Mouse T-cell Research Articles

Tissue-resident Lachnospiraceae family bacteria protect against colorectal carcinogenesis by promoting tumor immune surveillance

The intestinal microbiota plays an important role in colorectal cancer (CRC) progression. However, the effect of tissue-resident commensal bacteria on CRC immune surveillance remains poorly understood. Here, we analyzed the intratissue bacteria from CRC patient colon tissues. We found that the commensal bacteria belonging to the Lachnospiraceae family, including Ruminococcus gnavus (Rg), Blautia producta (Bp), and Dorea formicigenerans (Df), were enriched in normal tissues, while Fusobacterium nucleatum (Fn) and Peptostreptococcus anaerobius (Pa) were abundant in tumor tissues. Tissue-resident Rg and Bp reduced colon tumor growth and promoted the activation of CD8+ Tcells in immunocompetent mice. Mechanistically, intratissue Rg and Bp degraded lyso-glycerophospholipids that inhibited CD8+ Tcell activity and maintained the immune surveillance function of CD8+ Tcells. Lyso-glycerophospholipids alone promoted tumor growth that was abrogated with Rg and Bp injection. Collectively, intratissue Lachnospiraceae family bacteria facilitate the immune surveillance function of CD8+ Tcells and control colorectal cancer progression.

Abstract PD12-05: PD12-05 Adipocyte directed vaccination to reduce the risk of breast cancer

Abstract Background: Obesity is an important risk factor for breast cancer and women with metabolic syndrome may be at the highest risk. Infiltration of CD8 T-cells into fat is an early event in obesity. Type I cytokines secreted by CD8 T-cells upregulate costimulatory molecules on enlarged adipocytes. The adipocytes, now antigen presenting cells, further stimulate Type I T-cell activation. The resulting T-cells compete for glucose and fatty acids which leads to metabolic dysfunction in both the adipose tissue and the T-cells themselves. The T-cells are not able to maintain tumor immune surveillance and secretion of adipokines promotes malignant transformation. Immunologic memory prevents inflammation from resolving even if an individual becomes normal weight. Strategies to increase Type II (anti-inflammatory) T-cells in inflamed adipose could have clinical benefit. Methods: We developed a method of CD4 epitope identification that includes functional screening for Th1 or Th2 epitopes. We use a multi-algorithm approach to ensure responsiveness across diverse HLA alleles. We identified Th2 selective epitopes associated with high IL-10 secretion for 6 adipocyte associated antigens that become overexpressed in inflamed adipocytes (IGF-IR, HIF-1a, DUSP1, FABP4, PAI-1 and ATGL). The epitopes were highly homologous between mouse and man (median 100% (range-82-100%). When the epitopes were used to immunize mice, all antigens generated a significant IL-10 response compared to control (p< 0.05). We questioned whether our “adipocyte directed” vaccine (ADVac) could prevent the development of breast cancer in obese mice. Results: First, C57BL/6 mice were fed a high fat high sucrose (HFHS) diet or normal chow. When mice became obese, vaccination with ADVac or adjuvant alone (Alum) was initiated. Four weeks after the final vaccine, visceral adipose tissue showed significantly fewer CD8 T-cells in the obese mice immunized with ADVac as compared to the control, p=0.0011. The decrease in CD8 T-cells was specific for adipose tissue as no change was observed in matched spleen. There was a significant increase in T-regulatory cells in the adipose tissue of mice immunized with ADVac as compared to control, p=0.031. Two weeks after the final vaccine, a glucose tolerance test (GTT) and insulin tolerance (ITT) showed blood glucose concentrations were significantly lower at all time points for the ADVac-immunized obese mice as compared to the control obese mice (p< 0.01 for all). TgMMTV-neu develop aggressive breast cancer when made obese. Ten-week old TgMMTV-neu mice were fed a HFHS diet for 4 weeks, then randomized into 2 cohorts when obese, one cohort receiving the adjuvant only (Alum) and one receiving ADVac. Mice were sacrificed at 31 weeks of age when all controls had developed tumor. ITT showed the glucose levels in the blood were significantly lower in the ADVac group as compared to control (p< 0.0001). Fewer CD8 T-cells were observed in mammary adipose tissue of AdVac immunized mice compared to control (p=0.001). There was significantly less leptin detected in the serum of ADVac vaccinated mice compared to Alum immunized, p=0.024. The median age of tumor development was 25 weeks in controls and 29 weeks in the immunized group (p=0.009). Sixty percent (9/15) of the vaccinated mice were tumor free at study termination, whereas 100% of the control mice had developed tumor. Conclusions: ADVac represents the first vaccine to lower breast cancer risk in obesity. Vaccination corrected metabolic dysfunction as evidenced by reversal of diabetes and prevented breast cancer in the majority of obese ADVac immunized mice. Further studies are ongoing evaluating the systemic distribution of ADVac specific T-cells and the safety of the approach. Citation Format: Mary Disis, Lauren Corulli, Erin R. Rodmaker, Denise Cecil. PD12-05 Adipocyte directed vaccination to reduce the risk of breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD12-05.

