RAG-2 KO Mice Research Articles

Tissue Engineering Scaffolds for Minimally-Invasive Immunosurveillance of Acute Cellular Graft Rejection

<h3>Purpose</h3> As there is no assay to predict the risk of acute cellular allograft rejection (ACAR), clinicians rely on protocol graft biopsy and aggressive, <i>one-size-fits-all</i> immunosuppression. Immunosuppression protects grafts from ACAR but increases systemic toxicities. Endomyocardial biopsy (EMB) histology suffers from variability and lags behind molecular biomarkers. <b>A minimally invasive surveillance method is urgently needed to quantify early risk of rejection for minimizing graft biopsy and personalizing treatment.</b> <h3>Methods</h3> Biomaterial scaffolds accumulate immune cells producing biomarkers of ACAR as an engineered immunological niche. We implanted subcutaneous poly-caprolactone scaffolds in murine heart or skin transplants to determine if they could identify ACAR. Rag2KO mice received full MHC mismatch allografts or syngeneic controls, and ACAR was initiated as a tunable event by C57BL/6 T cell transfer. Scaffolds were explanted and analyzed via histology and differential gene expression by RNA sequencing. <h3>Results</h3> The scaffolds provided a novel measure of graft health. We algorithmically identified <b>11 differentially-expressed genes as biomarkers</b> that distinguish mice with rejecting grafts from those with healthy grafts. <i>Importantly, the scoring system detects oncoming ACAR prior to clinical signs of rejection.</i> Also, histology of these niches identified changes associated with ACAR progression. <h3>Conclusion</h3> Gene expression in the cell-capture scaffold identified a biomarker signature of early ACAR, without invasive biopsy. <b>Scaffold cell capture technology provides a leading indicator of ACAR by monitoring tissue-specific immune responses associated with allograft rejection.</b> This implantable scaffold enables minimally invasive histological evaluation and molecular calculation of the early risk of rejection to reduce the frequency of routine EMB and personalize immune suppression that ultimately could prolong transplant life while minimizing risk.

Skin and heart allograft rejection solely by long-lived alloreactive TRM cells in skin of severe combined immunodeficient mice.

Whether induced tissue-resident memory T (TRM) cells in nonlymphoid organs alone can mediate allograft rejection is unknown. By grafting alloskin or heart into severe combined immunodeficient or Rag2KO mice in which a piece of induced CD4+ and/or CD8+ TRM cell–containing MHC-matched or syngeneic skin was transplanted in advance, we addressed this issue. The induced CD4+ TRM cells in the skin alone acutely rejected alloskin or heart grafts. RNA-seq analysis showed that induced CD4+ TRM cells in skin favorably differentiated into TH17-like polarization during the secondary immune response. Inhibition of the key TH17 signaling molecule RORγt attenuated TRM cell–mediated graft rejection. Thus, we offer a unique mouse model to specifically study TRM cell–mediated allograft rejection without the involvement of lymphocytes in lymphoid organs and tissues. Our study provides strong evidence supporting the hypothesis that long-lived alloreactive TRM cells resident in other organs/tissues substantially contribute to organ allograft rejection.

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.

Evolution of the Tumor Microenvironment throughout Progression and Transformation of EZH2 Mutant Follicular Lymphoma

