GITR Ligation Improves Anti-PD1-Mediated Restoration of Human MMR-Proficient Colorectal Carcinoma Tumor-Derived T Cells.

BACKGROUND & AIMS: In contrast to mismatch repair deficient colorectal carcinoma (CRC), MMR proficient (pMMR) CRC does not respond to immune checkpoint blockade. We studied immune checkpoint stimulation via glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) on ex vivo functionality of human tumor-infiltrating lymphocytes (TIL) isolated from pMMR primary CRC and liver metastases (CRLM). METHODS: Using lymphocytes from resected tumor, adjacent tissues, and peripheral blood mononuclear cells (PBMC) of 132 pMMR primary CRC or CRLM patients, we determined GITR expression and the in vitro T-cell agonistic activity of recombinant GITR ligation. RESULTS: Here, we show that GITR was overexpressed on TIL when compared with other stimulatory immune checkpoints (4-1BB, OX40). Its expression was enhanced in TIL compared with PBMC and adjacent tissues. Among CD4+ TIL, GITR expression was primarily expressed by CD45RA- FoxP3hi activated regulatory T cells. Within CD8+ TIL, GITR was predominantly expressed on functionally exhausted and putative tumor-reactive CD103+ CD39+ TIL. Strikingly, recombinant GITRL reinvigorated ex vivo TIL responses by significantly enhancing CD4+ and CD8+ TIL numbers. Dual treatment with GITRL and nivolumab (anti-PD1) enhanced CD8+ TIL expansion compared with GITRL monotherapy. Moreover, GITRL/anti-PD1 dual therapy further improved anti-PD1-mediated reinvigoration of interferon gamma secretion by exhausted CD8 TIL from primary CRC. CONCLUSIONS: GITR is overexpressed on CD4+ and CD8+ TIL from pMMR CRC and CRLM. Agonistic targeting of GITR enhances ex vivo human TIL functionality and may therefore be a promising approach for novel monotherapy or combined immunotherapies in primary pMRR CRC and CRLM.

GITR/GITRL reverse signalling modulates the proliferation of hepatic progenitor cells by recruiting ANXA2 to phosphorylate ERK1/2 and Akt.

Hepatic stem/progenitor cells are the major cell compartment for tissue repair when hepatocyte proliferation is compromised in chronic liver diseases, but the expansion of these cells increases the risk of carcinogenesis. Therefore, it is essential to explore the pathways restricting their expansion and abnormal transformation. The ligand of glucocorticoid-induced tumour necrosis factor receptor (GITRL) showed the most highly increased expression in hepatic progenitor cells treated with transforming growth factor (TGF)-ß1. If overexpressed by hepatic progenitor cells, GITRL stimulated cell proliferation by activating the epithelial-mesenchymal transition pathway and enhancing ERK1/2 and Akt phosphorylation via GITRL binding to ANXA2. However, GITR, the specific GITRL receptor, suppressed the epithelial-mesenchymal transition pathway of GITRL-expressing cells and decreased their growth by dissociating ANXA2 from GITRL and reducing downstream ERK1/2 and Akt phosphorylation. This study identifies GITR/GITRL reverse signalling as a cross-interaction pathway between immune cells and hepatic stem/progenitor cells that restricts the expansion of hepatic stem/progenitor cells and reduces the possibility of carcinogenesis.

TNF Receptor-Associated Factor 5 Limits IL-27 Receptor Signaling in CD4+ T Lymphocytes.