Cellular T-cell immune response profiling by tetravalent dengue subunit vaccine (DSV4) candidate in mice.

While most vaccines aim to develop a solid humoral and neutralizing antibody response against the pathogen, an effective vaccine candidate should be able to stimulate both the B-cell mediated humoral immunity, and T-cell mediated cellular immunity. The focus of vaccinology is rapidly gaining to generate T cell responses, which can mediate pathogen clearance and help B cells leading to protective antibody responses. Here we evaluate the cellular immune response of the pre-clinical tetravalent dengue subunit vaccine candidate, DSV4, in mice. While we have shown previously that DSV4 induces type-specific neutralizing antibody responses in mice, in this study, we show that the vaccine candidate DSV4 well induces dengue-specific T- cell responses evaluated by their ability to produce IFN-γ. In addition to IFN-γ secretion by both CD4+ and CD8+ T-cells in immunized mice, we observed that DSV4 also induces a higher frequency and cytokine functions of follicular CD4+ helper T-cells (TFH). These cytokines lead to an efficient germinal center reaction and potent B cell antibody response. Apart from TFH response, DSV4 stimulated Type 1 T helper cells (TH1) which is characteristic of a viral infection leading to secretion of pro-inflammatory cytokines and phagocyte-dependent protective immune responses. Our study highlights that DSV4 can mediate both arms of adaptive immunity-humoral and cell-mediated immunity in mice. By elucidating vaccine-specific T cell response, our work has implications in showing DSV4 as an effective, type-specific and safe dengue vaccine candidate.

OX40 agonism enhances PD-L1 checkpoint blockade by shifting the cytotoxic T cell differentiation spectrum

Immune checkpoint therapy (ICT) has the power to eradicate cancer, but the mechanisms that determine effective therapy-induced immune responses are not fully understood. Here, using high-dimensional single-cell profiling, we interrogate whether the landscape of Tcell states in the peripheral blood predict responses to combinatorial targeting of the OX40 costimulatory and PD-1 inhibitory pathways. Single-cell RNA sequencing and mass cytometry expose systemic and dynamic activation states of therapy-responsive CD4+ and CD8+ Tcells in tumor-bearing mice with expression of distinct natural killer (NK) cell receptors, granzymes, and chemokines/chemokine receptors. Moreover, similar NK cell receptor-expressing CD8+ Tcells are also detected in the blood of immunotherapy-responsive cancer patients. Targeting the NK cell and chemokine receptors in tumor-bearing mice shows the functional importance of these receptors for therapy-induced anti-tumor immunity. These findings provide a better understanding of ICT and highlight the use and targeting of dynamic biomarkers on Tcells to improve cancer immunotherapy.

Reprogramming the tumor microenvironment leverages CD8+ T cell responses to a shared tumor/self antigen in ovarian cancer

Tumor antigen-driven responses to weakly immunogenic self-antigens and neoantigens directly affect treatment efficacy following immunotherapy. Using orthotopically grown SV40T antigen+ ovarian carcinoma in antigen-naive wild-type or TgMISIIR-TAg-Low transgenic mice expressing SV40T antigen as a self-antigen, we investigated the impact of CXCR4-antagonist-armed oncolytic virotherapy on tumor progression and antitumor immunity. Immunostaining and single-cell RNA sequencing analyses of the peritoneal tumor microenvironment of untreated tumors in syngeneic wild-type mice revealed the presence of SV40T antigen-specific CD8+ Tcells, a balanced M1/M2 transcriptomic signature of tumor-associated macrophages, and immunostimulatory cancer-associated fibroblasts. This contrasted with polarized M2 tumor-associated macrophages, immunosuppressive cancer-associated fibroblasts, and poor immune activation in TgMISIIR-TAg-Low mice. Intraperitoneal delivery of CXCR4-antagonist-armed oncolytic vaccinia virus led to nearly complete depletion of cancer-associated fibroblasts, M1 polarization of macrophages, and generation of SV40T antigen-specific CD8+ Tcells in transgenic mice. Cell depletion studies revealed that the therapeutic effect of armed oncolytic virotherapy was dependent primarily on CD8+ cells. These results demonstrate that targeting the interaction between immunosuppressive cancer-associated fibroblasts and macrophages in the tolerogenic tumor microenvironment by CXCR4-A-armed oncolytic virotherapy induces tumor/self-specific CD8+ Tcell responses and consequently increases therapeutic efficacy in an immunocompetent ovarian cancer model.

