RAG-2 KO Research Articles

A Feasibility Study of [18F]F-AraG Positron Emission Tomography (PET) for Cardiac Imaging-Myocardial Viability in Ischemia-Reperfusion Injury Model.

Myocardial infarction (MI) with subsequent inflammation is one of the most common heart conditions leading to progressive tissue damage. A reliable imaging marker to assess tissue viability after MI would help determine the risks and benefits of any intervention. In this study, we investigate whether a new mitochondria-targeted imaging agent, 18F-labeled 2'-deoxy-2'-18F-fluoro-9-β-d-arabinofuranosylguanine ([18F]F-AraG), a positron emission tomography (PET) agent developed for imaging activated T cells, is suitable for cardiac imaging and to test the myocardial viability after MI. To test whether the myocardial [18F]-F-AraG signal is coming from cardiomyocytes or immune infiltrates, we compared cardiac signal in wild-type (WT) mice with that of T cell deficient Rag1 knockout (Rag1 KO) mice. We assessed the effect of dietary nucleotides on myocardial [18F]F-AraG uptake in normal heart by comparing [18F]F-AraG signals between mice fed with purified diet and those fed with purified diet supplemented with nucleotides. The myocardial viability was investigated in rodent model by imaging rat with [18F]F-AraG and 2-deoxy-2[18F]fluoro-D-glucose ([18F]FDG) before and after MI. All PET signals were quantified in terms of the percent injected dose per cc (%ID/cc). We also explored [18F]FDG signal variability and potential T cell infiltration into fibrotic area in the affected myocardium with H&E analysis. The difference in %ID/cc for Rag1 KO and WT mice was not significant (p = ns) indicating that the [18F]F-AraG signal in the myocardium was primarily coming from cardiomyocytes. No difference in myocardial uptake was observed between [18F]F-AraG signals in mice fed with purified diet and with purified diet supplemented with nucleotides (p = ns). The [18F]FDG signals showed wider variability at different time points. Noticeable [18F]F-AraG signals were observed in the affected MI regions. There were T cells in the fibrotic area in the H&E analysis, but they did not constitute the predominant infiltrates. Our preliminary preclinical data show that [18F]F-AraG accumulates in cardiomyocytes indicating that it may be suitable for cardiac imaging and to evaluate the myocardial viability after MI.

Antigen-driven T cell-macrophage interactions mediate the interface between innate and adaptive immunity in histidyl-tRNA synthetase-induced myositis.

Previous work in humans has demonstrated that both innate and adaptive immune signaling pathways contribute to the pathogenesis of idiopathic inflammatory myopathy (IIM), a systemic autoimmune disease targeting muscle as well as extra-muscular organs. To better define interactive signaling networks in IIM, we characterized the cellular phenotype and transcriptomic profiles of muscle-infiltrating cells in our established murine model of histidyl-tRNA synthetase (HRS)-induced myositis. Myositis was induced in wild type (WT) and various congenic/mutant strains of C57BL/6 mice through intramuscular immunization with recombinant HRS. Histopathological, immunohistochemical, flow cytometric, and transcriptomic assessments were used to characterize the functional relationship between muscle-infiltrating cell populations in these strains lacking different components of innate and/or adaptive immune signaling. RAG1 KO mice developed markedly reduced muscle inflammation relative to WT mice, demonstrating a key requirement for T cells in driving HRS-induced myositis. While the reduction of mononuclear cell infiltrates in CD4-Cre.MyD88fl/fl conditional knockout mice and OT-II TCR transgenic mice highlighted roles for both innate and TCR-mediated/adaptive immune signaling in T cells, diminished inflammation in Lyz2-Cre.MyD88fl/fl conditional knockout mice underscored the importance of macrophage/myeloid cell populations in supporting T cell infiltration. Single cell RNA sequencing-based clustering of muscle-infiltrating subpopulations and associated pathway analyses showed that perturbations of T cell signaling/function alter the distribution and phenotype of macrophages, fibroblasts, and other non-lymphoid cell populations contributing to HRS-induced myositis. Overall, HRS-induced myositis reflects the complex interplay between multiple cell types that collectively drive a TH1-predominant, pro-inflammatory tissue phenotype requiring antigen-mediated activation of both MyD88- and TCR-dependent T cell signaling pathways.

