range 7-126 days), assessed as unlikely related or unrelated to vaccine based on pre-existing GVHD and timing of onset. Median time of follow-up is 23 months post-transplant (range 11-59 months). Amongst 19 vaccinated patients, 14 remained in CR and median overall survival estimate is 49 months post transplant. 3 died of relapsed disease, 2 died of GVHD and/or infection, and 1 died of unknown cause. Preliminary results of PB CyTOF from 4 patients in remission and 1 patient that relapsed indicate higher proportions of T cells and DCs in patients in CR. Interferon expression was higher in a patient with durable remission while absent from a patient with early relapse. Conclusions Vaccination using host-derived leukemia cells and donor derived DC was safe and feasible. Adverse events post vaccination have included injection site reactions as well as acute and chronic graft versus host disease. Vaccination may be a promising strategy to reduce relapse as 14 of 19 vaccinated patients remain relapse free. Albeit this approach is limited to patients who have not had early relapse or early GVHD. Immunologic response by interferon release upon lysate stimulation, CyTOF and single cell RNA sequencing of the immune milieu will be reported.

Expression of IFN-Gamma is significantly reduced during severity of covid-19 infection in hospitalized patients.

Cytokines play an important role in SARS-CoV-2 infection progression and severity. A number of inflammatory cytokines have been directly associated with disease severity including IL-6 (interleukin-6), IL-10, TNF-α (tumor necrosis factor alpha), IFN-γ (interferon-gamma). Here, in this study, the aim was to better understand the interplay between host immune response mediated by cytokines and severity of SARS-CoV-2 infection by assessing cytokine expression. Therefore, we measured expression levels of a total of 12 genes (IFNA-1, IFN-γ, IL-1α, IL-1β, IL-4, IL-6, IL-7, IL-10, IL-11, IL-13, IL-15, and IL-27) encoding inflammatory, anti-inflammatory and regulatory cytokines using QRT-PCR in hospitalized patients with severe infection compared to mildly infected. IFN-γ was identified as a potent marker of disease severity as indicated previously. Moreover, levels of IL-7 were also found to be partially reduced in patients compared to the healthy controls and linked negatively to disease severity. Identification of these cytokines may be helpful in not only understanding disease pathogenesis but also in better management of the patients after covid infection.

Efficacy of STC-1010, a new allogenic cancer vaccine in colorectal cancer models.

e14661 Background: Colorectal cancer (CRC) is the second cause of cancer-related death worldwide. Recent tremendous progresses have installed immunotherapy as a treatment option and opened multiple ways to address therapeutic challenges for MSI or MSS CRC patients. Accordingly, Brenus-Pharma developed a therapeutic cancer vaccine, the STC-1010, basing on the technology by stimulating tumor cells (STC) with physical (irradiation and heat shock) and chemical (via standard-of-care (SoC) chemotherapy) stress, coupled with the haptenization for overexpressing tumor-related antigens. Methods: The mouse version of STC-1010 (mSTC-1010) vaccine prepared using two CRC mouse cell lines (CT26 and CMT93) and one pancreatic cancer mouse cell line (LTPA), previously showed encouraging results in pre-clinical studies on MSS CT26 ‘cold-tumor’ and anti-PD1 resistant MSI MC38 ‘hot-tumor’ murine models. mSTC-1010 associated with immunostimulant (cyclophosphamide and GM-CSF at low doses), combined or not with chemotherapy (FOLFOX or FOLFIRI) led to a significant decrease of tumor volume, a M1-oriented macrophage response (immunohistochemical, iNOS/CD163 > 1), and an increase of T lymphocyte infiltration. Results: We confirmed these proof-of-concept efficacy with the human STC-1010 using three CRC human cell lines (HCT116, HT29 and Lovo). STC-1010 favored ex vivo an immune stimulation on human dendritic cells (DCs) which further primed CD8+ T cells to induce massive apoptosis of CRC cells (versus control). In HT29 in-ovo chicken embryo Chorio-Allantoid Membrane (CAM) assay, STC-1010 significatively increased IL-12, IL-2 and IFN-gamma expression (versus control) (p < 0.02). Results from this immune reactive model showed significant increase of tumor necrosis (p = 0,0267), metastasis regression (-49%), and increase infiltration of CD4+, CD8+ compared to control group. Conclusions: The good tolerability of the STC-1010 vaccine in different models allows to plan a first-in-human phase I/II clinical trial with MSS and MSI-H metastatic CRC (mCRC) patients. A dose-escalation phase Ia (STC-1010 plus immunostimulant, associated to mFOLFOX6 w/o bevacizumab) will be performed with MSS mCRC patients to evaluate the safety and the recommended phase 2 trial dose (RP2D). A subsequent expansion phase IIa part will assess STC-1010 efficiency, associated with SoC in first-line setting for MSS patients, and in second-line setting after immunotherapy for MSI-H, dMMR or Lynch syndrome mCRC patients.