PS-BPB03-3: MODIFIED ANGIOTENSIN II PEPTIDE VACCINE FOR HYPERTENSION

We and others recently reported that an angiotensin II vaccine for hypertension successfully attenuated elevated blood pressures in an animal model without any immunogenic side effects. In this system, an immunogenic molecule (i.e., KLH) with adjuvants provides an antigen that supports the activation of helper T cells. We recently developed a novel peptide, AJP001, that possesses both a mouse T-cell epitope and adjuvant action. Direct conjugation to the antigen is useful for peptide vaccines without the addition of adjuvants. In this study, the efficacy of an angiotensin II and AJP001-conjugated peptide vaccine (AJP001- angiotensin II) was evaluated in mice. The anti-angiotensin II antibody titer was measured in Balb/C mice following three injections of AJP001-angiotensin II at 2-week intervals. SBP was measured during vaccination of Balb/C mice treated with angiotensin II infusion. AJ-angiotensin II treatment resulted in an increase in the anti-angiotensin II antibody titer in a dose-dependent manner without the addition of adjuvants. In the analysis of the humoral immune response, AJP001-angiotensin II mainly elicited IL-4 and IL-10 production, as measured by an enzyme-linked immune absorbent spot assay, which suggests the induction of a Th2 response. Importantly, cotreatment with purified antibodies attenuated angiotensin II-induced extracellular signal-regulated kinase phosphorylation in cultured vascular smooth muscle cells. The SBP in immunized mice was significantly lower than that in nonimmunized mice (135.9 ± 8.5 vs. 154.9 ± 16.8 mmHg, P = 0.02). Furthermore, angiotensin II-induced perivascular fibrosis in the heart was significantly attenuated in immunized mice, which also exhibited decreased mRNA expression of collagen I/III and transforming growth factor-beta. It may be a simple and useful therapeutic peptide vaccine, and we now further modify the T cell epitope for clinical trials.

Identification of TRDV-TRAJ V domains in human and mouse T-cell receptor repertoires.

Here, we describe the identification of two T-cell receptors (TRs) containing TRDV genes in their TRA chains, the first one in human and the second one in mouse. First, using 5'RACE on a mixed lymphocyte-tumor cell culture (MLTC), we identified TRDV1 5'-untranslated region (UTR) and complete coding sequence rearranged productively to TRAJ24. Single-cell TR RNA sequencing (RNA-seq) of the MLTC, conducted to identify additional clonotypes, revealed that the analysis software detected the hybrid TRDV-TRAJ TRA (TRA) chain but excluded it from the final results. In a separate project, we performed TR sequencing of tumor-infiltrating lymphocytes (TILs) in a murine tumor model. Here, the predominant clonotype contained a TRA chain with a TRDV2-2-TRAJ49 rearrangement. Again, the hybrid TRA chain was not reported in the final results. Transfection of both TR cDNAs resulted in cell surface localization of TR together with CD3, suggesting a productive protein in both cases. Tumor recognition of the Homo sapiens (Homsap) TRDV1-containing TR could be demonstrated by IFN Gamma ELISA ELISpot kit, whereas the Mus musculus (Musmus) TR did not recognize a tumor-derived cell line. To determine whether the TRDV-containing TRA chains we detected were rare events or whether TRDV genes are commonly incorporated into TRA chains, we queried the NCBI Sequence Read Archive for TR single-cell RNA-seq data and analyzed 21 human and 23 murine datasets. We found that especially Homsap TRDV1, Musmus TRDV1, and to some extent Musmus TRDV2-2 are more commonly incorporated into TRA chains than several TRAV genes, making those TRDV genes a relevant contribution to TRA diversity. TRDV-containing TRA chains are currently excluded from the final results of V-(D)-J dataset analyses with the CellRanger software. We provide a work-around to avoid exclusion of those hybrid TRA chains from the final analysis results.

Non-standard viral genome-derived RNA activates TLR3 and type I IFN signaling to induce cDC1-dependent CD8+ T-cell responses during vaccination in mice

There is a critical need to develop vaccine adjuvants that induce robust immune responses able to protect against intracellular pathogens, including viruses. Previously, we described defective viral genome-derived oligonucleotides (DDOs) as novel adjuvants that strongly induce type 1 immune responses, including protective Th1 CD4+ T-cells and effector CD8+ T-cells in mice. Here, we unravel the early innate response required for this type 1 immunity induction. Upon DDO subcutaneous injection, type 1 conventional dendritic cells (cDC1s) accumulate rapidly in the draining lymph node in a Toll-like receptor 3 (TLR3)- and type I interferon (IFN)-dependent manner. cDC1 accumulation in the lymph node is required for antigen-specific CD8+ T-cell responses. Notably, in contrast to poly I:C, DDO administration resulted in type I IFN expression at the injection site, but not in the draining lymph node. Additionally, DDOs induced an inflammatory cytokine profile distinct from that induced by poly I:C. Therefore, DDOs represent a powerful new adjuvant to be used during vaccination against intracellular pathogens.