The genetic hallmarks of follicular lymphomas include BCL2 translocations and somatic mutations of epigenetic modifier genes such as EZH2. Histologically, FLs typically feature a rich microenvironment, most notably featuring extensive follicular dendritic cell (FDC) networks with dendrites making extensive contact with lymphoma cells. In recent work we showed that the main effect of EZH2 gain-of-function mutations in GC B-cells is to enable them to become less dependent of T-cell help and strengthen their immune synapse formation with FDCs, which induces aberrant proliferation and survival of GC centrocytes and hence formation of a unique lymphoma-permissive immune niche. However, it is still unknown how interactions between EZH2 mutant GC B-cells and other immune cells change throughout the progression of the disease.To evaluate the evolution of the tumor microenvironment (TME) throughout EZH2 mutant lymphoma progression, we developed a genetically engineered mouse model designed for conditional expression of gain-of-function Ezh2 mutant and BCL2 in GC B-cells, Rosa26 LSL.BCL2.IRES.GFP;Ezh2 Y641F;Cγ1Cre (hereafter BCL2/Ezh2 Y641F), in which Cγ1-driven Cre excision of a STOP cassette in the Rosa26 locus leads to overexpression of BCL2 and GFP, and expression of the endogenous Ezh2 mutant Y641F. This mouse model develops low-grade follicular-like lymphoma, characterized by expanded follicles composed largely of centrocyte neoplastic GC B-cells and extensive FDC meshwork, along with presence of CD4 +, TFH and regulatory T cells (FOXP3 +), as depicted by multiparametric in situ imaging and multiparametric flow cytometry. At cytological level, cells are predominantly small with condensed chromatin, irregular nuclei, scant cytoplasm, and inconspicuous nucleoli, without sheets of large cells. Over time, these low grade FLs progress to advanced grade, characterized by disruption of follicle structures, expansion of centroblast-like large tumor cells and reduced CD4 + T cell infiltration and FDC meshwork.Furthermore, we have developed a murine transformed FL cell line by sequential passages of an original BCL2/Ezh2 Y641F low-grade FL into immunodeficient Rag1KO mice. We have transduced this BCL2/Ezh2 Y641F cell line with luciferase, and it successfully engrafted and homed to lymphoid organs when injected i.v. into immunocompetent C57BL6 mice. The cell line consists of high proliferative GC B-cells with multiple and irregular nuclei, open chromatin and prominent nucleoli that resemble DLBCL. The microenvironment of tumors developed in C57BL6 mice is characterized by disruption of follicle structures, severe reduction or absence of FDC meshwork, decreased CD4 +, CD8 + and Tregs, downregulation of MHC-I and MHC-II. Therefore, our mouse model also recapitulates progression and transformation stages.Since MYC translocations are frequent events that occur during histological transformation of FL, we modeled this more aggressive EZB MYC subtype of DLBCL. For that, we further crossed our BCL2/Ezh2 Y641F mice to Rosa26 LSL.MYC.IRES.hCD2. We generated cohorts of transplanted mice by injecting bone marrow cells from BCL2, BCL2/Ezh2 Y641F, BCL2/MYC, and BCL2/Ezh2 Y641F/MYC mice into lethally irradiated C57BL6 recipients. Recipient mice (n=5 per group) were immunized with SRBC to induce formation of GCs and sacrificed 5.5 months post bone marrow transplant. All genotypes showed expansion of FAS +CD38 -BCL2 GFP+ GC B-cells in spleen and lymph nodes; however, the proportion of GC B-cells in Ezh2 Y641F was significantly increased compared with Ezh2 WT, in both presence and absence of MYC. In contrast, FAS -CD38 +BCL2 GFP+ and CD138 +BCL2 GFP+ cells were significantly increased in MYC + mice and decreased in Ezh2 Y641F, suggesting that Ezh2 mutation is required to maintain the GC phenotype. The acquisition of Ezh2 Y641F in BCL2/MYC mice induced a reduction of CD4 + and CD8 + in the TME, and decreased TFH without changes in TFR proportions in GCs. Strikingly, FDC meshwork was significantly shrank in MYC + cases, and partially restored by acquisition of Ezh2 Y641F, indicating that MYC overexpression may contribute to acquire FDC independency for the survival of lymphoma cells.Our results indicate that progression of FL critically affects the TME and acquisition of additional mutations such as MYC alters the interactions between lymphoma cells and tumor supportive immune cells in TME. DisclosuresMelnick: Epizyme: Consultancy; Janssen: Research Funding; KDAC Therapeutics: Current holder of individual stocks in a privately-held company; Celgene Corporation: Consultancy; Constellation Pharmaceuticals: Consultancy; Astra Zeneca: Consultancy; Daiichi Sankyo: Consultancy; Exo Therapeutics: Membership on an entity's Board of Directors or advisory committees; Sanofi US Services: Research Funding.