T Cell CEACAM1–TIM-3 Crosstalk Alleviates Liver Transplant Injury in Mice and Humans

Carcinoembryonic antigen-related cell adhesion molecule 1 (CC1) acts through homophilic and heterophilic interactions with T cell immunoglobulin domain and mucin domain-containing protein 3 (TIM-3), which regulates innate immune activation in orthotopic liver transplantation (OLT). We investigated whether cluster of differentiation (CD) 4+ T cell-dependent CC1-TIM-3 crosstalk may affect OLT outcomes in mice and humans. Wild-type (WT) and CC1-deficient (CC1 knock-out [KO]) mouse livers were transplanted into WT, CC1KO, or T-cell TIM-3 transgenic (TIM-3Tg)/CC1KO double-mutant recipients. CD4+ T cells were adoptively transferred into T/B cell-deficient recombination activating gene 2 protein (Rag2) KO recipients, followed by OLT. The perioperative liver-associated CC1 increase was analyzed in 50 OLT patients. OLT injury in WT livers deteriorated in CC1KO compared with CC1-proficient (WT) recipients. The frequency of TIM-3+CD4+ T cells was higher in WT than CC1KO hosts. Reconstitution of Rag2KO mice with CC1KO-T cells increased nuclear factor (NF)-κB phosphorylation and OLT damage compared with recipients repopulated with WT T cells. T-cell TIM-3 enhancement in CC1KO recipients (WT→ TIM3Tg/CC1KO) suppressed NF-κB phosphorylation in Kupffer cells and mitigated OLT injury. However, TIM-3-mediated protection was lost by pharmacologic TIM-3 blockade or an absence of CC1 in the donor liver (CC1KO→ TIM-3Tg/CC1KO). The perioperative CC1 increase in human OLT reduced hepatocellular injury, early allograft dysfunction, and the cumulative rejection rate. This translational study identifies T cell-specific CC1 signaling as a therapeutic means to alleviate OLT injury by promoting T cell-intrinsic TIM-3, which in turn interacts with liver-associated CC1 to suppress NF-κB in Kupffer cells. By suppressing peritransplant liver damage, promoting T-cell homeostasis, and improving OLT outcomes, recipient CC1 signaling serves as a novel cytoprotective sentinel.

Deficiency of Pink1 promotes the differentiation of Th1 cells

Previous studies have found that Pink1 is crucial for T cell activation and the function of Treg cells. However, the effect of Pink1 on inflammatory Th1 cells is largely unknown. In the process of Th1 differentiation from human naïve T cells, we found a reduction of Pink1 and Parkin. We then focused our attention on the Pink1 KO mice. Although there was no difference in the baseline of the T cell subset of Pink1 KO mice, Th1 differentiation from Pink1 KO naïve T cells in vitro showed a significant increase. Subsequently, we transferred naïve CD4+ T cells into Rag2 KO mice to establish a T-cell colitis mouse model and found that CD4+ T cells in mesentery lymph nodes of mice receiving Pink1 KO cells increased significantly, especially Th1 cells. Intestinal IHC staining also showed that the transcription factor T-bet of Th1 increased. Treatment of CD4+ T cells from lupus-like mice with mitophagy agonist urolithin A, a reduction of Th1 cells was observed, suggesting the clinical value of using mitophagy agonists to suppress Th1-dominated disease in the future.

Abstract 610: Modeling tumor immunoediting coupled with T-cell repertoire dynamic

Abstract As tumors evolve, cancer cells acquire somatic mutations and these mutations generate tumor specific neoantigens that are different from self and can be recognized by T-cells, inducing anti-tumor immune responses. Such immunogenic antigens can be effective targets for neoantigen-based cancer vaccines. However, the identification of immunogenic neoantigens remains limited with the current prediction models. Here we designed mouse experiments to model tumor evolution under immune pressure and its association with T-cell clones to identify these antigens agnostically and improve current prediction models. We injected three lung cancer cell lines (KPA, KPC, and HKP1) in wildtype immune-competent C57BL/6 mice and immune-deficient Rag1 defective (Rag1 KO) mice, respectively. The three tumor cell lines have common driver mutations, Kras G12D/+ and p53 flox/flox. Two weeks post tumor implantation, whole exome sequencing and RNA-seq was performed on tumor samples. We also performed T cell receptor (TCR) sequencing to profile tumor infiltrated TCR clones. Despite the shared driver mutations, tumor growth patterns in vivo over time are very distinct for each cell line. Interestingly, KPA tumors show differential evolutionary patterns in wild type mice versus Rag1 KO mice, regressing in the former but rapidly progressing in the latter, suggesting that the difference can be attributed to immunoediting of KPA specific neoantigens. HKP1 tumors do not show substantial differences in that aggressive tumor progression is observed in both types of mice. Using WES data, we estimated the cancer cell fraction of each mutation based on phylogeny re-construction. We found that evolved KPA tumors (immunogenic) had significantly lower mutation burden in wild type mice compared to Rag1 KO mice (3410 vs 4199 on average), and the fraction of cell line mutations edited in vivo is significantly higher in wild type than in Rag1 KO (26% vs 14% on average), suggesting again the active on-going immunosurveillance in immune competent mice. In contrast, HKP1 tumors (non-immunogenic) do not show significant differences in mutation burden nor fraction of mutations edited between wild type and Rag1 KO mice. Furthermore, TCR-seq data show that the clonal expansion of T-cells is strongly associated with tumor immunogenicity. KPA tumor samples show significantly higher TCR clustering but with less diversity, quantified by modified Simpson’s Index, that is strongly correlated with the suppression of mutational burden (r=0.74). In summary, we found that tumor evolution in immune competent mice is dependent on mutational context of the tumor. By analyzing longitudinal tumor and TCR sequencing data, we showed that the immunogenicity of tumors is associated with significant decrease in mutational burden and higher T-cell clustering that is indicative of tumor mutation and TCR clone expansion co-evolution in immune competent mice. Citation Format: Jayon Lihm, Hyejin Choi, Mariam M. George, Yuval Elhanati, David Hoyos, Marta Luksza, Taha Merghoub, Benjamin D. Greenbaum. Modeling tumor immunoediting coupled with T-cell repertoire dynamic [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 610.