Intradermal Immunization of SARS-CoV-2 Original Strain Trimeric Spike Protein Associated to CpG and AddaS03 Adjuvants, but Not MPL, Provide Strong Humoral and Cellular Response in Mice.

Despite the intramuscular route being the most used vaccination strategy against SARS-CoV-2, the intradermal route has been studied around the globe as a strong candidate for immunization against SARS-CoV-2. Adjuvants have shown to be essential vaccine components that are capable of driving robust immune responses and increasing the vaccination efficacy. In this work, our group aimed to develop a vaccination strategy for SARS-CoV-2 using a trimeric spike protein, by testing the best route with formulations containing the adjuvants AddaS03, CpG, MPL, Alum, or a combination of two of them. Our results showed that formulations that were made with AddaS03 or CpG alone or AddaS03 combined with CpG were able to induce high levels of IgG, IgG1, and IgG2a; high titers of neutralizing antibodies against SARS-CoV-2 original strain; and also induced high hypersensitivity during the challenge with Spike protein and a high level of IFN-γ producing CD4+ T-cells in mice. Altogether, those data indicate that AddaS03, CpG, or both combined may be used as adjuvants in vaccines for COVID-19.

Development, analysis, and preclinical evaluation of inactivated vaccine candidate for prevention of Covid-19 disease

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in the sprawling capital of Central China’s Hubei province Wuhan in 2019. Since late 2019, SARS-CoV-2 has been responsible for a global pandemic that has resulted in 405,961,201 confirmed cases of infection and 5,789,567 deaths as of February 11, 2022, according to the World Health Organization (WHO). Here we report the development of an inactivated SARS-CoV-2 vaccine candidate and show that its efficacy and safety in preclinical studies warrant further clinical evaluation. Efficiency and possible side effects of vaccine candidate on different doses were evaluated using histopathology, K18 hACE2 transgenic mice challenge, T-cell response, and serum neutralization test in Balb/c mice. All pilot production and propagation experiments of SARS-CoV-2 were performed in the Level 3 Negative Pressure Biosafety Facility (BSL3/ABSL-3) according to COVID-19 interim guidance of WHO. All analytical studies have shown that the quality control parameters of the vaccine candidate meet the requirements for inactivated viral vaccines according to European Pharmacopeia. The vaccine candidate (KOCAK-19) has been authorized by the Turkish Medicines and Medical Devices Agency and has now entered phase 2 clinical development (NCT04838080). Abbreviations: SARS-CoV-2: Severe Acute Respiratory Syndrome Coronavirus 2.; MERS-CoV: Middle East Respiratory Syndrome Coronavirus.; qRT-PCR: Quantitative Real-Time Polymerase Chain Reaction.; TEM: Transmissible Electron Microscopy.; SEC: Size Exclusion.; AEX: Anion exchange.; BPL: β-Propiolactone.; CPE: Cytopathic Effect.; TCID50: 50% Tissue Culture Infectious Dose.; hACE-2: Human Angiotensin-Converting Enzyme 2.; mRNA: Messenger RNA.; RBD: Receptor binding domain.; Nab: Neutralizing antibody.; MOI: Multiply of Infection. HIGHLIGHTS A new inactivated vaccine candidate to protect against COVID −19 SARS-CoV-2 inactive vaccine development in disposable wave bioreactor systems with microcarrier technologies and analytical analysis Preclinical toxicity and immunity trials for COVID-19 inactive vaccine candidate in Balb/c mice, K18 transgenic mice and, ferrets.

The effect of “early” proteins E2, E6 and E7 of papillomavirus of high-risk cancerogenous type HPV16 on cancer HeLa cells, inducing tumour growths in mice lungs