KS01.5.A Allergic airway inflammation impacts tumor take and delays experimental glioblastoma progression

Abstract BACKGROUND Numerous epidemiological studies have highlighted the protective role of immunoglobulin E-mediated allergic diseases on glioblastoma (GBM) susceptibility and prognosis. However, the mechanistic explanations behind these phenomena remain unexplored. Our objective was to set up a preclinical model and investigate the mechanisms underlying such protection to improve our understanding of the crosstalk between immune system and brain tumor development. MATERIAL AND METHODS A mouse model of allergic airway inflammation (AAI) induced by repeated nasal instillation of House Dust Mite extract was initiated before intracranial implantation of GL261 glioma cells, in both immunocompetent (C57BL/6) and immunodeficient (RAG-KO) mice. Tumor take and tumor growth were monitored by MRI. Central (microglia) and peripheral (spleen, bone marrow) immune cells were characterized by flow cytometry. The response of microglia was further assessed by RNA sequencing. Impact of candidate genes on patient survival was characterized by Cox regression analysis using data from TCGA and CGGA. RESULTS Following AAI induction in C57BL/6 mice, engraftment of GL261 cells in the brain was delayed and tumor growth rate was reduced. This correlated with an increase in survival of the mice and was accompanied by increased effector memory T-cells in the circulation. Of note, the survival benefit was lost in RAG-KO mice devoid of adaptive immunity. At the level of the brain, we observed enhanced secretion of TNFα and IL6 in microglia ex vivo. AAI induced a transcriptional reprogramming of microglia towards a pro-inflammatory-like state. We identified an allergy-related microglia gene signature that is associated with improved prognosis of glioma patients. CONCLUSION Our results demonstrate that AAI limits both tumor take and GBM progression in mice, providing a preclinical model to study the role of allergic inflammation in GBM susceptibility and prognosis, respectively. At the functional level, we identify a potentiation of microglial and adaptive anti-tumoral immunity. Further investigations are warranted to shed light on the reciprocal crosstalk between microglial reprogramming and peripheral immunity in the context of allergies and brain tumors.

Repeated Stimulation of Toll-Like Receptor 2 and Dectin-1 Induces Chronic Pancreatitis in Mice Through the Participation of Acquired Immunity

Stimulation of Toll-like receptor 3 (TLR3) induces autoimmune-mediated pancreatitis in susceptible mice, whereas stimulation of TLR4 causes nonautoimmune-mediated pancreatitis. However, the effects of TLR2 stimulation on the pancreas are unknown. We investigated the role of TLR2 stimulation on pancreatic damage by repeatedly stimulating mice with TLR2 ligands. Wild-type (WT) and interleukin 10-deficient (IL-10-knockout (KO)) mice were administered zymosan and lipoteichoic acid (LTA) intraperitoneally at various doses twice weekly for 4weeks. Syngeneic T-cell-deficient mice, B-cell-deficient mice, recombination activating gene 2-deficient (RAG2-KO) mice and RAG2-KO mice that had been reconstituted with CD4+ or CD8+ T cells isolated from WT mice were treated with zymosan similarly. Mice were killed, the severity of pancreatitis was graded histologically, and serum cytokine levels were measured. Repeated administration of zymosan induced pancreatitis dose dependently in both WT and IL-10-KO mice. Administration of LTA induced pancreatitis only in IL-10-KO mice. Adoptive transfer of splenocytes obtained from IL-10-KO mice with pancreatitis did not cause pancreatitis in recipient RAG2-KO mice. Pancreatitis was scarcely observed in RAG2-KO mice and was attenuated in T-cell-deficient and B-cell-deficient mice compared with WT mice. A single administration of zymosan significantly increased the serum level of monocyte chemoattractant protein 1 (MCP-1) in WT mice. Repeated stimulation of TLR2 and dectin-1 induced nonautoimmune-mediated pancreatitis in mice. Participation of acquired immunity seems to play an important role in the pathogenesis of pancreatitis in association with the increase in serum MCP-1 level.