The Effects of Mesenchymal Stem Cells on the Gene Expression of TGF-beta and IFN-gamma in Patients with Rheumatoid Arthritis.

The therapeutic and immunomodulatory potential of mesenchymal stem cells (MSCs) in rheumatoid arthritis (RA) has attracted considerable scientific attention in recent decades. This study aimed to evaluate the expression of genes encoding interleukin (IL)4 and IL10, as well as interferon-gamma (IFNG) and transforming growth factor beta (TGFB1) in refractory RA patients following intravenous injection of autologous bone marrow-derived MSCs (BM-MSCs). This study was registered in Iranian Registry of Clinical Trials (IRCT) (2015102824760N1) and ClinicalTrials.gov (identifier: NCT03333681). Blood samples were taken from 13 patients before and 1 and 6 months after the MSC injection to evaluate the clinical manifestations, paraclinical factors, and expression of IL4, IL10, IFNG, and TGFB1 genes employing the SYBR Green real-time reverse-transcriptase polymerase chain reaction (RT-PCR) technique. There was a significant increase in the expression of TGFB1 at 1 and 6 months after the MSC injection compared to that in the baseline, while the expression of IL4 and IL10 did not change significantly. On the other hand, the expression of IFNG increased significantly after 1 month but decreased significantly at 6 months compared to 1 month after the intervention. Nevertheless, it showed no significant decrease compared to the baseline. A significant decrease was observed for the expression of IFNG 6 months after the injection compared to that after 1 month, which was in concordance with the rise in the expression of the TGFB1 gene. A significant change in the gene expression of TGFB1 and IFNG in our study wasconsistent with the amelioration of clinical manifestations, suggesting a mechanism of action for MSCs in the treatment of RA.

Blocking activation of CD4−CD8− T cells modulates their cytotoxic potential and decreases the expression of inflammatory and chemotactic receptors

CD4−CD8− (double negative – DN) T cells represent a small fraction of circulating T lymphocytes but are a major source of pro-inflammatory cytokines in patients with infectious diseases, including chronic Chagas cardiomyopathy (CCC), one of the deadliest cardiopathies known. Chagas disease is caused by an infection with the protozoan parasite Trypanosoma cruzi and can lead to either an asymptomatic form or a high-mortality cardiac disease. While circulating DN T cells represent a major inflammatory cytokine-expressing cell population in Chagas disease, their potential to be recruited to the heart and to perform cytotoxicity has not been determined. Our previous studies showed that blocking DN T cell activation decreases the expression of IFN-gamma, a cytokine involved in the severity of CCC. Here, studying a well-characterized cohort of Chagas patients with CCC or the asymptomatic form of Chagas disease (indeterminate form, IND), we evaluated the expression of cytotoxic molecules, cytokine and chemokine receptors in γδ+ and αβ+ DN T cells by multiparameter flow cytometry, and investigated whether blocking the activation of DN T cells influences the expression of these molecules. We observed that DN T cells from CCC display a higher expression of granzyme A, perforin, inflammatory molecules, and inflammatory chemokine receptors than cells from IND. Messenger RNA coding for these molecules is also upregulated in the heart of CCC patients. Importantly, blocking the activation of DN T cells from CCC modulates their cytotoxic potential and the expression of inflammatory and of chemokine receptors, suggesting that targeting DN T cell activation may be a valid strategy to reduce recruitment to the heart, inflammation, cytotoxicity and, thereby diminish CCC progression and severity.