291 High Potency STING Agonists Induce Adaptive Immunity-Dependent Curative Responses in an Immune Checkpoint Blockade-Refractory Glioblastoma Model

OBJECTIVES/GOALS: Glioblastoma (GBM) is the most common and aggressive adult primary brain malignancy. Clinically, GBM is refractory to T cell immune checkpoint blockade (ICB), in part due to its dense immune suppressive myeloid stroma. Here we show that myeloid-targeting STING agonists can repolarize the GBM microenvironment to cure ICB-refractory GBM models. METHODS/STUDY POPULATION: Using the synthetic cyclic di-nucleotide STING agonist IACS-8803 (8803) we treated orthotopic ICB-refractory QPP8 orthotopic murine GBM tumors intratumorally. We then analyzed survival and performed high parameter flow cytometry profiling of the tumor immune microenvironment following STING agonist treatment. To assess the contribution of adaptive immunity to STING agonist therapeutic efficacy, we treated orthotopic QPP8 tumors implanted in RAG1 KO mice and monitored survival. RESULTS/ANTICIPATED RESULTS: We found that STING agonist therapy cured murine orthotopic QPP8 tumors, in contrast to ICB that showed no survival benefit. In RAG1-/- mice bearing QPP8 tumors STING agonist therapy extended survival, however, the curative effect observed in wild-type mice was lost in the absence of adaptive immunity. STING agonist-treated QPP8 tumors displayed increased counts of CD8 T cells and NK cells, and decreased CD8 T cell PD1 expression. Infiltration of STING-treated gliomas by Ly6C+ F4/80+ Mono-MDSC substantially increased; however, these cells expressed reduced CD206 and CD163, suggestive of reduced immuno-suppression. Finally, in the cervical LN of QPP8-treated mice the frequency and CD80/CD86 expression of cDC1 cells increased.​ DISCUSSION/SIGNIFICANCE: ICB has failed in GBM, and the suppressive myeloid stroma remains a major barrier to generating anti-GBM T cell responses. Our work shows that STING activation, which primarily targets innate immunity myeloid cells 'upstream' of T cells in the antitumor immunity cycle, can cure ICB-refractory GBM tumors in an adaptive immunity-dependent manner.

Gut inflammation and adaptive immunity amplify acetaminophen toxicity in bowel and liver.

Prevention of liver failure arising from accidental or deliberate paracetamol (acetaminophen [APAP]) overdose remains a vexed health problem despite well-publicized guidelines for its early detection and treatment. It is recognized that the gut may aggravate liver pathology, via the gut-liver axis. The main aim of this study was to assess the role of the colon in APAP-induced liver toxicity. Liver necrosis and colitis were studied following sublethal doses of APAP administered intraperitoneally to C57Bl/6 wild-type (WT) mice, as well as to C57Bl/6 Winnie mice, which develop a spontaneous colitis caused by a SNP in Muc2, and WT mice with acute DSS-induced colitis. Repeated APAP exposure was studied in WT and Rag1 ko mice that lack mature T and B lymphocytes. APAP overdose resulted in significant colonic injury in WT mice (P<0.05), which resolved by 24h. Underlying colitis was not associated with liver necrosis, but colitis exacerbated APAP-induced liver injury and extended APAP-colonic injury. Prior APAP exposure exacerbated both APAP-liver and APAP-colonic injury more so in WT than Rag1 ko mice. APAP impaired barrier function with increased intestinal permeability and associated bacterial translocation to the liver and spleen in mice with the Winnie phenotype. This study identifies novel roles for APAP in causing colitis, the amplification of APAP-liver toxicity where there is underlying colitis, and involvement of immune memory in APAP-toxicity. The latter could be key for decoding the poorly understood but important clinical entity of chronic APAP liver failure.

Pseudomonas Aeruginosa Lung Infection Subverts Lymphocytic Responses through IL-23 and IL-22 Post-Transcriptional Regulation.