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.

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.

Intracellular PD-L1 regulates DNA damage checkpoints and suppresses Chk1 and PARP inhibitor synthetic lethality

Abstract Tumor PD-L1 mediates non-canonical, intracellular signals including the DNA damage response (DDR), of unclear significance and mechanisms. We show that genetic PD-L1 depletion (PD-L1KO) destabilized the Chk2 protein, a DNA damage response (DDR) factor, resulting in ATM/Chk2 pathway defects but not ATR/Chk1 in melanoma, bladder, breast, and ovarian cancer. Consistent with ATM/Chk2 defects, PD-L1 KO led to DNA damage (γH2AX) and impaired homologous recombination (HR) (p-RPA32, BRCA1, Rad51 foci). Thus, PD-L1 KO vs control cells were significantly more sensitive to DDR inhibitors (DDRi) against ATR, Chk1, and PARP in vitro and in vivo in NSG mice. Chk2 regulation by PD-L1 was independent of PD-L1 cytoplasmic tail yet required intracellular (vs surface) PD-L1 suggesting physical PD-L1/Chk2 interaction supported by IP and imaging. PD-L1 stabilized Chk2 protein by preventing its lysosomal degradation without altering Chek2 mRNA. αPD-L1 is thought to work by protecting PD-L1 induced anti-tumor T cell suppression via PD-1, but PD-L1 DDR effects were PD-1-independent. Intracellular PD-L1 suppressed DDRi induced cGAS/STING activation by immunoblots and qRT-PCR of type 1 IFN genes. In vivo in WT mice, genetic PD-L1 depletion but not αPD-L1, sensitized highly immunotherapy resistant 4T1 breast cancer to PARPi. Strikingly, PARPi had reduced effect on PD-L1KO tumors in RAG2KO mice despite treating WT mice, indicating a strong immune component to DDRi efficacy. Our work implicates a novel role of intracellular PD-L1 in DDR and tumor immunogenicity and identifies related therapeutic vulnerabilities exposed by intracellular PD-L1 targeting. Surface vs intracellular PD-L1, and specific DDR signals could be treatment response biomarkers.

Anti-pseudomonas antibodies contribute to lung disease in cystic fibrosis.

Abstract Cystic fibrosis (CF) is an inherited lethal disease caused by mutations in CFTR. Approximately 60% of patients have homozygous F508del mutation, but their lung function is quite variable. Gene modifier studies identified I MHC II alleles are associated with the age of first Pseudomonas aeruginosa (PA) infection and lung function. Thus, we assessed serological responses to PA among CF subjects with poor lung function (FEV1&amp;lt;70%) versus good lung function (FEV1&amp;gt;90%). Immunoprecipitation-proteomics using outer membrane proteins (OMPs) from PA and human serum identified OprI as a potential important B cell antigen. We found increased anti-mucoid PA antibodies in CF and higher titers against OMPs and recombinant OprI in patients with poor lung function. Mice that were immunized with either recombinant OprI or inactivated whole PA subcutaneously developed high IgG titers in serum. Immunized mice that were challenged with mucoid PA had more weight loss and more severe lung pathology compared to unimmunized controls. There was minimal weight loss in PA immunized Rag2KO mice (impaired T and B cells), suggesting a role of adaptive immunity in the pathology. We observed more sensitive responses in FABP-hCFTR mice (CFTR knockout except GI tract) that OprI-immunized group exhibited the highest anti-OprI IgG titer and the most significant weight loss after challenge. These data suggest that one mechanism by which class II MHC is a modifier gene in CF is through antigen presentation and determining the types of anti-PA antibodies are generated in CF. Anti-PA antibodies may contribute to CF lung disease that we are conducting systems serology studies to understand which antibody functions are exacerbating disease.

Adenosine A3 agonists reverse neuropathic pain via T cell-mediated production of IL-10.