Abstract 835: Translational nanotechnology-based cancer vaccine to re-educate host immune response against metastatic cancers

Abstract Sixty percent of melanoma patients present brain metastases, having low response rates to current systemic therapies, which do not prolong survival. New therapeutic options to address this clinical need are urgent. Here, we report the development of a polymeric nanoparticle as a cancer nanovaccine capable of selectively co-targeting toll-like receptor function and PD-L1 expression to improve antigen presentation and subsequent effector immune cell function within brain microenvironment. Mannose-poly(lactic-co-glycolic acid)/poly(lactic acid) (man-PLGA/PLA) nanoparticles (NP) were prepared by a modified double emulsion solvent evaporation method, to deliver combinations of melanoma neoantigens, toll-like receptor ligands and regulators of the PD-1/PD-L1 axis. NP physicochemical properties were fully characterized, including size, surface charge and morphology. The amount of melanoma neoantigens and immune regulators entrapped within NP was determined by HPLC. Immature dendritic cells (DC) were used to evaluate the impact of NP on cell viability, and to assess NP uptake kinetics by flow cytometry. NP ability to target and trigger the activation of DC and T cells, was assessed in the lymph nodes and spleen of immunized mice. The NP anti-tumor immune-mediated effect was evaluated in vivo in two primary melanoma-bearing immunocompetent mouse models (B16F10 and B16MO5), and in a melanoma brain metastasis (MBM) mouse model (B16F10), which also included the immune profiling within tumor site by flow cytometry, before and after treatment.NP presented an average diameter of 180 nm, narrow polydispersity index, surface charge close to neutrality, spherical morphology, and high loadings of the neoantigens and immunoregulators. NP did not affect DC viability and were extensively internalized by immature DC. NP were preferentially taken-up in vivo by DC, increasing the expression of activation/maturation markers at DC surface, such as CD80 and CD86. NP elicited antigen-specific T-cell responses, by the significant increase in the expression of TNF-alpha and IFN-gamma (TH1-guided response). In vivo combination of NP with anti-PD-L1 induced a potent immune-mediated anti-tumor response in both primary and preclinical models of MBM, overcoming tumor development with an increased overall survival. This combination re-shaped immune and stroma cell populations in primary tumor and MBM microenvironment, which presented a marked infiltration of cytotoxic T cells that correlated with a decreased expression of PD-1 and PD-L1 within the TME. The synergy between our nanovaccine and anti-PD-L1 provide essential insights to devise alternative combinations regiments to improve the efficacy of immune checkpoint inhibitors in metastatic melanoma, thus opening new line for this unmet medical need. Citation Format: Barbara Carreira, Rita A. Acúrcio, Sabina Pozzi, Ron Kleiner, Liane I. Moura, Ana I. Matos, Daniela vaskovich, Carina Peres, Adelaide Fernandes, Sara Xapelli, Ronit Satchi-Fainaro, Helena F. Florindo. Translational nanotechnology-based cancer vaccine to re-educate host immune response against metastatic cancers [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 835.

Abstract 6388: High dose acetaminophen with n-acetylcysteine rescue inhibits M2 polarization of tumor associated macrophages

Abstract Background: High dose acetaminophen (AAP) with n-acetylcysteine (NAC) rescue is among the few treatments that has shown a signal of activity in phase I trials (21% response rate, 3/14 assessable patients) without achieving dose limiting toxicity that has not progressed to evaluation in later line studies. We have previously shown that high dose AAP selectively depletes glutathione in the liver but not the tumor, suggesting that NAC, the FDA-approved antidote for AAP toxicity and a glutathione precursor, may selectively rescue the normal liver but not the tumor from high dose AAP. While the anti-tumor effects of AAP/NAC appear to not be mediated by glutathione depletion and free radical injury, the mechanism of anti-tumor effects of AAP/NAC has not been definitively characterized. Methods: In vitro the effects of Vehicle, AAP, or AAP/NAC was evaluated on bone marrow derived macrophages polarized to M2 and M1 phenotypes using IL-4 or IFN/LPS, respectively. Tumor cell viability was assessed with MTT assay. Effects of AAP on IL-4/STAT6 (M2) or IFN/LPS/STAT1 (M1) signaling, and downstream gene and protein expression was studied. In vivo, effects of AAP/NAC on tumor size and tumor associated macrophage polarization were characterized in orthotopic triple negative breast tumor models 4T1 and EF43.fgf4 using cytokine elisa of tumor lysate and flow cytometry. Results: NAC reversed AAP toxicity in the normal liver but did not reverse AAP cytotoxicity against tumor cells in vitro. AAP/NAC selectively inhibited IL-4-induced STAT6 phosphorylation but not IFN/LPS-induced STAT1 phosphorylation. Downstream, AAP/NAC inhibited IL-4 induction of M2-associated genes (such as CCL24, YM1 and arginase) and proteins (arginase, PD-L2, and CD206). However, AAP/NAC did not inhibit IFN/LPS induction of M1-associated genes (IL-6, TNF, IL-12) and proteins (INOS, PD-L1, MHC I, and CD64). In vivo, AAP/NAC inhibited tumor growth in EF43.fgf4 and 4T1 triple negative breast tumors. Flow cytometry of tumor associated macrophages revealed that AAP/NAC selectively inhibited M2 but not M1 polarization. ELISA of tumor lysate demonstrated relatively stable expression of IFN gamma but markedly suppressed expression of IL-10 in the tumor immune microenvironment. Conclusion: Our study is the first to show that at high doses AAP/NAC has profound effects on the tumor immune microenvironment that may facilitate immune-mediated inhibition of tumor growth. Citation Format: Alexander Neuwelt, Allyn Bryan, Won Lee, Pavani Pingali, Howard Li, Bhaumik Patel. High dose acetaminophen with n-acetylcysteine rescue inhibits M2 polarization of tumor associated macrophages [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 6388.