TNF receptor-associated factor 5 (TRAF5) restrains early signaling activity of the IL-6 receptor in naive CD4+ T cells by interacting with the shared gp130 chain, although TRAF5 was initially discovered as a cytoplasmic adaptor protein to activate signaling mediated by TNF receptor family molecules. This leads to the question of whether TRAF5 limits signaling via the receptor for IL-27, which is composed of gp130 and WSX-1. The aim of this study is to clarify the role of TRAF5 in IL-27 receptor signaling and to understand the differential role of TRAF5 on cytokine receptor signaling. We found that Traf5 -/- CD4+ T cells displayed significantly higher levels of phosphorylated STAT1 and STAT-regulated genes Socs3 and Tbx21, as early as 1 h after IL-27 exposure when compared with Traf5 +/+ CD4+ T cells. Upon IL-27 and TCR signals, the Traf5 deficiency significantly increased the induction of IL-10 and promoted the proliferation of CD4+ T cells. Traf5 -/- mice injected with IL-27 displayed significantly enhanced delayed-type hypersensitivity responses, demonstrating that TRAF5 works as a negative regulator for IL-27 receptor signaling. In contrast, IL-2 and proliferation mediated by glucocorticoid-induced TNF receptor-related protein (GITR) and TCR signals were significantly decreased in Traf5 -/- CD4+ T cells, confirming that TRAF5 works as a positive regulator for cosignaling via GITR. Collectively, our results demonstrate that TRAF5 reciprocally controls signals mediated by the IL-27 receptor and GITR in CD4+ T cells and suggest that the regulatory activity of TRAF5 in gp130 is distinct from that in TNF receptor family molecules in a T cell.

Activated but functionally impaired memory Tregs are expanded in slow progressors to type 1 diabetes.

AIMS/HYPOTHESIS: Slow progressors to type 1 diabetes are individuals positive for multiple pancreatic islet autoantibodies who have remained diabetes-free for at least 10 years; regulation of the autoimmune response is understudied in this group. Here, we profile CD4+ regulatory T cells (Tregs) in a small but well-characterised cohort of extreme slow progressors with a median age 43 (range 31-72 years), followed up for 18-32 years. METHODS: Peripheral blood samples were obtained from slow progressors (n = 8), age- and sex-matched to healthy donors. One participant in this study was identified with a raised HbA1c at the time of assessment and subsequently diagnosed with diabetes; this donor was individually evaluated in the analysis of the data. Peripheral blood mononuclear cells (PBMCs) were isolated, and to assess frequency, phenotype and function of Tregs in donors, multi-parameter flow cytometry and T cell suppression assays were performed. Unsupervised clustering analysis, using FlowSOM and CITRUS (cluster identification, characterization, and regression), was used to evaluate Treg phenotypes. RESULTS: Unsupervised clustering on memory CD4+ T cells from slow progressors showed an increased frequency of activated memory CD4+ Tregs, associated with increased expression of glucocorticoid-induced TNFR-related protein (GITR), compared with matched healthy donors. One participant with a raised HbA1c at the time of assessment had a different Treg profile compared with both slow progressors and matched controls. Functional assays demonstrated that Treg-mediated suppression of CD4+ effector T cells from slow progressors was significantly impaired, compared with healthy donors. However, effector CD4+ T cells from slow progressors were more responsive to Treg suppression compared with healthy donors, demonstrated by increased suppression of CD25 and CD134 expression on effector CD4+ T cells. CONCLUSIONS/INTERPRETATIONS: We conclude that activated memory CD4+ Tregs from slow progressors are expanded and enriched for GITR expression, highlighting the need for further study of Treg heterogeneity in individuals at risk of developing type 1 diabetes.

Cyclophosphamide enhances the antitumor potency of GITR engagement by increasing oligoclonal cytotoxic T cell fitness.

Only a subset of cancer patients responds to checkpoint blockade inhibition in the clinic. Strategies to overcome resistance are promising areas of investigation. Targeting glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) has shown efficacy in preclinical models, but GITR engagement is ineffective in controlling advanced, poorly immunogenic tumors, such as B16 melanoma, and has not yielded benefit in clinical trials. The alkylating agent cyclophosphamide (CTX) depletes regulatory T cells (Tregs), expands tumor-specific effector T cells (Teffs) via homeostatic proliferation, and induces immunogenic cell death. GITR agonism has an inhibitory effect on Tregs and activates Teffs. We therefore hypothesized that CTX and GITR agonism would promote effective antitumor immunity. Here we show that the combination of CTX and GITR agonism controlled tumor growth in clinically relevant mouse models. Mechanistically, we show that the combination therapy caused tumor cell death, clonal expansion of highly active CD8+ T cells, and depletion of Tregs by activation-induced cell death. Control of tumor growth was associated with the presence of an expanded population of highly activated, tumor-infiltrating, oligoclonal CD8+ T cells that led to a diminished TCR repertoire. Our studies show that the combination of CTX and GITR agonism is a rational chemoimmunotherapeutic approach that warrants further clinical investigation.