The A3 adenosine receptor (A3AR) has emerged as a therapeutic target with A3AR agonists to tackle the global challenge of neuropathic pain, and investigation into its mode of action is essential for ongoing clinical development. Immune cell A3ARs, and their activation during pathology, modulate cytokine release. Thus, the use of immune cells as a cellular substrate for the pharmacological action of A3AR agonists is enticing, but unknown. The present study discovered that Rag-KO mice lacking T and B cells, as compared with WT mice, are insensitive to the anti-allodynic effects of A3AR agonists. Similar findings were observed in interleukin-10 and interleukin-10 receptor knockout mice. Adoptive transfer of CD4+ T cells from WT mice infiltrated the dorsal root ganglion (DRG) and restored A3AR agonist-mediated anti-allodynia in Rag-KO mice. CD4+ T cells from Adora3-KO or Il10-KO mice did not. Transfer of CD4+ T cells from WT mice, but not Il10-KO mice, into Il10-KO mice or Adora3-KO mice fully reinstated the anti-allodynic effects of A3AR activation. Notably, A3AR agonism reduced DRG neuron excitability when cocultured with CD4+ T cells in an IL-10-dependent manner. A3AR action on CD4+ T cells infiltrated in the DRG decreased phosphorylation of GluN2B-containing N-methyl-D-aspartate receptors at Tyr1472, a modification associated with regulating neuronal hypersensitivity. Our findings establish that activation of A3AR on CD4+ T cells to release IL-10 is required and sufficient evidence for the use of A3AR agonists as therapeutics.

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.

Splenic erythroid progenitors decrease TNF-α production by macrophages and reduce systemic inflammation in a mouse model of T cell-induced colitis.

In inflammatory bowel disease (IBD), inflammation can occur beyond the intestine and spread systemically causing complications such as arthritis, cachexia, and anemia. Here, we determine the impact of CD45, a pan-leukocyte marker and tyrosine phosphatase, on IBD. Using a mouse model of T cell transfer colitis, CD25- CD45RBhigh CD4+ T cells were transferred into Rag1-deficient mice (RAGKO) and CD45-deficient RAGKO mice (CD45RAGKO). Weight loss and systemic wasting syndrome were delayed in CD45RAGKO mice compared to RAGKO mice, despite equivalent inflammation in the colon. CD45RAGKO mice had reduced serum levels of TNF-α, and reduced TNF-α production by splenic myeloid cells. CD45RAGKO mice also had increased numbers of erythroid progenitors in the spleen, which had previously been shown to be immunosuppressive. Adoptive transfer of these erythroid progenitors into RAGKO mice reduced their weight loss and TNF-α expression by splenic red pulp macrophages. In vitro, erythroid cells suppressed TNF-α expression in red pulp macrophages in a phagocytosis-dependent manner. These findings show a novel role for erythroid progenitors in suppressing the pro-inflammatory function of splenic macrophages and cachexia associated with IBD.

Immune Landscape of Pituitary Tumors Reveals Association Between Macrophages and Gonadotroph Tumor Invasion.

Pituitary neuroendocrine tumors (PitNETs) are frequent intracranial neoplasms that present heterogenic characteristics. Little is known about the immune cell network that exists in PitNETs and its contribution to their aggressive behavior. Here we combined flow cytometry, t-SNE analysis, and histological approaches to define the immune landscape of surgically resected PitNETs. Xenografts of rodent pituitary tumor cells and resected PitNETs were performed in Rag2KO mice, in combination with in vitro analysis aimed at dissecting the role of pituitary tumor-cells in monocyte recruitment. We report that gonadotroph PitNETs present an increased CD68+ macrophage signature compared to somatotroph, lactotroph, and corticotroph PitNETs. Transcriptomic and histological characterizations confirmed gonadotroph infiltrating macrophages expressed CD163, MRC-1, ARG1, and CSF1R M2 macrophage markers. Use of growth hormone (GH)3/GH4 somatotroph and LβT2/αT3.1 gonadotroph cells drove THP1 macrophage migration through respective expression of CCL5 or CSF1. Although both LβT2 and GH3 cells recruited F4/80 macrophages following their engraftment in mice, only LβT2 gonadotroph cells showed a capacity for M2-like polarization. Similar observations were performed on patient-derived xenografts from somatotroph and gonadotroph tumors. Analysis of clinical data further demonstrated a significant correlation between the percentage of CD68+ and CD163+ infiltrating macrophages and the invasive character of gonadotroph tumors. Gonadotroph tumor drive the recruitment of macrophages and their subsequent polarization to an M2-like phenotype. More importantly, the association between infiltrating CD68+/CD163+ macrophages and the invasiveness of gonadotroph tumors points to macrophage-targeted immunotherapies being a potent strategy to limit the progression of gonadotroph PitNETs.