Pseudomonas aeruginosa (P.a) is a pathogen causing significant morbidity and mortality, particularly in hospital patients undergoing ventilation and in individuals with cystic fibrosis. Although we and others have investigated mechanisms used by P.a to subvert innate immunity, relatively less is known about the potential strategies used by this bacterium to fight the adaptive immune system and, in particular, T cells. Here, using RAG KO (devoid of ‘classical’ αβ and γδ TCR T lymphocytes) and double RAG γC KO mice (devoid of T, NK and ILC cells), we demonstrate that the lymphocytic compartment is important to combat P.a (PAO1 strain). Indeed, we show that PAO1 load was increased in double RAG γC KO mice. In addition, we show that PAO1 down-regulates IL-23 and IL-22 protein accumulation in the lungs of infected mice while up-regulating their RNA production, thereby pointing towards a specific post-transcriptional regulatory mechanism not affecting other inflammatory mediators. Finally, we demonstrate that an adenovirus-mediated over-expression of IL-1, IL-23 and IL-7 induced lung neutrophil and lymphocytic influx and rescued mice against P.a-induced lethality in all WT, RAG γC KO and RAG γC KO RAG-deficient mice, suggesting that this regimen might be of value in ‘locally immunosuppressed’ individuals such as cystic fibrosis patients.

P704 Bifidobacterium longum species protect murine colitis through different activation repertoires of immune cells

Abstract Background Inflammatory bowel disease (IBD) is a chronic disease that causes an inappropriate immune response due to genetic and environmental factors. The scientific interest in mechanisms of probiotics are world-widely growing. Bifidobacterium plays an essential role in the maturation of the immune system, and the prevention of colitis and tumor. However, only limited reports on bifidobacterium isolated to prevent colitis and their exact mechanisms of action remain poorly understood. This study identifies the different anticolitic effects between B. breve strain and Bifidobacterium longum (B. longum) strain, the most abundant probiotic strains in lactating babies. We explore the different activation repertoire of the immune cell according to Bifidobacterium species and the mechanisms in these processes. Methods We administrated B. breve and B. longum ATCC by oral gavage 1×109 CFU to RAG1 KO mice. Colitis was induced with 2.5% DSS for 7 days. To assess the direct role of B. longum on ILCs, ILCs from B. breve- and B. longum-treated wild-type mice were transferred to RAG1 KO mice. We observed daily weight loss, stool consistency, and rectal bleeding of mice and transformed to the disease activity index (DAI) scores. Macrophages and T-cell subsets in splenocytes, lamina propria cells were analyzed by flow cytometry. Gene expression and cytokine profiles were determined using qRT-PCR. We performed whole-genome sequencing of reactive oxygen species (ROS)-resistant B. longum species using DNA full-sequencing analysis. Results The two functional strains of B. breve and B. longum ameliorate gut immunopathology during colitis in RAG1 knockout mice. However, B. longum but not B. breve, drastically restored body weight loss, DAI, and colon length compared to B. breve-treated group. We found that the anticolitic effect of B. longum is specialized by modulation of ILCs. Newly identified B. longum strain from the feces of healthy adults has ROS resistance even though it is anaerobic bacteria. The new B. longum strain exhibits ANI values of 94.92% with respect to B. longum ATCC 15697. The new B. longum strain has a strong colonization ability and improves DSS-induced colitis in the wild-type mice than other strains. Our data revealed genus-specific effects of bifidobacteria on colonization in the colon, and ILCs, thus discriminating a differential degree of modulation of immune cells. Conclusion We explore the different activation repertoire of the immune cell according to Bifidobacterium species and the mechanisms in these processes. These findings suggest that B. longum can be used as a potent immunomodulatory of ILCs, which has significant implications for IBD treatment.

Myelin-Independent Therapeutic Potential of Canine Glial-Restricted Progenitors Transplanted in Mouse Model of Dysmyelinating Disease.

Background: Dysfunction of glia contributes to the deterioration of the central nervous system in a wide array of neurological disorders, thus global replacement of glia is very attractive. Human glial-restricted precursors (hGRPs) transplanted intraventricularly into neonatal mice extensively migrated and rescued the lifespan in half of the studied mice, whereas mouse GRPs (mGRPs) presented no therapeutic benefit. We studied in the same experimental setting canine GRPs (cGRP) to determine whether their therapeutic potential falls between hGRPs and mGRPs. Additional motivation for the selection of cGRPs was a potential for use in veterinary medicine. Methods: cGRPs were extracted from the brain of dog fetuses. The cells were transplanted into the anterior or posterior aspect of the lateral ventricle (LV) of neonatal, immunodeficient, dysmyelinated mice (Mbpshi, Rag2 KO; shiv/rag2). Outcome measures included early cell biodistribution, animal survival and myelination assessed with MRI, immunohistochemistry and electron microscopy. Results: Grafting of cGRP into posterior LV significantly extended animal survival, whereas no benefit was observed after anterior LV transplantation. In contrast, myelination of the corpus callosum was more prominent in anteriorly transplanted animals. Conclusions: The extended survival of animals after transplantation of cGRPs could be explained by the vicinity of the transplant near the brain stem.