GITR Promotes the Polarization of TFH-Like Cells in Helicobacter pylori-Positive Gastritis.

Gastric CD4+T cells contribute to Helicobacter pylori (H. pylori)-induced gastritis by amplifying mucosal inflammation and exacerbating mucosal injuries. However, the pathogenic CD4+ T cell subset involved in gastritis and the potential regulators are still unclear. Here we identified an IL-21-producing gastric CD4+T cell subset, which exhibited tissue-resident CXCR5-BTLA-PD-1hi TFH-like phenotype in H. pylori-positive gastritis patients. Meanwhile, we identified glucocorticoid-induced tumor necrosis factor receptor (GITR) as an important regulator to facilitate IL-21 production by CD4+T cells and accelerate mucosal inflammation in gastritis patients with H. pylori infection. Moreover, GITR expression was increased in gastric CD4+T cells of gastritis patients compared to healthy controls, along with the upregulated expression of its ligand GITRL in mucosal macrophages (Mϕ) of gastritis patients. Further observations showed that the activation of GITR/GITRL signal promoted the IL-21 production of CD4+T cells via the STAT3 pathway. Besides this, IL-21 from CD4+T cells induced the proliferation of B cell and promoted the production of inflammatory cytokines IL-1ß and IL-6 and chemokines MIP-3α and CCL-25 as well as matrix metalloproteinase (MMP)-3 and MMP-9 by human gastric epithelial cells, suggesting the facilitating effect of IL-21-producing CD4+T cells on mucosal inflammation and injuries. Taking these data together, we revealed that GITR/GITRL signal promoted the polarization of mucosal IL-21-producing CD4+T cells in H. pylori-positive gastritis, which may provide therapeutic strategies for the clinical treatment of H. pylori-induced gastritis.

Atypical TNF-TNFR superfamily binding interface in the GITR-GITRL complex for T cell activation.

Glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) is a critical regulatory molecule in modulation of T cell immune responses. Here we report the mouse GITR (mGITR) and mGITR ligand (mGITRL) complex structure and find that the binding interface of mGITR and mGITRL is distinct from the typical tumor necrosis factor superfamily (TNFSF)/TNF receptor superfamily (TNFRSF) members. mGITR binds to its ligand with a single domain, whereas the binding interface on mGITRL is located on the side, which is distal from conserved binding sites of TNFSF molecules. Mutational analysis reveals that the binding interface of GITR/GITRL in humans is conserved with that in the mouse. Substitution of key interacting D93-I94-V95 (DIV) in mGITR with the corresponding K93-F94-S95 (KFS) in human GITR enables cross-recognition with human GITRL and cross-activation of receptor signaling. The findings of this study substantially expand our understanding of the interaction of TNFSF/TNFRSF superfamily molecules and can benefit the future design of biologics by targeting GITR/GITRL.

Multiple mechanisms for TRAF3-mediated regulation of the T cell costimulatory receptor GITR.

Tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3) plays context-specific roles in multiple receptor-mediated signaling pathways in different cell types. Mice lacking TRAF3 in T cells display defective T-cell-mediated immune responses to immunization and infection and demonstrate defective early signaling via the TCR complex. However, the role of TRAF3 in the function of GITR/TNFRSF18, an important costimulatory member of the TNFR superfamily, is unclear. Here we investigated the impact of T cell TRAF3 status on both GITR expression and activation of specific kinases in the GITR signaling pathway in T cells. Our results indicate that TRAF3 negatively regulates GITR functions in several ways. First, expression of GITR protein was elevated in TRAF3-deficient T cells, resulting from both transcriptional and posttranslational regulation that led to greater GITR transcript levels, as well as enhanced GITR protein stability. TRAF3 associated with T cell GITR in a manner dependent upon GITR ligation. TRAF3 also inhibited several events of the GITR mediated early signaling cascade, in a manner independent of recruitment of phosphatases, a mechanism by which TRAF3 inhibits signaling through several other cytokine receptors. These results add new information to our understanding of GITR signaling and function in T cells, which is relevant to the potential use of GITR to enhance immune therapies.