Spontaneous onset of TNFα-triggered colonic inflammation depends on functional T lymphocytes, S100A8/A9 alarmins, and MHC H-2 haplotype.

Recently, we established a doxycycline-inducible human tumor necrosis factor alpha (TNFα)-transgenic mouse line, ihTNFtg. Non-induced young and elderly mice showed low but constitutive expression of hTNFα due to promoter leakiness. The persistently present hTNFα stimulated endogenous pro-inflammatory mouse mS100A8/A9 alarmins. Secreted mS100A8/A9 in turn induced the expression and release of mouse mTNFα. The continuous upregulation of pro-inflammatory mTNFα and mS100A8/A9 proteins, due to their mutual expression dependency, gradually led to increased levels in colon tissue and blood. This finally exceeded the threshold levels tolerated by the healthy organism, leading to the onset of intestinal inflammation. Here, recombinant hTNFα functioned as an initial trigger for the development of chronic inflammation. Crossing ihTNFtg mice with S100A9KO mice lacking active S100A8/A9 alarmins or with Rag1KO mice lacking T and B lymphocytes completely abrogated the development of colonic inflammation, despite the still leaky hTNFα promoter. Furthermore, both the intensity of the immune response and the strength of immunosuppressive Treg induction was found to depend on the major histocompatibility complex (MHC) genetic composition. In summary, the onset of intestinal inflammation in elderly mice depends on at least four factors that have to be present simultaneously: TNFα upregulation, S100A8/A9 protein expression, functional T lymphocytes and genetic composition, with the MHC haplotype being of central importance. Only joint action of these factors leads to chronic intestinal inflammation, while absence of any of these determinants abrogates the development of the autoimmune disorder. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

2056-P: In Vivo Human Islet Compensation and Dysfunction to Diet-Induced Obesity: A Phase 0 Preclinical Model

A translational discrepancy exists between animal models used for diabetes research and diabetes in man, thus human relevant models must become a priority. We showed that collectively human islet grafts from 26 donors statistically significantly adapted their function and endocrine and β-cell mass to HFD (high fat diet) in vivo in 233 RAG2KO mice. Human islet compensation occurs at 6 weeks and metabolic dysfunction upon chronic ≥10 weeks HFD. Herein, gene expression studies (GeneChip® Human Gene 2.0 ST Arrays) identified molecular pathways involved. Indeed, human islets compensate to HFD by upregulating hormones, transcription factors, antioxidants, and unfolded protein response genes, and then become dysfunctional at 10 wks -by downregulating these genes. Herein we show Krebs cycle and Mitochondrial electron transport chain was targeted with a transient increase at 6wk and then a drop-in expression at 10 wks; whereas uncouplinggene expression UCP2 and 3 increased. Glycolysis remained upregulated. Explanations for expanded islet and beta mass in HFD (vs. CTL) may be found in cell cycle heatmap. Lastly, a marker of activated stellate cells TAGLN, and matrix genes rose during islet dysfunction (fibrosis?). Conclusion: This human-based model system progressively depicts defects identified in human pre and T2DM that could serve as a “phase 0” in vivo model to test new targets and develop effective therapies in man. Disclosure J.A. Kerr-Conte: None. J. Thevenet: None. G. Pasquetti: None. P. Petit: None. C. Clabaut: None. V. Gmyr: None. C. Bonner: None. S. Gargani: None. F. Pattou: None. Funding JDRF; Programme d’investissements d’avenir; LabEx; European Genomic Institute for Diabetes (ANR-10-LABX-46)