The goal of investigation was the development of oral therapeutic vaccine against cancer on basis of antigenic regulatory protein HPV16 E2 encoded by the “early” hpv16 E2 gene.The aim. To study tumour growths in mice lungs inoculated with cancer HeLa cells and then vaccinated with vaccine material of transgenic tomato with the gene/protein HPV16 E2, and to study the activation of the immunogenesis of T-lymphocytes in mice after oral vaccination with HPV16 E2.Materials and methods. Proliferating cancer HeLa cells, mice peripheric blood mononuclear cells, splenocytes, intact and isolated lungs of mice were used in the study. Elispot was used for the evaluation of increasing of immunogenicity.Results. “Early” proteins HPV16 E6 or HPV16 E7 did not reveal any effect on HeLa cells. The regulatory antigenic “early” protein HPV16 E2 drastically degraded HeLa cells recorded by trypane blue. Four types of tumours were found in mice lung lobes placed in the suspension of cancer HeLa cells for 2–5 days: 1 – peripheral round tumours on epithelium, 2 – central tumours in the area of tracheal carina, 3 – pneumonialike peripheral cancer on lung lobes which was very similar to small-round-cell lung sarcoma with hyperchromic nuclei and 4 – Pancoast-like cancer in apical parts of lung lobe. Patterns of normal developed lung tissues were viewed on slices of lungs infected with HeLa in the presence of E2 simultaneously. The very high contents of γ‑interferon, CD4/CD8 T lymphocytes, T cell receptor and apoptotic enzymes: granzyme B, perforin and granulysine – were detected in blood and splenocytes of mice vaccinated with HPV16 E2.Conclusion. The study is promising for the development of an oral therapeutic vaccine based on a plant expression system (tomatoes) with the HPV16 E2 antigenic protein against lung cancer, cervical cancer and other types of cancer.

Diversity-Oriented Synthesis Catalyzed by Diethylaminosulfur-Trifluoride-Preparation of New Antitumor Ecdysteroid Derivatives.

Fluorine represents a privileged building block in pharmaceutical chemistry. Diethylaminosulfur-trifluoride (DAST) is a reagent commonly used for replacement of alcoholic hydroxyl groups with fluorine and is also known to catalyze water elimination and cyclic Beckmann-rearrangement type reactions. In this work we aimed to use DAST for diversity-oriented semisynthetic transformation of natural products bearing multiple hydroxyl groups to prepare new bioactive compounds. Four ecdysteroids, including a new constituent of Cyanotis arachnoidea, were selected as starting materials for DAST-catalyzed transformations. The newly prepared compounds represented combinations of various structural changes DAST was known to catalyze, and a unique cyclopropane ring closure that was found for the first time. Several compounds demonstrated in vitro antitumor properties. A new 17-N-acetylecdysteroid (13) exerted potent antiproliferative activity and no cytotoxicity on drug susceptible and multi-drug resistant mouse T-cell lymphoma cells. Further, compound 13 acted in significant synergism with doxorubicin without detectable direct ABCB1 inhibition. Our results demonstrate that DAST is a versatile tool for diversity-oriented synthesis to expand chemical space towards new bioactive compounds.

Toll-like receptor agonist combinations augment mouse T-cell anti-tumor immunity via IL-12- and interferon ß-mediated suppression of immune checkpoint receptor expression

ABSTRACT We previously found that activated CD8+ T-cells increase expression of PD-1, which can be attenuated in the presence of specific Toll-like receptor (TLR) agonists, mediated by IL-12 secreted by professional antigen-presenting cells. While these CD8+ T-cells had greater anti-tumor activity, T-cells stimulated by different TLR had different gene expression profiles. Consequently, we sought to determine whether combinations of TLR agonists might further affect the expression of T-cell checkpoint receptors and improve T-cell anti-tumor immunity. Activation of CD8+ T-cells in the presence of specific TLR ligands resulted in decreased expression of PD-1, LAG-3, and CD160, notably with combinations of TLR1/2, TLR3, and TLR9 agonists. Immunization of E.G7-OVA or TRAMP-C1 tumor-bearing mice with peptide or DNA vaccines, co-administered with combination of TLR3 and TLR9 agonists, showed greater suppression of tumor growth. The anti-tumor effect of TLR1/2 and/or TLR9, but not TLR3, was abrogated in IL-12KO mice. RNA sequencing of TLR-conditioned CD8+ T-cells revealed IL-12 pathway activation, and type 1 IFN pathway activation following TLR3 stimulation. Our results provide a mechanistic rationale for the choice of optimal combinations of TLR ligands to use as adjuvants to improve the efficacy of anti-tumor vaccines.