Targeted Alpha-Particle Radiotherapy and Immune Checkpoint Inhibitors Induces Cooperative Inhibition on Tumor Growth of Malignant Melanoma.

Simple SummaryRadiation therapy and immune checkpoint inhibitors (ICIs) have been demonstrated to cooperatively activate adaptive anti-tumor immunity with curative potential in preclinical models of melanoma. Receptor-targeted radionuclide therapy can be systemically injected to selectively deliver ionizing radiation to tumor sites throughout the body, potentially rendering all tumor sites more susceptible to anti-tumor immune response. In this study, we demonstrated the feasibility of delivering alpha-particle radiation to murine melanoma tumors using a 212Pb radiolabeled peptide [212Pb]VMT01 that targets the melanocortin 1 receptor (MC1R). Our data showed anti-tumor cooperation between [212Pb]VMT01 and ICIs in melanoma, mediated by induction of tumor-specific immunity. The immunogenicity of [212Pb]VMT01 in melanoma was also evidenced by enhanced tumor infiltrating lymphocytes and tumor vaccination assays.Radiotherapy can facilitate the immune recognition of immunologically “cold” tumors and enhance the efficacy of anti-PD-1 and anti-CTLA-4 immune checkpoint inhibitors (ICIs) in melanoma. Systemic administration of receptor-targeted radionuclide therapy has the potential to selectively deliver radionuclides to multiple tumors throughout the body in metastatic settings. By triggering immunologic cell death and increasing the immune susceptibility of surviving tumor cells in these locations, targeted radionuclide therapies may overcome resistance to ICIs and render immunologically “cold” tumors throughout the body responsive to ICIs and immunologically “hot”. Here, we show the anti-tumor cooperation of targeted α-particle radionuclide therapy (α-TRT) and ICIs in preclinical models of melanoma. Melanocortin 1 receptor (MC1R)-targeted radiopeptide [212Pb]VMT01 was employed to deliver α-radiation to melanoma tumors in mice. A single injection of 4.1 MBq [212Pb]VMT01 significantly slowed the tumor growth of B16-F10 melanoma and the combination of [212Pb]VMT01 and ICIs induced a cooperative anti-tumor effect leading to 43% complete tumor response with no sign of malignancy on autopsy. Animals with complete response developed anti-tumor immunity to reject further tumor inoculations. This therapeutic cooperation was completely abolished in RAG1 KO mice, which are deficient in T-cell maturation. In addition, the anti-tumor cooperation was compromised when fractionated [212Pb]VMT01 was used in the combination. We also demonstrated that [212Pb]VMT01 induced immunogenic cell death in tumor vaccination assays and in vitro exposure to [212Pb]VMT01 sensitized immunotolerant melanoma to ICIs treatment in vivo. Enhanced tumor infiltrating CD3+, CD4+, CD8+ lymphocytes were observed following injection of 1.4 MBq [212Pb]VMT01. Overall, we demonstrated anti-tumor cooperation between α-TRT and ICIs in melanoma that is mediated by tumor specific immunity.

Trichinella spiralis Paramyosin Induces Colonic Regulatory T Cells to Mitigate Inflammatory Bowel Disease.

Helminth infection modulates host regulatory immune responses to maintain immune homeostasis. Our previous study identified Trichinella spiralis paramyosin (TsPmy) as a major immunomodulatory protein with the ability to induce regulatory T cells (Tregs). However, whether TsPmy regulates gut Tregs and contributes to intestinal immune homeostasis remains unclear. Here we investigated the therapeutic effect of recombinant TsPmy protein (rTsPmy) on experimental colitis in mice, and elucidated the roles and mechanisms of colonic Tregs induced by rTsPmy in ameliorating colitis. Acute colitis was induced by dextran sodium sulfate (DSS) in C57BL/6J mice, and chronic colitis was induced by naïve T cells in Rag1 KO mice. Mice with colitis were pre-treated with rTsPmy intraperitoneally, and clinical manifestations and colonic inflammation were evaluated. Colonic lamina propria (cLP) Tregs phenotypes and functions in DSS-induced colitis were analyzed by flow cytometry. Adoptive transfer of cLP Tregs treated by rTsPmy into Rag1 KO chronic colitis was utilized to verify Tregs suppressive function. rTsPmy ameliorated the disease progress of DSS-induced colitis, reduced pro-inflammatory responses but enhanced regulatory cytokines production in DSS-induced colitis. Moreover, rTsPmy specifically stimulated the expansion of thymic-derived Tregs (tTregs) rather than the peripherally derived Tregs (pTregs) in the inflamed colon, enhanced the differentiation of effector Tregs (eTregs) with higher suppressive function and stability in colitis. This study describes the mechanisms of colonic Tregs induced by the Trichinella-derived protein rTsPmy in maintaining gut immune homeostasis during inflammation. These findings provide further insight into the immunological mechanisms involved in the therapeutic effect of helminth-derived proteins in inflammatory bowel diseases.