Blocking the GITR-GITRL pathway to overcome resistance to therapy in sarcomatoid malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive neoplasm originating from the pleura. Non-epithelioid (biphasic and sarcomatoid) MPM are particularly resistant to therapy. We investigated the role of the GITR-GITRL pathway in mediating the resistance to therapy. We found that GITR and GITRL expressions were higher in the sarcomatoid cell line (CRL5946) than in non-sarcomatoid cell lines (CRL5915 and CRL5820), and that cisplatin and Cs-137 irradiation increased GITR and GITRL expressions on tumor cells. Transcriptome analysis demonstrated that the GITR-GITRL pathway was promoting tumor growth and inhibiting cell apoptosis. Furthermore, GITR+ and GITRL+ cells demonstrated increased spheroid formation in vitro and in vivo. Using patient derived xenografts (PDXs), we demonstrated that anti-GITR neutralizing antibodies attenuated tumor growth in sarcomatoid PDX mice. Tumor immunostaining demonstrated higher levels of GITR and GITRL expressions in non-epithelioid compared to epithelioid tumors. Among 73 patients uniformly treated with accelerated radiation therapy followed by surgery, the intensity of GITR expression after radiation negatively correlated with survival in non-epithelioid MPM patients. In conclusion, the GITR-GITRL pathway is an important mechanism of autocrine proliferation in sarcomatoid mesothelioma, associated with tumor stemness and resistance to therapy. Blocking the GITR-GITRL pathway could be a new therapeutic target for non-epithelioid mesothelioma.

Structures of mouse and human GITR-GITRL complexes reveal unique TNF superfamily interactions.

Glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) and GITR ligand (GITRL) are members of the tumor necrosis superfamily that play a role in immune cell signaling, activation, and survival. GITR is a therapeutic target for directly activating effector CD4 and CD8 T cells, or depleting GITR-expressing regulatory T cells (Tregs), thereby promoting anti-tumor immune responses. GITR activation through its native ligand is important for understanding immune signaling, but GITR structure has not been reported. Here we present structures of human and mouse GITR receptors bound to their cognate ligands. Both species share a receptor-ligand interface and receptor-receptor interface; the unique C-terminal receptor-receptor enables higher order structures on the membrane. Human GITR-GITRL has potential to form a hexameric network of membrane complexes, while murine GITR-GITRL complex forms a linear chain due to dimeric interactions. Mutations at the receptor-receptor interface in human GITR reduce cell signaling with in vitro ligand binding assays and minimize higher order membrane structures when bound by fluorescently labeled ligand in cell imaging experiments.

Platelet-expressed immune checkpoint regulator GITRL in breast cancer.

Owing to their key role in several diseases including cancer, activating and inhibitory immune checkpoint molecules are increasingly exploited as targets for immunotherapy. Recently, we demonstrated that platelets, which largely influence tumor progression and immune evasion, functionally express the ligand of the checkpoint molecule GITR. This immunoreceptor modulates effector functions of T cells and NK cells with its function varying dependent on cellular context and activation state. Here, we provide a comparative analysis of platelet-derived GITRL (pGITRL) in breast cancer patients and healthy volunteers. The levels of pGITRL were found to be higher on platelets derived from cancer patients and appeared to be specifically regulated during tumor progression as exemplified by several clinical parameters including tumor stage/grade, the occurrence of metastases and tumor proliferation (Ki67) index. In addition, we report that pGITRL is upregulated during platelet maturation and particularly induced upon exposure to tumor-derived soluble factors. Our data indicate that platelets modulate the GITR/GITRL immune checkpoint in the context of malignant disease and provide a rationale to further study the GITR/GITRL axis for exploitation for immunotherapeutic intervention in cancer patients.