The effect of spermidine on autoimmunity and beta cell function in NOD mice

Spermidine is a natural polyamine which was shown to prolong lifespan of organisms and to improve cardiac and cognitive function. Spermidine was also reported to reduce inflammation and modulate T-cells. Autophagy is one of the mechanisms that spermidine exerts its effect. Autophagy is vital for β-cell homeostasis and autophagy deficiency was reported to lead to exacerbated diabetes in mice. The effect of spermidine in type 1 diabetes pathogenesis remains to be elucidated. Therefore, we examined the effect of spermidine treatment in non-obese diabetic (NOD) mice, a mouse model for type 1 diabetes. NOD mice were given untreated or spermidine-treated water ad libitum from 4 weeks of age until diabetes onset or 35 weeks of age. We found that treatment with 10 mM spermidine led to higher diabetes incidence in NOD mice despite unchanged pancreatic insulitis. Spermidine modulated tissue polyamine levels and elevated signs of autophagy in pancreas. Spermidine led to increased proportion of pro-inflammatory T-cells in pancreatic lymph nodes (pLN) in diabetic mice. Spermidine elevated the proportion of regulatory T-cells in early onset mice, whereas it reduced the proportion of regulatory T-cells in late onset mice. In summary spermidine treatment led to higher diabetes incidence and elevated proportion of T-cells in pLN.

Time isn't kind to female T-cells.

While investigating sex-differences in T-cell aging, Mkhikian et al., identified a role for excessive IL-7 signaling and N-glycan branching in age-related mouse and human female T-cell dysfunction. These findings point to the increasingly-recognized importance of the impact of biological sex on immune aging and delineate new targetable pathways in age-related immune dysfunction.

IQCELL: A platform for predicting the effect of gene perturbations on developmental trajectories using single-cell RNA-seq data.

The increasing availability of single-cell RNA-sequencing (scRNA-seq) data from various developmental systems provides the opportunity to infer gene regulatory networks (GRNs) directly from data. Herein we describe IQCELL, a platform to infer, simulate, and study executable logical GRNs directly from scRNA-seq data. Such executable GRNs allow simulation of fundamental hypotheses governing developmental programs and help accelerate the design of strategies to control stem cell fate. We first describe the architecture of IQCELL. Next, we apply IQCELL to scRNA-seq datasets from early mouse T-cell and red blood cell development, and show that the platform can infer overall over 74% of causal gene interactions previously reported from decades of research. We will also show that dynamic simulations of the generated GRN qualitatively recapitulate the effects of known gene perturbations. Finally, we implement an IQCELL gene selection pipeline that allows us to identify candidate genes, without prior knowledge. We demonstrate that GRN simulations based on the inferred set yield results similar to the original curated lists. In summary, the IQCELL platform offers a versatile tool to infer, simulate, and study executable GRNs in dynamic biological systems.

Abstract P041: Simultaneous checkpoint inhibition and immune cell activation that is safely localized to solid tumors

Abstract Unlike other checkpoint inhibitors, our targeted immunotherapeutic localizes to any solid tumor and simultaneously shields an agent of immunosuppression while presenting a signal for immunostimulation. Phosphatidylserine (PS) exposure on the extracellular surface of living tumor cells and their vasculatures provides one avenue by which the tumor microenvironment promotes immunosuppression. Extracellular surface PS is inherent to a tumor and its vasculature, even for inoperable tumors, and its expression cannot be mutated nor affected by acquired drug resistance. Annexin A5 (AnxA5) is a direct, high-affinity PS-binding protein that localizes to cells with PS exposed on the outer plasma membrane. In our studies, we conjugated a proprietary modified AnxA5, lacking cellular internalization, to TNFα (AnxA5MOD-TNFα) to convert the immunosuppresive environs of a murine 4T1 triple negative breast cancer (TNBC) into an immunostimulated one. This strategy localized the immune response to the tumor and minimized side effects, as evidenced by a lack of toxicity for up to 7 days in non-tumor bearing Balb/c female mice given up to 1 mg/kg. Proper assembly and functionality of AnxA5MOD-TNFα was verified simultaneously by ellipsometry, an optical technique similar to plasmon resonance. Fully assembled constructs were tested for binding to PS coated slides. The degree of light polarization is proportional to the amount of PS bound by the AnxA5 complex. Samples could be further incubated with TNF receptors to verify TNFα activity. Based on dose escalation studies in 4T1 tumor-bearing mice where the TNBC tumors were grown in the mammary fat pads, optimal dosages were determined for AnxA5MOD-TNFα (18 µg) and AnxA5MOD alone as a control (180 µg). These doses were further tested in a 4T1 growth inhibition study. Tumor size was tracked by caliper in two groups of mice (n=5/group) receiving drug treatment on days 12, 14 and 16 and a repeated measures ANOVA was conducted on measurements taken before, during and post-treatment. While median tumor size did not differ between control and drug treatment groups during the pre-treatment interval (p=0.84), there was a significant difference post-treatment (p<0.001) with mice receiving AnxA5MOD-TNFα having much smaller TNBC tumors. Tumors from the study were embedded in paraffin, sectioned (5 µm) and the overall immune cell content determined by H&E staining. Once it was evident there was a greater quantity of immune cells in AnxA5MOD-TNFα treated tumors vs. controls, sections were stained with validated antibodies to identify and count the immunoactivated T-cells, NK-cells and macrophages. There was a 3X greater mean percentage of CD8 and CD4 T-cells in mice receiving drug vs. control (p=0.03) along with 2.5X and 5X increases in NK-cells and M1 immunoactive macrophages, respectively. Conclusion: Our AnxA5MOD-TNFα inhibits the PS inhibitor while simultaneously activating TNF activators! Citation Format: Richard A. Richieri, Navneet Narula, Cynthia A. Loomis, Valeria Mezzano, John Billimek, Glenn T. Reynolds, Chris Reutelingsperger, Andries Zijlstra, Missag H. Parseghian. Simultaneous checkpoint inhibition and immune cell activation that is safely localized to solid tumors [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P041.

