Transcription Factor TBX21 Research Articles

Deciphering the immune modulation through deep transcriptomic profiling and therapeutic implications of DNA damage repair pattern in hepatocellular carcinoma

AimsDNA damage repair (DDR) plays a pivotal role in hepatocellular carcinoma (HCC), driving oncogenesis, progression, and therapeutic response. However, the mechanisms of DDR mediated immune cells and immuno-modulatory pathways in HCC are yet ill-defined. MethodsOur study introduces an innovative deep machine learning framework for precise DDR assessment, utilizing single-cell RNA sequencing (scRNA-seq) and bulk RNA-seq data. Single-cell RNA sequencing data were obtained and in total 85,628 cells of primary or post-immunotherapy cases were analyzed. Large-scale HCC datasets, including 1027 patients in house together with public datasets, were used for 101 machine-learning models and a novel DDR feature was derived at single-cell resolution (DDRscore). Druggable targets were predicted using the reverse phase protein array (RPPA) proteomic profiling of 169 HCC patients and RNA-seq data from 22 liver cancer cell lines. ResultsOur investigation reveals a dynamic interplay of DDR with natural killer cells and B cells in the primary HCC microenvironment, shaping a tumor-promoting immune milieu through metabolic programming. Analysis of HCC post-immunotherapy demonstrates elevated DDR levels that induces epithelial-mesenchymal transition and fibroblast-like transformation, reshaping the fibrotic tumor microenvironment. Conversely, attenuated DDR promotes antigen cross-presentation by dendritic cells and CD8+ T cells, modulating the inflammatory tumor microenvironment. Regulatory network analysis identifies the CXCL10-CXCR3 axis as a key determinant of immunotherapeutic response in low DDR HCC, potentially regulated by transcription factors GATA3, REL, and TBX21. Using machine learning techniques by combining bulk RNA-seq data in house together with public datasets, we introduce DDRscore, a robust consensus DDR scoring system to predict overall survival and resistance to PD-1 therapy in HCC patients. Finally, we identify BRAF as a potential therapeutic target for high DDRscore patients. ConclusionOur comprehensive findings advance our understanding of DDR and the tumor microenvironment in HCC, providing insights into immune regulatory mechanisms mediated via DDR pathways.

Doxorubicin induced epigenetic regulation of dendritic cell maturation in association with T cell activation facilitates tumor protective immune response in non-small cell lung cancer (NSCLC)

BackgroundNSCLC is one of the leading causes of death and is often diagnosed at late stages with no alternative therapeutic approach. DCs are professional antigen-presenting cells and DC-based immunotherapy has been under the spotlight for its anti-cancer properties. Epigenetic modifications including DNA methylation and histone modification in DCs play a crucial role in regulating their functions such as maturation and activation,innate immune responses, T cell priming, antigen presentation, and cytokine production. In the current study, we investigated the anti-cancer properties of Doxorubicin at a noncytotoxic concentration that could be extrapolated as an epigenetic regulator for DC maturation to elicit anti-tumor activity. MethodologiesPBMCs from normal and NSCLC blood samples were isolated and treated with growth factors. DCs were matured with low dose Doxorubicin and the DC maturation markers were checked by using flow-cytometry. Further, ELISA was performed and low dose Doxorubicin-induced DCs were pulsed with LCA (Lung Cancer Antigen) and primed with CD4 +T helper (Th) cells for cytotoxicity assessment. Further, epigenetic markers of T: DC conjugation were immunofluorescently visualized under a microscope. ChIP-qPCR and Invitro assays such as histone methylation, DNA methylation, and m6A methylation were performed to study the epigenetic changes under low dose Dox treatment. IL-12 neutralization assay was performed to check for the IL-12 dependency of DCs and their effect under Dox at low dose treatment. This was further followed by a Western Blotting analysis for histone and non-histone proteins. ResultsLow dose Doxorubicin induces epigenetic changes in DCs to elicit an anti-tumor response in NSCLC through the generation of CTLs with a concomitant increase in the extracellular secretions of anti-inflammatory cytokines. We also found that low dosage of Doxorubicin matured DCs when pulsed with LCA and primed with CD4 +T helper cells, secrete IFN-γ which is important in orchestrating adaptive immunity by activating CD8 + cytotoxic T-lymphocytes. Also, the secretions of IL-12 help us infer that protective immunity is also induced via Th1 response which triggered selectively the translocation of PKCθ to immunological synapse in between DC and Th. Further, methylation and acetylation markers H3K4me3 and H3K14Ac respectively upregulated whereas levels of STAT5, NFkB, NOTCH1, and DNAPKcs were downregulated. DNA and RNA methylation assays then lead to confirmations about the epigenetic changes caused by low dose Dox treatment. DNA methylation was reduced which resulted in the activation of tumor suppressor gene p53 and Th1-associated transcription factor TBX21. On the other hand, both absolute and relative RNA methylation quantification increased in the presence of Dox at a low dose. ConclusionFrom this study, we understand that non-cytotoxic concentration of Doxorubicin increases the Ag-presenting ability of DCs via an IL-12-dependent mechanism and causes epigenetic modifications in NSCLC.