T cells modulate IL-4 expression by eosinophil recruitment within decellularized scaffolds to repair nerve defects

Decellularized nerve, or acellular nerve allografts (ANAs), are an increasingly used alternative to nerve autografts to repair nerve gaps to facilitate regeneration. The adaptive immune system, specifically T cells, plays a role in promoting regeneration upon these ANA scaffolds. However, how T cells promote regeneration across ANAs is not clear. Here, we show that T cells accumulate within ANAs repairing nerve gaps resulting in regulation of cytokine expression within the ANA environment. This in turn ultimately leads to robust nerve regeneration and functional recovery. Nerve regeneration across ANAs and functional recovery in Rag1KO mice was limited compared to wild-type (WT) mice. Prior to appreciable nerve regeneration, ANAs from Rag1KO mice contained fewer eosinophils and reduced IL-4 expression compared to ANAs from WT mice. During this period, both T cells and eosinophils regulated IL-4 expression within ANAs. Eosinophils represented the majority of IL-4 expressing cells within ANAs, while T cells regulated IL-4 expression. Finally, an essential role for IL-4 during nerve regeneration across ANAs was confirmed as nerves repaired using ANAs had reduced regeneration in IL-4 KO mice compared to WT mice. Our data demonstrate T cells regulate the expression of IL-4 within the ANA environment via their effects on eosinophils. Statement of significanceThe immune system has been emerging as a critical component for tissue regeneration, especially when regeneration is supported upon biomaterials. The role of T cells, and their roles in the regeneration of nerve repaired with biomaterials, is still unclear. We demonstrated that when nerves are repaired with decellularized nerve scaffolds, T cells contribute to regeneration by regulating cytokines. We focused on their regulation of cytokine IL-4. Unexpectedly, T cells do not produce IL-4, but instead regulate IL-4 by recruiting eosinophils, which are major cellular sources of IL-4 within these scaffolds. Thus, our work demonstrated how IL-4 is regulated in a model biomaterial, and has implications for improving the design of biomaterials and understanding immune responses to biomaterials.

Efficacy and safety of anti-CD45–saporin as conditioning agent for RAG deficiency

Background:Mutations in the recombinase-activating genes cause severe immunodeficiency, with a spectrum of phenotypes ranging from severe combined immunodeficiency to immune dysregulation. Hematopoietic stem cell transplantation is the only curative option, but a high risk of graft failure and poor immune reconstitution have been observed in the absence of myeloablation.Objectives:Our aim was to improve multilineage engraftment; we tested nongenotoxic conditioning with anti-CD45 mAbs conjugated with saporin CD45 (CD45-SAP).Methods:Rag1-KO and Rag1-F971L mice, which represent models of severe combined immune deficiency and combined immune deficiency with immune dysregulation, respectively, were conditioned with CD45-SAP, CD45-SAP plus 2 Gy of total body irradiation (TBI), 2 Gy of TBI, 8 Gy of TBI, or no conditioning and treated by using transplantation with lineage-negative bone marrow cells from wild-type mice. Flow cytometry and immunohistochemistry were used to assess engraftment and immune reconstitution. Antibody responses to 2,4,6-trinitrophenyl–conjugated keyhole limpet hemocyanin were measured by ELISA, and presence of autoantibody was detected by microarray.Results:Conditioning with CD45-SAP enabled high levels of multilineage engraftment in both Rag1 mutant models, allowed overcoming of B- and T-cell differentiation blocks and thymic epithelial cell defects, and induced robust cellular and humoral immunity in the periphery.Conclusions:Conditioning with CD45-SAP allows multilineage engraftment and robust immune reconstitution in mice with either null or hypomorphic Rag mutations while preserving thymic epithelial cell homeostasis.