IRAK1-regulated IFN-\u03b3 signaling induces MDSC to facilitate immune evasion in FGFR1-driven hematological malignancies

BackgroundStem Cell leukemia/lymphoma syndrome (SCLL) presents as a myeloproliferative disease which can progress to acute myeloid leukemia and is associated with the coincident development of B-cell and T-cell lymphomas. SCLL is driven by the constitutive activation of fibroblast growth factor receptor-1 (FGFR1) as a result of chromosome translocations with poor outcome. Mouse models have been developed which faithfully recapitulate the human disease and have been used to characterize the molecular genetic events that are associated with development and progression of the disease.MethodsCRISPR/Cas9 approaches were used to generate SCLL cells null for Interleukin receptor associated kinase 1 (IRAK1) and interferon gamma (IFNG) which were introduced into syngeneic hosts through tail vein injection. Development of the disease and changes in immune cell composition and activity were monitored using flow cytometry. Bead-based immunoassays were used to compare the cytokine and chemokine profiles of control and knock out (KO) cells. Antibody mediated, targeted depletion of T cell and MDSCs were performed to evaluate their role in antitumor immune responses.ResultsIn SCLL, FGFR1 activation silences miR-146b-5p through DNMT1-mediated promoter methylation, which derepresses the downstream target IRAK1. IRAK1 KO SCLL cells were xenografted into immunocompetent syngeneic mice where the typical rapid progression of disease was lost and the mice remained disease free. IRAK1 in this system has no effect on cell cycle progression or apoptosis and robust growth of the KO cells in immunodeficient mice suggested an effect on immune surveillance. Depletion of T-cells in immunocompetent mice restored leukemogenesis of the KO cells, and tumor killing assays confirmed the role of T cells in tumor clearance. Analysis of the immune cell profile in mice transplanted with the IRAK1 expressing mock control (MC) cells shows that there is an increase in levels of myeloid-derived suppressor cells (MDSCs) with a concomitant decrease in CD4+/CD8+ T-cell levels. MDSC suppression assays and depletion experiments showed that these MDSCs were responsible for suppression of the T cell mediated leukemia cell elimination. Immuno-profiling of a panel of secreted cytokines and chemokines showed that activation of IFN-γ is specifically impaired in the KO cells. In vitro and in vivo expression assays and engraftment with interferon gamma receptor-1 (IFNGR1) null mice and IFNG KO SCLL cells, showed the leukemia cells produced IFN-γ directly participating in the induction of MDSCs to establish immune evasion. Inhibition of IRAK1 using pacritinib suppresses leukemogenesis with impaired induction of MDSCs and attenuated suppression of CD4+/CD8+ T-cells.ConclusionsIRAK1 orchestrates a previously unknown FGFR1-directed immune escape mechanism in SCLL, through induction of MDSCs via regulation of IFN-γ signaling from leukemia cells, and targeting IRAK1 may provide a means of suppressing tumor growth in this syndrome by restoring immune surveillance.

Зміни ендокринної функції тимуса, макрофагів і Т-лімфоцитів головного мозку в мишей різного віку після введення нейротоксину купризону і цитокіну