The effect of glatiramer acetate, IFNβ-1a, fingolimod, and dimethyl fumarate on the expression of T-bet, IFN-γ, and MEG3 in PBMC of RRMS patients

ObjectiveMultiple sclerosis (MS) is a progressing neurodegenerative disease marked by chronic central nervous system inflammation and degeneration.This study investigates gene expression profiles of T-box transcription factor TBX21 (T-bet), interferon-gamma (IFN-γ), and long non-coding RNA MEG3 in peripheral blood mononuclear cells (PBMCs) from treatment-naïve Relapsing-Remitting Multiple Sclerosis patients (RRMS), healthy controls, and RRMS patients on different Disease Modifying Therapies (DMTs). The aim is to understand the role of T-bet, IFN-γ, and MEG3 in MS pathogenesis and their potential as diagnostic and therapeutic targets.ResultsElevated T-bet expression is observed in treatment-naïve RRMS patients compared to healthy individuals. RRMS patients treated with Interferon beta-1alpha (IFNβ-1a) and fingolimod exhibit downregulated T-bet and MEG3 expression levels, respectively, with more pronounced effects in females. Healthy individuals show a moderate positive correlation between T-bet and MEG3 and between IFN-γ and T-bet. In RRMS patients treated with Glatiramer Acetate (GA), a strong positive correlation is observed between MEG3 and IFN-γ. Remarkably, RRMS patients treated with Dimethyl Fumarate (DMF) exhibit a significant positive correlation between T-bet and MEG3. These findings underscore the diagnostic potential of T-bet in RRMS, warranting further exploration of MEG3, T-bet, and IFN-γ interplay in RRMS patients.

Expression patterns of T cells-specific long noncoding RNAs in systemic lupus erythematosus patients carrying HLA risk/nonrisk alleles.

Long noncoding RNAs (LncRNAs) play key roles in the regulation of gene expression and subsequently in the pathogenesis of several autoimmune diseases. This study aimed to explore the peripheral expression levels of T-cells-specific LncRNAs and transcription factors in systemic lupus erythematosus (SLE) patients carrying either human leukocyte antigens (HLA) risk or non-risk alleles. Genotypes of HLA-DRB1 and HLA-DQB1 loci for 106 SLE patients were determined by PCR-SSP. In the next step, patients were stratified based on the presence of HLA-DRB1*03 and/or DRB1*16 allele groups (HLA risk alleles positive or HLA-RPos) or carrying other DRB1 allele groups (HLA-RNeg). Then, transcript levels of LncRNAs (IFNG-AS1, RMRP, Th2LCR, and DQ786243) and mRNAs for transcription factors (Foxp3, Gata3, and Tbx21) were measured using qRT-PCR and compared between two subgroups of patients. Totally, 47 cases were classified as HLA-RPos and 59 cases as HLA-RNeg patients. The HLA-RPos patients showed decreased transcript levels of DQ786243 (p = .001) and elevated expression of IFNG-AS1 (p = .06) and T-bet mRNA (p = .03) compared to the HLA-RNeg group. We observed significantly lower expression of Th2LCR (p < .0001) and DQ786243 (p = .001) and higher expression of Tbx21 (p = .009) and Foxp3 (p = .02) in DR3-positive versus DR3-negative patients. Likewise, decreased transcript levels of DQ786243 (p = .02) and RMRP (p = .003) were observed in DR16-positive versus DR16-negative patients. ROC curve analysis revealed the potential of DQ786243 and RMRP as biomarkers in SLE disease based on the carriage of HLA risk alleles. Our results indicate that the contribution of multiple T cell subsets in SLE disease progression as judged by expression analysis of LncRNAs and transcription factors can be inspired by the inheritance of HLA risk/nonrisk alleles is SLE patients.