Characterization of an Expanded IL-10-Producing-Suppressive T Cell Population Associated with Immune Tolerance.

An increase in the number of glucocorticoid-induced tumor necrosis factor receptor-family related gene/protein (GITR)+CD25- (or fork-head box protein 3: Foxp3-) CD4+ T cells, after treating a mouse model of arthritis with fingolimod (FTY720), and a pathogenic antigen may play a key role in the establishment of immune tolerance. In this study, we characterized a specific expanded T cell subset in this population. Mice with glucose-6-phosphate isomerase peptide (GPI325-339)-induced arthritis were treated with FTY720 (1 mg/kg, per os) and GPI325-339 (10 µg/mouse, intravenously) for five days, starting from the onset of symptoms. The expanded GITR+CD25- (or Foxp3-) CD4+ T cell population and its cytokine production were examined using flow cytometry. Furthermore, time-dependent changes in T-bet and/or early growth response gene 2 (Egr-2) expression in this T cell subset were examined. The density of T cell immunoreceptors with immunoglobulin (Ig) and immunoreceptor tyrosine-based inhibition motif domains (TIGIT)+CD39+ cell subset in the GITR+Foxp3-CD4+ T cell population was significantly increased only in the combined treatment group, compared to that in the untreated and single-treatment groups. In the TIGIT+CD39+GITR+Foxp3-CD4+ T cell population, T-bet+Egr-2+/T-bet+Egr-2- cell ratio increased in the latter stage of the treatment. Furthermore, this T cell subset, which corresponded to a T helper 1 (Th1) response, produced high levels of both interleukin (IL)-10 and interferon (IFN)-γ. In conclusion, expanded TIGIT+CD39+GITR+Foxp3-CD4+ T cells shifted from an effector Th1 to IL-10-producing-suppressor T cell phenotype, which may promote an immune-tolerant state.

Adaptive Immune Response Signaling Is Suppressed in Ly6Chigh Monocyte but Upregulated in Monocyte Subsets of ApoE-/- Mice - Functional Implication in Atherosclerosis.

Rationale: Inflammatory monocyte (MC) subset differentiation is a major feature in tissue inflammatory and atherosclerosis. The underlying molecular mechanism remains unclear. Objective: This study aims to explore molecule targets and signaling which determinate immunological features in MC subsets. Methods and Results: Blood Ly6Chigh and Ly6Clow MC subsets from control and ApoE-/- mice were isolated by flow cytometry sorting and subjected for bulk high-throughput RNA-sequencing. Intensive bioinformatic studies were performed by analyzing transcriptome through four pairs of comparisons: A) Ly6Chigh vs Ly6Clow in control mice; B) Ly6Chigh vs Ly6Clow in ApoE-/- mice; C) ApoE-/- Ly6Chigh vs control Ly6Chigh MC; D) ApoE-/- Ly6Clow vs control Ly6Clow MC. A total of 80 canonical pathways and 16 enriched pathways were recognized by top-down analysis using IPA and GSEA software, and further used for overlapping analysis. Immunological features and signaling were assessed on four selected functional groups, including MHCII, immune checkpoint, cytokine, and transcription factor (TF). Among the total 14578 significantly differentially expressed (SDE) genes identified though above four comparison, 1051 TF and 348 immunological genes were discovered. SDE immunological genes were matched with corresponding upstream SDE TF by IPA upstream analysis. Fourteen potential transcriptional axes were recognized to modulate immunological features in the Ly6C MC subset. Based on an intensive literature search, we found that the identified SDE immune checkpoint genes in Ly6Chigh MC are associated with pro-inflammatory/atherogenic balance function. Immune checkpoint genes GITR, CTLA4, and CD96 were upregulated in Ly6Clow MC from all mice and presented anti-inflammatory/atherogenic features. Six cytokine genes, including Ccl2, Tnfsf14, Il1rn, Cxcl10, Ccl9, and Cxcl2, were upregulated in Ly6Chigh MC from all mice and associated with pro-inflammatory/atherogenic feature. Cytokine receptor gene Il12rb2, Il1r1, Il27ra, Il5ra, Ngfr, Ccr7, and Cxcr5 were upregulated in Ly6Clow MC from all mice and presented anti-inflammatory/atherogenic features. MHCII genes (H2-Oa, H2-DMb2, H2-Ob, H2-Eb2, H2-Eb1, H2-Aa, and Cd74) were elevated in Ly6Clow MC from all mice. ApoE-/- augmented pro-atherogenic/inflammatory and antigen-presenting cells (APC) feature in both subsets due to elevated expression of cytokine genes (Cxcl11, Cntf, Il24, Xcl, Ccr5, Mpl, and Acvr2a) and MHCII gene (H2-Aa and H2-Ea-ps). Finally, we modeled immunological gene expression changes and functional implications in MC differentiation and adaptive immune response for MC subsets from control and ApoE-/- mice. Conclusions: Ly6Chigh MC presented pro-inflammatory/atherogenic features and lower APC potential. Ly6Clow MC displayed anti-inflammatory/atherogenic features and higher APC potential. ApoE-/- confers upon both subsets with augmented pro-atherogenic/inflammatory function and APC potential.