Актуальність. Відома участь макрофагів і Т­лімфоцитів в ушкодженні нервових клітин головного мозку. Гормон тимуса тимулін виявляє імуномодулюючі й антизапальні властивості. Вікові зміни активності макрофагів, Т­лімфоцитів головного мозку та тимуса можуть впливати на їх відповідь на дію токсичних та регуляторних факторів. Мета — дослідження змін рівня в крові тимуліну та кількості макрофагів і Т­лімфоцитів у головному мозку молодих і старіючих мишей, яким вводили нейротоксин купризон і рекомбінантний лейкемія­інгібіторний фактор людини (rhLIF). Матеріали та методи. Миші лінії 129/Sv отримували з їжею щоденно купризон упродовж 3 тижнів. З 8­ї доби купризонової дієти щоденно вводили rhLIF у дозі 50 мг/кг. Результати. Підвищення рівня в крові тимуліну через 7 діб прийому купризону інтенсивніше в молодих мишей, ніж у старіючих. Через 3 тижні купризонової дієти рівень гормона зменшувався тільки в молодих мишей. Кількість макрофагів у головному мозку молодих мишей збільшувалась вже через 7 діб прийому купризону, а у старіючих — тільки через 3 тижні. У мишей обох вікових груп із купризоновою дієтою (особливо в молодих) збільшувалась кількість Т­клітин у головному мозку. Ін’єкції rhLIF підвищували рівень у крові тимуліну і зменшували кількість Т­клітин у головному мозку молодих мишей, тоді як у старіючих мишей спостерігалось зменшення кількості макрофагів у головному мозку. Висновки. Дія як купризону, так і rhLIF на ендокринну функцію тимуса та кількість макрофагів і Т­лімфоцитів у головному мозку мишей залежить від віку.

Combining the IAP Antagonist Tolinapant with a DNA Hypomethylating Agent Enhances Immunogenic Cell Death in Preclinical Models of T-Cell Lymphoma

Introduction: Tolinapant is a potent, non-peptidomimetic antagonist of cIAP1, cIAP2 and XIAP. In ongoing Phase 2 trial (NCT02503423), tolinapant has shown activity against highly pre-treated peripheral and cutaneous T-cell lymphoma (Samaniego et al., Hematological Oncology, 2019). Hypomethylating agents (HMAs) have also shown clinical responses in some subsets of PTCL (Lemonnier et al., Blood, 2019). Both HMAs and IAP antagonists show immunomodulatory anti-cancer potential in pre-clinical studies. A Phase 1 clinical study investigating the combination of tolinapant and ASTX727 (oral decitabine) in AML is currently in progress (NCT04155580). Here we have undertaken a biomarker-driven approach to understand the potential for induction of immunogenic forms of cell death (ICD), such as necroptosis, by rational combination of our clinical compounds in pre-clinical models of T-cell lymphoma (TCL).Methods: On-target effects of decitabine and tolinapant were measured by analysing levels of DNMT1 and cIAP1, respectively, by Western blotting in mouse and human cell lines. Levels of key apoptosis, necroptosis or pyroptosis biomarkers were also monitored by Western blotting to provide evidence of lytic cell death contributing to a potential immune response. RIPK3- or MLKL-knockout cell lines were generated by CRISPR to demonstrate involvement of necroptosis in drug-induced cell death in a T-cell lymphoma cell line (BW5147.G.1.4) in vitro. Cell death was monitored by viability (CellTiterGlo) or real-time microscopy (IncuCyte) assays. Levels of key inflammatory mediators or DAMPS were measured in tissue culture supernatants and mouse plasma by Luminex assay (Ampersand).Results: Combined treatment of tolinapant and decitabine led to depletion of cIAP1 and DNMT1 in TCL cell lines, demonstrating on-target activity of tolinapant and decitabine, respectively. The combination of tolinapant and decitabine acted synergistically in mouse and human T-cell lymphoma cell lines to reduce viability in proliferation assays.Necroptosis was induced by decitabine or tolinapant alone in mouse TCL cell lines with robust activation of the RIPK1/RIPK3/MLKL necroptosis pathway when caspase activity was inhibited, and the combination of both agents enhanced loss of viability.Furthermore, we demonstrated decitabine treatment led to re-expression of both RIPK3 and MLKL in mouse cell lines, supporting published evidence that methylation can silence these key biomarkers (Koo et al., Cell Research, 2015; Koch et al., Neoplasia, 2021). Enhanced release of chemokine, cytokine and DAMPs was demonstrated with the combination of agents in vitro and in vivo.By removal of key necroptosis pathway components using CRISPR, we confirmed the importance of this lytic cell death pathway by demonstrating that RIPK3 -/- and MLKL -/- T-cell lymphoma (BW5147.G.1.4) cell lines had reduced necroptosis potential after treatment with tolinapant or decitabine alone or in combination; and demonstrate reduced release of inflammatory mediators in vitro.Finally, our in vivo evaluation of the combination of agents in mouse syngeneic models suggested that increased anti-tumour activity and immune-potentiating systemic biomarker modulation can be achieved with a tolerated dosing regimen of both compounds.Conclusion: These data demonstrate that decitabine enhances immunogenic cell death induced by tolinapant through the re-expression of genes in the necroptotic pathway. This finding provides strong rationale to explore this combination clinically. DisclosuresSims: Astex Pharmaceuticals: Current Employment. Davis: Astex Pharmacueticals: Current Employment. Smyth: Astex Pharmaceuticals: Current Employment.