P032 MiR-511 deficiency aggravates T cell transfer colitis in mice

Abstract Background MiR-511 is embedded in intron region 5 of the CD206/MRC1 gene, expressed by macrophage and dendritic cell populations. In this study, we aimed to investigate the effect of MiR-511 deficiency on intestinal inflammation in a murine T cell transfer colitis model. Methods A double MiR-511- and Rag-1 (knockout) KO mouse was generated and a T cell transfer colitis was induced by intraperitoneal injection of naïve T cells from donor WT mice. Since these mice lack mature T and B cells, first signs of inflammation appeared at week 3 after T cell injection. An endoscopy score was obtained to determine inflammation at week 3 and 5, respectively. The experiment was terminated at week 5 and severity of inflammation was assessed on the basis of weight loss, colon weight/length ratio, histology score, spleen weight and disease activity index. In addition, flow cytometry was performed for analysing immune cell populations (monocyte, macrophages, dendritic cells, neutrophils) in the colons of both control and colitis groups and T cells in the spleens of colitis group, respectively. Results Following the induction of T cell transfer colitis, colon weight/length ratio, spleen weight and endoscopic score were significantly increased in the double KO mice compared to Rag-1 KO control mice. A higher histology score and disease activity index in the double KO with no change in weight loss compared to Rag-1 KO control mice was observed. A significant increase in monocyte population in the colons of double KO was seen and increased numbers of monocytes was also observed in the double KO control group with no inflammation. Also, a higher influx of T cells in the double KO mice with a significant increase in Foxp3 and IL4 population was observed in the group with colitis. Conclusion MiR-511 deficiency aggravates intestinal inflammation compared to Rag-1 KO control mice. Also, a higher presence of monocyte as well as T cell populations were observed in these mice. Together these data show that MiR-511 is involved in the regulation of intestinal health. Future research will focus on underlying mechanisms.

Invariant NKT cells promote expansion of novel CXCR3+CCR4+CD8+ T cells in the liver following allogeneic hepatocyte transplant

Abstract Hepatocyte transplant (HcTx) is a treatment for metabolic diseases and a bridge to liver transplant but is limited by immunogenicity. Our prior work identified the critical role of CD4-dependent and CD4-independent CD8+ T cells in HcTx rejection. Herein, we evaluated the influence of invariant natural killer T cells (iNKTs), abundant in the liver, upon CD8+ T cell responses in the presence and absence of CD4+ T cells. To investigate this, C57BL/6 (wild-type; WT) or iNKT deficient Jα18 KO (H-2b) received FVB/N (H-2q) HcTx. Cohorts were depleted of CD4+ T cells and/or reconstituted with iNKTs. Recipients were evaluated for cytotoxicity, CD8+ T cell phenotype, and HcTx rejection. By comparing CD4-sufficient WT and Jα18 KO recipients, we found that the presence of iNKTs significantly enhanced in vivo CD8-mediated cytotoxicity (83±3% vs 23±3%; p&amp;lt;0.001) but did not affect survival (10 days vs 14 days; p=ns). However, in CD4-deficient recipients, iNKTs were critical, since without CD4+ T cells and iNKTs, CD8-mediated in vivo cytotoxicity was completely abrogated (31±3% vs 2±1%; p&amp;lt;0.001) and HcTx survival was significantly prolonged (14 days vs 56 days; p&amp;lt;0.001). iNKTs enhanced the quantity of a novel CD8+ T cell subset, CD44+IFN-γ+CXCR3+CCR4+CD8+ T cells, in the liver (2-fold) in all HcTx recipients (p&amp;lt;0.03 for all comparisons). CXCR3+CCR4+CD8+ T cells, when compared to CXCR3+CCR4−CD8+ T cells, express higher IFN-γ (32±3% vs 9±1%; p&amp;lt;0.001) and TNF-α (37±3% vs 6±1%; p&amp;lt;0.001). They also exhibit greater in vivo cytotoxicity (13±1% vs 5±1%; p&amp;lt;0.001) and mediate rapid HcTx rejection in RAG1 KO recipients (5 days vs 16 days; p=0.02). We show that iNKTs enhance the expansion of highly cytotoxic CXCR3+CCR4+CD8+ T cells that mediate rapid rejection of HcTx.