Abstract 6645: A systematic approach for detection of tumor genomic alterations which alter the immune microenvironment

Abstract Objectives: Leveraging whole exome and transcriptome data from large cancer datasets, we sought to develop a computational approach to nominate candidate genomic alterations that are associated with hot or cold tumor. We anticipate this tool will identify candidate novel drug targets and biomarkers for immunotherapy in cancer. Methods: The user selects a gene of interest (GOI) as input for the pipeline. The pipeline searches TCGA (The Cancer Genome Atlas) mutation and structural variant outputs to collect hits in the gene of interest. Each sample with an alteration in the GOI is annotated with the alteration’s predicted biological function (loss-of-function or LOF vs gain-of-function or GOF) using gene set enrichment analysis and pathway analysis from RNA sequencing. After annotating by sample, the alterations themselves are scored and recorded. Next, Quantiseq and Trust4 are employed to estimate features of the immune microenvironment in both the LOF and GOF samples. These results along with cancer type distributions, genomic position maps, and oncoplots are delivered to the user for further investigation. We are creating an online application to provide these results to the public. Simultaneously, a command line tool is being developed for application to any data source. Results: We will present findings for the pipeline using positive controls including genomic alterations in ADAR, MTAP, IDH1, and CD274 (PD-L1). Preliminarily, we identified candidate GOF mutations (n=81) in PD-L1 that have inflamed tumor immune microenvironments like those of samples with known GOF structural variations in PD-L1. These samples have an overall “hot” immune microenvironment with significantly (p<.05) increased M1 macrophages, increased expression of IFN-gamma, and decreased M2 macrophages. Conclusion: Currently there are no systematic approaches to interrogate individual genomic alterations and their association with an altered immune microenvironment. Our approach is pragmatic for any gene of interest, can be applied to any database, and identifies clinically actionable targets. This approach will identify heretofore unstudied connections between genomic alterations and immune microenvironment and support personalized drug development strategies in immuno-oncology. Citation Format: Raven Vella, Emily L. Hoskins, Julie W. Reeser, Michele R. Wing, Eric Samorodnitsky, Anoosha Paruchuri, Amy Smith, Leah Stein, Thuy Dao, Zachary Risch, Sameek Roychowdhury. A systematic approach for detection of tumor genomic alterations which alter the immune 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 6645.

Human umbilical cord mesenchymal stem cell-derived TGFBI attenuates streptozotocin-induced type 1 diabetes mellitus by inhibiting T-cell proliferation

MSCs have been demonstrated to have a great benefit for type 1 diabetes mellitus (T1DM) due to their strong immunosuppressive and regenerative capacity. However, the comprehensive mechanism is still unclear. Our previous study indicated that transforming growth factor beta induced (TGFBI) is highly expressed in human umbilical cord-derived mesenchymal stem or stromal cells (hUC-MSCs), which are also implicated in T1DM. In this study, we found that infusion of TGFBI knockdown hUC-MSCs displayed impaired therapeutic effects in T1DM mice and decreased immunosuppressive capability. TGFBI knockdown hUC-MSCs could increase the proportion of T-cell infiltration while increasing the expression of IFN-gamma and interleukin-17A in the spleen. In addition, we also revealed that hUC-MSC-derived TGFBI could repress activated T-cell proliferation by interfering with G1/S checkpoint CyclinD2 expression. Our results demonstrate that TGFBI plays a critical role in MSC immunologic regulation. TGFBI could be a new immunoregulatory molecule controlling MSC function for new treatments of T1DM.Graphical Schematic Representation of the Immunosuppression capacity of hUC-MSC by TGFBI

A Personal Perspective on My Scientific Career

A Personal Perspective on My Scientific Career

Detailed phenotypic and functional characterization of CMV-associated adaptive NK cells in rhesus macaques.