Sectm1a Facilitates Protection against Inflammation-Induced Organ Damage through Promoting TRM Self-Renewal.

Tissue-resident macrophages (TRMs) are sentinel cells for maintaining tissue homeostasis and organ function. In this study, we discovered that lipopolysaccharide (LPS) administration dramatically reduced TRM populations and suppressed their self-renewal capacities in multiple organs. Using loss- and gain-of-function approaches, we define Sectm1a as a novel regulator of TRM self-renewal. Specifically, at the earlier stage of endotoxemia, Sectm1a deficiency exaggerated acute inflammation-induced reduction of TRM numbers in multiple organs by suppressing their proliferation, which was associated with more infiltrations of inflammatory monocytes/neutrophils and more serious organ damage. By contrast, administration of recombinant Sectm1a enhanced TRM populations and improved animal survival upon endotoxin challenge. Mechanistically, we identified that Sectm1a-induced upregulation in the self-renewal capacity of TRM is dependent on GITR-activated T helper cell expansion and cytokine production. Meanwhile, we found that TRMs may play an important role in protecting local vascular integrity during endotoxemia. Our study demonstrates that Sectm1a contributes to stabling TRM populations through maintaining their self-renewal capacities, which benefits the host immune response to acute inflammation. Therefore, Sectm1a may serve as a new therapeutic agent for the treatment of inflammatory diseases.

Experimental Medicine Study to Measure Immune Checkpoint Receptors PD-1 and GITR Turnover Rates In Vivo in Humans.

Development of monoclonal antibodies (mAbs) targeting immune-checkpoint receptors (IMRs) for the treatment of cancer is one of the most active areas of investment in the biopharmaceutical industry. A key decision in the clinical development of anti-IMR mAbs is dose selection. Dose selection can be challenging because the traditional oncology paradigm of administering the maximum tolerated dose is not applicable to anti-IMR mAbs. Instead, dose selection should be informed by the pharmacology of immune signaling. Engaging an IMR is a key initial step to triggering pharmacologic effects, and turnover (i.e., the rate of protein synthesis) of the IMR is a key property to determining the dose level needed to engage the IMR. Here, we applied the stable isotope labeling mass spectrometry technique using 13 C6 -leucine to measure the in vivo turnover rates of IMRs in humans. The 13 C6 -leucine was administered to 10 study participants over 15 hours to measure 13 C6 -leucine enrichment kinetics in 2 IMR targets that have been clinically pursued in oncology: GITR and PD-1. We report the first measurements of GITR and PD-1 median half-lives associated with turnover to be 55.6 and ≥ 49.5 hours, respectively. The approach outlined here can be applied to other IMRs and, more generally, to protein targets.

Proteomic profiles of unilateral cryptorchidism in pigs at different ages using MALDI-TOF mass spectrometry and in-gel digestion coupled with mass spectrometry (GeLC-MS/MS) approaches.