Immunopathology and Meningitis During Late-Onset Gut-Originating Neonatal Sepsis

Abstract Late-onset neonatal sepsis (LOS) is a bacterial bloodstream infection that occurs during the first two months of life, and remains a significant contributor to neonatal mortality. In addition to the number of clinical challenges these patients face in the short term, neonates who survive LOS can experience a number of long-term adverse outcomes, such as impaired cognitive development. A major obstacle in LOS research remains the lack of an animal model that successfully recapitulates clinical LOS. We have developed a mouse model that replicates the clinical finding that a subset of LOS infections results from enterally-derived pathogens translocating from the intestine. In this model, pups develop sepsis-like disease upon oral gavage of virulent E. coli species. This translocation results in immune cell infiltration and bacterial presence in many peripheral sites, including the meninges. Specifically, we found infiltration of monocytes, and neutrophils into the meninges, along with increased numbers and activation status of both αβ+ and γδ+ T cells. Meningitis could be replicated by LPS alone, suggesting systemic bacterial presence contribute to infiltration into the meninges. Interestingly, mice older than 10 days did not develop meningitis in response to LPS, suggesting neonatal pups are uniquely susceptible to bacterial-initiated meningitis. Furthermore, this meningitis is induced partially by adaptive immune cell components, as no neutrophil or monocyte infiltration was seen in RAG KO mice following LPS injection. This study provides novel insights into the clinical reported complication of meningitis during LOS and suggests a unique contribution of adaptive lymphocytes in the neonate during this systemic infection.

CD122-SELECTIVE IL-2 COMPLEXES TREAT OVARIAN CARCINOMAS, INDUCE TREG FRAGILITY AND PROMOTE T CELL STEM CELLS

Abstract Regulatory T cells (Treg) engage IL-2 by high affinity CD25, and anti-tumor effector T cells (Teff) use intermediate affinity CD122. We studied IL-2 complexes (IL-2c) that selectively activate CD122+ Teff over CD25+ Tregs. We found IL-2c but not aPD-L1 potently inhibits ID8agg, an aggressive mouse ovarian cancer (OC) model. IL-2c decreased ascites Treg functional markers (CD25, GranzymeB) while upregulating them on Teffs. IL-2c inhibited Treg suppressive function in ascites but not TDLN. Ascites Tregs after IL-2c showed a fragile phenotype (increased PD-1, T-bet, and IFNg with maintained FoxP3) known to contribute to better immunotherapy response. These data suggest IL-2c inhibits Treg in the ascites through inducing fragile Tregs. Neither CD8+ T cells nor gd T cells is required for IL-2c induced Treg fragility. Studies testing other populations are ongoing. CD8+TCF-1+T cell stem cells (TCSC) improve immune checkpoint blockade in many cancer types. In ID8agg tumors, IL-2c induces CD8+TCF-1+ TCSC with tumor inhibitory capacity when transferred into Rag KO mice. tSNE analysis revealed IL-2c-induced TCSC are CXCR5+PD-1− express CCR2 CXCR3 and produce TNFa, while aPD-L1 induced TCSC are CXCR5+ and PD-1+, consistent with previous reports. These data suggest IL-2c induces beneficial CD8+TCSC distinct from aPD-L1. Although aPD-L1 is not efficacious alone in ID8agg, it significantly promoted IL-2c efficacy. Mice treated with IL-2c+αPD-L1 are resistant to tumor re-challenge even 6 weeks after final dose, suggesting durable immune memory. In sum, IL-2c is a novel OC immunotherapy that targets distinct immune pathways simultaneously, including inhibiting Tregs, boosting TCSCs, and inducing immune memory when combined with aPD-L1.

TFF3 mediates the NF-κB/COX2 pathway to regulate PMN-MDSCs activation and protect against necrotizing enterocolitis.

Intestinal trefoil factor 3 (TFF3) plays an important role in repairing the intestinal mucosa. However, the detailed mechanism regarding immune regulation by TFF3 is not well defined. Here, we reported that treatment of mouse BM cells and human peripheral blood mononuclear cells from healthy volunteers with TFF3 activated polymorphnuclear myeloid-derived suppressor cells (PMN-MDSCs) in vitro. We also found that prostaglandin E2 is a major TFF3-mediated MDSC target, and that NF-κB/COX2 signaling was involved in this process. Moreover, TFF3 treatment or transfer of TFF3-derived PMN-MDSCs (TFF3-MDSCs) to experimental necrotizing enterocolitis (NEC) mice caused PMN-MDSC accumulation in the lamina propria (LP), which was associated with decreased intestinal inflammation, permeability, bacterial loading, and prolonged survival. Interestingly, no NEC severity remission was observed in Rag1 KO mice that were given TFF3-MDSCs, but coinjection with CD4+ T cells significantly relieved NEC inflammation. Overall, TFF3 mediates the NF-κB/COX2 pathway to regulate PMN-MDSC activation and attenuates NEC in a T-cell-dependent manner, which suggests a novel mechanism in preventing NEC occurrence.