Previous research on adaptive NK cells in rhesus macaques suffered from the lack of specific antibodies to differentiate between inhibitory CD94/NKG2A and stimulatory CD94/NKG2C heterodimeric receptors. Recently we reported an expansion of NKG2C receptor-encoding genes in rhesus macaques, but their expression and functional role on primary NK cells remained unknown due to this deficit. Thus, we established monoclonal antibodies 4A8 and 7B1 which show identical specificities and bind to both NKG2C-1 and NKG2C-2 but neither react with NKG2C-3 nor NKG2A on transfected cells. Using a combination of 4A8 and Z199 antibodies in multicolor flow cytometry we detected broad expression (4-73%) of NKG2C-1 and/or NKG2C-2 (NKG2C-1/2) on primary NK cells in rhesus macaques from our breeding colony. Stratifying our data to CMV-positive and CMV-negative animals, we noticed a higher proportion (23-73%) of primary NK cells expressing NKG2C-1/2 in CMV+ as compared to CMV- macaques (4-5%). These NKG2C-1/2-positive NK cells in CMV+ macaques are characterized by lower expression of IL12RB2, ZBTB16, SH2D1B, but not FCER1G, as well as high expression of IFNG, indicating that antibody 4A8 detects CMV-associated adaptive NK cells. Single cell RNA seq data of 4A8-positive NK cells from a rhCMV-positive macaque demonstrated that a high proportion of these adaptive NK cells transcribe in addition to NKG2C-1 and NKG2C-2 also NKG2C-3, but interestingly NKG2A as well. Remarkably, in comparison to NKG2A, NKG2C-1 and in particular NKG2C-2 bind Mamu-E with higher avidity. Primary NK cells exposed to Mamu-E-expressing target cells displayed strong degranulation as well as IFN-gamma expression of 4A8+ adaptive NK cells from rhCMV+ animals. Thus, despite co-expression of inhibitory and stimulatory CD94/NKG2 receptors the higher number of different stimulatory NKG2C receptors and their higher binding avidity to Mamu-E outreach inhibitory signaling via NKG2A. These data demonstrate the evolutionary conservation of the CMV-driven development of NKG2C-positive adaptive NK cells with particular molecular signatures in primates and with changes in gene copy numbers and ligand-binding strength of NKG2C isotypes. Thus, rhesus macaques represent a suitable and valuable nonhuman primate animal model to study the CMV-NKG2C liaison in vivo.

Immunization with live influenza virus induces greater polyfunctional CD4+ T cell response compared to inactivated virus

Previous work has shown that infection of dendritic cells with influenza virus results in more diverse and efficient antigen presentation on MHC-II compared to treatment with excess doses of killed influenza virus. Accordingly, immunization of mice with live influenza virus elicits a greater frequency and breadth of CD4+ T cells than killed virus, as determined by interferon (IFN)-gamma ELISpot. Here, we sought to more fully characterize the differences in CD4+ T cell functionality induced by live vs. killed virus vaccines. C57Bl/6 mice were immunized with live influenza virus A/Puerto Rico/8/1934 (PR8) or a large excess dose of inactivated PR8. Efforts were made to control for the antigen load and adjuvant effects. Influenza-specific CD4+ T cells were evaluated by peptide/MHC-II tetramer staining, intracellular cytokine staining after peptide restimulation, and flow cytometry. Greater frequencies of PR8-specific CD4+ T cells were elicited by live vs. killed virus, as measured by tetramer staining or expression of the cytokines IFN-gamma, TNF, or IL-2. Of particular note, live virus induced a significantly higher frequency of polyfunctional CD4+ T cells, producing two or more cytokines when stimulated with peptide. This result was evident when polyfunctional CD4+ T cells were expressed as a fraction of all CD4+ T cells or PR8 epitope-specific CD4+ T cells. The difference in polyfunctional CD4+ T cells was maintained when antigenically distinct influenza virus B/Lee/1940 was mixed with killed PR8 as an adjuvant or when killed PR8 was administered in a prime/boost series. The data are consistent with a model whereby endogenous antigen presentation on MHC-II transmits more potent activating signals to CD4+ T cells than exogenous presentation, leading to a greater level of type 1 helper cytokine production and potentially greater antiviral effects.