BACKGROUND: Cryptorchidism is a condition that occurs when one or both testes fail to descend into the scrotum. It is a common congenital disorder, causing economic loss in pig production. However, there have been only limited studies of differential protein expression profiles in undescended testes (UDTs) in the abdomen and descended testes (DTs) in cryptorchid pigs, especially at the peptidome and proteome levels. The present study aimed to analyze the peptidome of UDTs and DTs in unilateral cryptorchid pigs aged 1-2, 6, 15 and 20 weeks and in normal testes of healthy pigs aged 1-2 and 12 weeks, using peptide mass fingerprinting and three-dimensional principal component analysis (3D-PCA) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and to identify potential protein candidates, using in-gel digestion coupled with mass spectrometry (GeLC-MS/MS). Western blot analysis was used to verify protein expression. Protein sequence was affirmed by liquid chromatography-tandem mass spectrometry. RESULTS: A PCA plot showed a discrete cluster for each sample group. Peptide mass fingerprints (PMFs) demonstrated unique peptide fragments in UDTs at different ages. A number of markedly expressed proteins from GeLC-MS/MS were identified, including the multifunctional tumor necrosis factor receptor superfamily member 18 (TNFRSF18), in DTs at 1-2 and 6 weeks and in UDTs at 15 and 20 weeks of age. Using western blot analysis, high expression of TNFRSF18 was observed in the UDTs at 15 weeks. Using the STITCH database, this protein was found to be related to apoptosis, corresponding to the previous report in the UDTs at the same age. CONCLUSIONS: The present study revealed the specific PMFs and clusters for porcine cryptorchidism, and a novel protein, TNFRSF18, associated with the disease mechanism. These results could provide further insights into the pathogenesis of the disease.

GITR controls intestinal inflammation by suppressing IL-15-dependent NK cell activity.

Glucocorticoid-induced TNFR family related gene (GITR) is a member of the TNFR superfamily that is expressed on cells of the immune system. Although the protective and pathogenic roles of GITR in T cell immunity are well characterized, the role of GITR in innate immunity in the intestinal tissues has not been well clarified. In this study, using a dextran sulfate sodium (DSS)-induced colitis model in mice, we found that GITR-deficiency rendered mice more susceptible to acute intestinal inflammation and that a significantly higher number of activated natural killer (NK) cells was accumulated in the colonic lamina propria of Gitr-/- mice as compared to wild-type mice. Additionally, Rag2-/- Gitr-/- mice, which lack T cells but have NK cells, also displayed more severe colonic inflammation than Rag2-/- mice. In contrast, an anti-GITR agonistic antibody significantly alleviated colitis in Rag2-/- mice. Engagement of GITR inhibited IL-15-mediated activating signaling events in NK cells, which include cell activation and proliferation, and production of cytokines and cytotoxic granules. Taken together, our results provide the first evidence that GITR negatively controls intestinal inflammation through NK cell functions.

Two dimensional proteomic analysis of serum shows immunological proteins exclusively expressed in sulfur mustard exposed patients with long term pulmonary complications.

BACKGROUND: Despite more than 30 years after utilization of sulfur mustard or bis (2-chloroethyl) sulfide (SM) by Iraqi troops against Iranian military members and civilians, there are a lot of reported delayed complications for the exposed people. Nonetheless, the molecular mechanism of action from this chemical warfare agent is not recognized yet. MATERIAL AND METHOD: In this study, we employed two dimensional gel electrophoresis (2DE) technique to investigate the serum proteins from chemical exposed people compared to non-exposed individuals to provide an inside into molecular mechanism of this chemical agent. Each group was divided into two subgroups including individuals with, and without respiratory complications. For each group, 10 individuals were included after informed consent. RESULT: The results showed protein spots, which were exclusively/mainly expressed in chemical exposed patients with complications, including T cell receptor alpha, and hematopoietic cell signal transducer. Also there were protein spots that were expressed only in all exposed groups (with and without complications). On the other hand, we could identify protein spots that were exclusively expressed/altered only in non-exposed group with complications including Pre T-cell antigen receptor, CD40 ligand, and multidrug and toxin extrusion proteins. CONCLUSION: Our investigation could result in identification of proteins that are associated to chemical exposure, as well as those specific for respiratory complications irrespective of chemical exposure. These candidate proteins can be used as biomarker, as well as a base for understanding the molecular mechanism of this chemical agent.