The novel innate immune-antagonistic effects of the multifunctional ectromelia virus C15 protein

Abstract The success of poxviruses as pathogens depends upon their extensive antagonism of host immune responses by a large arsenal of immunomodulatory proteins. The C15 protein of ectromelia virus (ECTV, the agent of mousepox) is the largest of the ECTV immunomodulatory proteins and is a member of a well-conserved poxviral family previously studied as inhibitors of T cell activation. We have recently determined that C15 also facilitates early viral spread in vivo, suggesting a second non-adaptive function of C15. Accordingly, we sought to further investigate this new function and identify the cellular target. We first found that C15 maintains its replication-promoting effect in RAG KO but not RAG IL2RG double KO mice, implying the targeting of NK cells. Nevertheless, the impact of C15 is drastically less in RAG KO than WT mice, suggesting that C15 also targets a RAG-dependent immune component. Further studies in various immune deficient strains implicate type I and III interferons, perforin and the NK gene complex in this novel function. The results prompt further in vitro and in vivo studies into the impact of C15 on NK cell and γδ T cell function, as the RAG-independent and -dependent targets respectively, and lead us to propose that, unique among currently identified viral virulence factors, C15 selectively antagonizes both the adaptive and innate immune responses.

Protein Phosphatase 2A (PP2A) is essential for CD8+ T cell mediated anti-tumor immunity

Abstract PP2A controls a wide variety of cellular pathways responsible for proliferation, growth, differentiation, and apoptosis. However, its role in immune cells, particularly in CD8+ T cells, still remains a mystery. We therefore sought to characterize the influence of PP2A on CD8+ T cell function in anti-tumor response. T cell specific genetic ablation of PP2A in mouse resulted in aberrant anti-tumor activity evidenced by higher tumor volume in both skin melanoma B16F10 and lymphoma E.G7 tumor graft model. Similarly, inhibition of PP2A using small molecule inhibitor LB100 showed similar effect. In addition, lower expression of effector cytokines including IFN-g, TNF-a and Granzyme B was also observed in PP2A-deleted CD8+ T Cells isolated from tumor infiltrate. Importantly, the defect in anti-tumor response is due to deficiency in CD8+ T cell function, which was proven in adoptive transfer experiment, in which CD8+ T cells from both WT and PP2A KO mice into tumor bearing RAG1 KO mice. PP2A KO CD8+ T cells-transferred mice show markedly impaired anti-tumor response. Further mechanism study suggested a possible role of PP2A in regulating CD28 signaling. PP2A KO lead to lower AKT phosphorylation upon CD28 stimulation, along with lower proliferative capability of CD8+ T cells upon stimulation with anti-CD28 and suboptimal anti-CD3 concentration. Thus this study highlights the potentially important role of PP2A in CD8+ T cells and its therapeutic consideration for designing combinatorial immunotherapy.

A phosphotyrosine switch controls antitumor activity of estrogen receptor b

Abstract Recently, we identified a phosphotyrosine residue in human ERb (pY36), which is highly conserved in all mammalian ERb orthologs, but not present in ERa. In addition, this phosphotyrosine switch in ERb controls its tumor-intrinsic (tumor cells) activity. However, the weakness of the current literature concerns limited information about the ERb tumor-extrinsic antitumor activity. To investigate ERb tumor-extrinsic antitumor activity, we generated a whole-body knock-in (KI) mouse model in which the tyrosine residue Y55 of endogenous mouse ERb, which corresponds to Y36 of human ERb, is mutated to phenylalanine (Y55F). We found that B16 melanoma grew more robustly in KI recipient mice than in their WT counterparts, as well as MC38 and M-Wnt tumor models. These data demonstrate that the phosphotyrosine switch is important for ERb tumor-extrinsic antitumor activity in multiple tumor types and in both sexes of recipient mice. In the following mouse bone marrow chimera experiment, we found B16 melanoma growth was significantly faster in KI&amp;gt;WT chimeras versus male WT&amp;gt;WT control. This suggests that KI-derived immune cells poorly deterred tumor growth. Analysis with enriched subpopulations of immune cells from spleen, CD8 T cells have ERb protein expression. Adoptive transfer of CD8 T cells from WT donor but not KI donor significantly reduced tumor growth in Rag1 KO mice, a function attributed to reduced infiltration of KI CD8 T cells into tumors. Accordingly, increased infiltration of CD8 T cells was positively associated with ESR2 expression in various cancer types from TCGA datasets, including breast cancer and melanoma.