P06.08.B Radiation therapy enhances anti-tumor activity of a MET CAR T-based immunotherapy for glioblastoma

Abstract Background Glioblastoma is the most frequent primary brain tumor with dismal prognosis after standard treatment with surgery, and chemoradiation (the Stupp protocol). After a decade of failed clinical trials, tumor-treating fields have been first to show the added benefit of improved overall survival compared to the Stupp protocol (20.9 months vs 16.0 months). However, GBM remains a devastating disease, with almost inevitable recurrence, and limited options for second-line therapy. Radiation therapy (RT), is a standard therapy option for GBM, and it is used in most GBM cases affecting tumor through induction of DNA-damage. Recently, RT has been investigated as a mediator of T cell-based therapies in the context of immunosuppressive GBM microenvironment. The findings have shown promise in combination of T cell-based therapies, such as chimeric antigen receptor (CAR) T cell therapies, in improving the tumor infiltration, and penetration with immune cells. MET is a relevant oncogene in the context of GBM, being involved in stem-like properties, radiation response and resistance․ Hence, MET appeared to be a plausible target for combination with RT. In our research, we use MET-targeting CAR T cells (MET-CAR T cells) combined with radiation, and hypothesize synergistic interaction for GBM treatment. Material and Methods We used adherent (2D) and stem-like (3D) human GBM cell lines with different levels of MET expression. For MET-CAR T cell generation we did retrovirus-mediated transduction of activated human T cells and sorted the CAR-positive cells. We co-cultured MET-CAR T cells with GBM cells with or without RT, and assessed the killing and cytokine production in CAR T cells. Results Our results indicated that 5Gy radiation combined with MET-CAR T cells increases their potential in tumor cell killing. We observed increased CAR T cells effect at lower CAR T to target cells ratios when combined with radiation, even when radiation treatment did not lead to a significant decrease in viability. This phenomenon was similar across different types of cell lines (adherent, stem-like), different levels of MET expression, and different sensitivity to CAR T cells. We investigated the underlying mechanisms via intracellular cytokine measurement. We observed the most prominent response in TNF-α-expression. We also observed an increase in Granzyme B expression in co-culture with some of the GBM cell lines, especially in CD8+ subpopulation of CAR T cells. IFN-gamma expression increased in some adherent glioma cell lines but not in stem-like cell lines. Conclusion In conclusion, our data demonstrates the potency of MET-CAR T cells against GBM, and increased efficiency when combined with radiation. The suggested mechanism is the increased activation of T cells in TNF-α-dependent-manner. To validate these results we are testing our setup in an orthotopic mouse GBM model.

Sexual dimorphism modulates metabolic and cognitive alterations under HFD nutrition and chronic stress exposure in mice. Correlation between spatial memory impairment and BDNF mRNA expression in hippocampus and spleen

AimsThe accumulated evidence suggests that lifestyle - specifically dietary habits and stress exposure - plays a detrimental role in health. The purpose of the present study was to analyze the interplay of stress, diet, and sex in metabolic and cognitive alterations. Main methodsFor this purpose, one-month-old C57Bl/6J mice were fed with a standard diet or high-fat diet (HFD). After eight weeks, one subgroup of mice from each respective diet was exposed to 20 weeks of chronic mild stress (CMS), whilst the others were left undisturbed. Key findingsAfter 28 weeks of HFD feeding, mice from both sexes were overweight, with an increase in caloric intake and abdominal and subcutaneous fat pads. Stress exposure induced a decrease in body weight, related to a decrease in caloric efficiency in both males and females. Results indicate that males are more susceptible than the females in modulating metabolic and cognitive functions under HFD and CMS. Although both sexes demonstrated HFD-induced weight gain, fat accumulation, insulin resistance, high cholesterol, only males exposed to CMS but not females have (i) impaired glucose tolerance with higher glucose level; (ii) significant prolonged latency in Barnes test, suggesting cognitive impairment; (iii) increased IFN-gamma expression in hippocampus, suggesting greater neuroinflammatory response; (iv) poorer cognitive performance related to a decrease in hippocampal and spleen BDNF mRNA expression. SignificanceThe main finding in this study is the presence of a sexual dimorphism in modulating metabolic and cognitive functions under HFD and CMS, showing males are more susceptible than females. In addition, poorer cognitive performance was related to a decrease in hippocampal BDNF mRNA expression. Interestingly, these changes were observed in the spleen as well.