Identification of a Novel Antisepsis Pathway: Sectm1a Enhances Macrophage Phagocytosis of Bacteria through Activating GITR.

The inability to effectively control invading bacteria or other pathogens is a major cause of multiple organ dysfunction and death in sepsis. As the first-line defense of the immune system, macrophages play a crucial role in the removal of pathogens during sepsis. In this study, we define secreted and transmembrane 1A (Sectm1a) as a novel ligand of glucocorticoid-induced TNFR (GITR) that greatly boosts macrophage phagocytosis and bactericidal capacity. Using a global Sectm1a knockout (KO) mouse model, we observed that Sectm1a deficiency significantly suppressed phagocytosis and bactericidal activity in both recruited macrophages and tissue-resident macrophages, which consequently aggravated bacterial burden in the blood and multiple organs and further increased systemic inflammation, leading to multiple organ injury and increased mortality during polymicrobial sepsis. By contrast, treatment of septic mice with recombinant Sectm1a protein (rSectm1a) not only promoted macrophage phagocytosis and bactericidal activity but also significantly improved survival outcome. Mechanistically, we identified that Sectm1a could bind to GITR in the surface of macrophages and thereby activate its downstream PI3K-Akt pathway. Accordingly, rSectm1a-mediated phagocytosis and bacterial killing were abolished in macrophages by either KO of GITR or pharmacological inhibition of the PI3K-Akt pathway. In addition, rSectm1a-induced therapeutic effects on sepsis injury were negated in GITR KO mice. Taken together, these results uncover that Sectm1a may represent a novel target for drug development to control bacterial dissemination during sepsis or other infectious diseases.

Evaluation of 1,25(OH)2D3 Effects on FOXP3, ROR-γt, GITR, and CTLA-4 Gene Expression in the PBMCs of Vitamin D-Deficient Women with Unexplained Recurrent Pregnancy Loss (URPL).

Background: Vitamin D insufficiency and deficiency can be associated with adverse effects on fetus and pregnancy outcomes. This study aimed at evaluating the effect of 1,25VitD3 on specific transcription factor and markers of Tregs and T helper 17 (Th17) cells in peripheral blood mononuclear cells (PBMCs) of women with unexplained recurrent pregnancy loss (URPL) as a case group and PBMCs of healthy women as a control group. Methods: Samples from 20 non-pregnant patients with a history of URPL were compared to 20 normal non-pregnant women. PBMCs were divided into three wells for each subject in the presence of 1,25VitD3 (50 nM, for 16 hours), phytohemagglutinin (10 µM; positive control), and without any treatment (negative control). By Real-time PCR (Taqman assay), specific transcription factors of Tregs and Th17 cells, forkhead box P3 (FOXP3), retinoic acid-related orphan receptor γt (ROR-γt), glucocorticoid-induced tumor necrosis factor receptor-related (GITR), and CTLA-4 mRNA expressions in two groups were measured. Results: FOXP3/ROR-γt mRNA expression in PBMCs decreased significantly in women experiencing URPL compared to the control group (p = 0.0001). Although 1,25VitD3 (50 nM) increased FOXP3 gene expression (p = 0.0001), it did not significantly affect ROR-γt gene expression. Besides, 1,25VitD3 treatment significantly increased FOXP3/ROR-γt mRNA expression from baseline in PBMCs of the fetal loss group compared to that of the control group (p = 0.01). The 1,25VitD3 also increased GITR gene expression (p = 0.017) in PBMCs of URPL women compared to the controls. Conclusion: Vitamin D deficiency may be a contributor to recurrent pregnancy loss and suggests that the supplementation of women with Vitamin D pre-pregnancy may be protective against URPL via affecting Tregs signature genes, FOXP3 and GITR.