Network pharmacology and experimental validation identify the potential mechanism of sophocarpine for COVID-19.

Introduction. Coronavirus disease 2019 (COVID-19) has caused a serious threat to public health worldwide, and there is currently no effective therapeutic strategy for treating COVID-19.Hypothesis/Gap Statement. We propose that sophocarpine (SOP) might have potential therapeutic effects on COVID-19 through inhibiting the cytokine storm and the nuclear factor NF-κB signalling pathway.Aim. The objective was to elucidate the potential mechanism of SOP against COVID-19 through a network pharmacology analysis and its experimental validation.Methodology. The BATMAN-TCM database was used to identify the therapeutic targets of SOP, while the GeneCards and DisGeNET databases were used to identify the targets related to COVID-19. A protein-protein interaction (PPI) network was constructed from the STRING and analysed using Cytoscape software. Gene ontology (GO), Kyoto Encyclopaedia of Genes and Genomes (KEGG) and disease ontology (DO) enrichment analyses of the co-targets were performed using Metascape. Autodock 4.2.6 and Pymol software were applied for molecular docking. Levels of the proinflammatory cytokines IL-6, TNFα and IL-1β were measured by ELISA, while mRNA expression levels of intercellular adhesion molecule 1 (ICAM-1), vascular endothelial growth factor A (VEGFA) and IFN gamma (IFNG) were detected by real-time quantitative reverse transcription PCR. The protein levels of the molecules involved in the NF-κB signalling pathway were validated by western blot analysis.Results. A total of 65 co-targets of SOP and COVID-19 were determined. GO and KEGG enrichment analyses suggested that SOP affected COVID-19 by regulating the IL-17 signalling pathway, TNF signalling pathway and other signalling pathways. The PPI network and molecular docking showed that p65, ICAM-1 and VEGFA were key targets of SOP against COVID-19 and the underlying mechanism was validated in A549 cells in vitro. SOP attenuated the LPS-induced production of TNF-α and IL-6 and downregulated the LPS-induced mRNA expression of ICAM-1, VEGFA and IFNG. Mechanistically, SOP pretreatment inhibited the phosphorylation of p65 and facilitated the activation of Nrf2.Conclusions. SOP has a potential therapeutic effect on COVID-19 through multiple pathways and targets, and inhibits the production of pro-inflammatory cytokines and molecules involved in the NF-κB signalling pathway.

Expression pattern and clinical significance of microRNA-let-7a and IFN-gamma in placental tissue of patients with preeclampsia with severe features.

Preeclampsia with severe features (PECsf) isacommon disease in pregnant women. let-7a and IFN-gamma (interferon-gamma) are involved in diagnosis and prognosis of preeclampsia. This study explored effects of let-7a and IFN-gamma on PECsf patients. The placental tissue of 21PECsf, 19 preeclampsia without severe features (PEC), and 20 normal pregnant women were collected, and clinical data were recorded. let-7a and IFN-gamma expressions in placental tissue were detected. The correlation between let-7a/IFN-gamma expression and clinical indexes was analyzed. According to let-7a and IFN-gamma expressions, PECsf patients were assigned into Hlet-7a group (let-7a high expression group), Llet-7a group (let-7a low expression group), HIFN-gamma group (IFN-gamma high expression group) and LIFN-gamma group (IFN-gamma low expression group). The incidence of adverse prognosis was compared. let-7a and IFN-gamma were highly expressed in placental tissue of preeclampsia patients, with significant differences between PEC and PECsf. The high expressions of let-7a and IFN-gamma were positively correlated with mean arterial pressure, lactate dehydrogenase, and 24h urinary protein in placental tissues of PECsf patients. High let-7a and IFN-gamma expressions were correlated with adverse outcomes of PECsf. High let-7a and IFN-gamma expressions were correlated with clinical features, and could be used as biomarkers for treatment and poor prognosis of PECsf.