Induced regulatory T cells modified by knocking down T-bet in combination with ectopic expression of inhibitory cytokines effectively protect graft-versus-host disease

Induced regulatory T (iTreg) cells play a vital role in immune tolerance and in controlling chronic inflammation. Generated in the periphery, iTreg cells are suitable for responding to alloantigens and preventing transplant rejection. Nevertheless, their clinical application has been impeded by the plasticity and instability attributed to the loss of forkhead box protein 3 expression, raising concerns that iTreg may be converted to effector T cells and even exert a pathogenic effect. Herein, second-generation short hairpin RNAs loaded with 3 pairs of small interfering RNAs were utilized to target the T-box transcription factor TBX21. In addition, 2 immunosuppressive cytokines, namely, transforming growth factor beta and interleukin 10, were constitutively expressed. This novel engineering strategy allowed the generation of stably induced regulatory T (SI Treg) cells, which maintained the expression of forkhead box protein 3 even in an unfavorable environment and exerted potent immunosuppressive functions in vitro. Furthermore, SI Treg cells demonstrated an effector transcriptional profile. Finally, SI Treg cells showed a significant protective effect against graft-versus-host disease–related deaths in a xenotransplantation model. Collectively, these results signify that SI Treg cells hold great promise for future clinical application and offer a rational therapeutic approach for transplant rejection.

Single-Cell Multi-Omics Analysis Reveals the Role of CX3CR1+ GNLY+ CD8+ T Cells in Improving Survival of Patients with Relapse/Refractory Hematologic Malignancies after Haplo+Cord HSCT

Background Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment for hematological malignancies. Previous works have shown superior survival outcomes of sequential transplantation of haploidentical stem cell and unrelated cord blood (haplo+cord HSCT) among recipients with relapsed/refractory leukemia, compared with single cord HSCT. However, the underlying mechanisms of immune reconstitution remain largely unexplored. Methods we first performed scRNA-seq and scATAC-seq on bone marrow mononuclear cells (BMMCs) from nine patients with relapsed/refractory leukemia underwent haplo+cord or single cord HSCT to profile the immune landscapes at single cell resolution. Second, we compared the differences of gene expression and chromatin accessibility between the subsets of immune cells. Finally, by integrating multi-omics data, we explored the gene expression regulation mechanism behind the differences between two transplant group. Results We observed distinct composition and function of global immune landscapes (T, B, natural killer cells and monocytes) between two transplant strategies, where immune cells from haplo+cord HSCT involved in interferon signaling pathway and response to virus. Analysis on T cells revealed two cytotoxic CD8+ T cells populations, enriched in haplo+cord HSCT (expressing GNLY, GZMB and PRF1) and enriched in single cord HSCT (expressing CXCR4 and GZMK) respectively. Interestingly, the augmented expression of GNLY and CX3CR1 suggested a dual property of effector and memory of haplo+cord HSCT enriched CD8+ T cells that potentially offers long-term protection for patients post-transplantation. The transcription factor IRF7, IRF9 and TBX21 played the key role on regulating immune signal transduction of CD8+ T cell subsets after haplo+cord HSCT, which might be the mechanism of transcriptional regulation for cytotoxic CD8+T cell function. Finally, single cell multi-omics analysis delineate regulatory network controlling lineage-specific gene expression after HSCT, illustrated by the example of GNLY expression in CD8+ T cells regulated by an enhancer cluster within CTCF-mediated chromatin loop. Conclusions These data collectively constitute the transcriptomic and epigenetic landscape of bone marrow post-transplantation in hematopoietic malignancies at single-cell resolution, demonstrating that a CX3CR1+ GNLY+ CD8+ T cells characterized by effector and memory property served the improved survival of patients receiving haplo+cord HSCT. keywords Single cell multi-omics, CX3CR1+ GNLY+ CD8+T cells, haplo+cord HSCT, single cord HSCT, relapsed/refractory leukemia

Alzheimer's disease alters the transcriptomic profile of natural killer cells at single-cell resolution.

Alzheimer's disease (AD) is the most common dementia without an effective cure at least partially due to incomplete understanding of the disease. Inflammation has emerged as a central player in the onset and progression of AD. As innate lymphoid cells, natural killer (NK) cells orchestrate the initiation and evolution of inflammatory responses. Yet, the transcriptomic features of NK cells in AD remain poorly understood. We assessed the diversity of NK cells using web-based single-cell RNA sequencing data of blood NK cells from patients with AD and control subjects and flow cytometry. We identified a contraction of NK cell compartment in AD, accompanied by a reduction of cytotoxicity. Unbiased clustering revealed four subsets of NK cells in AD, i.e., CD56bright NK cells, CD56dim effector NK cells, adaptive NK cells, and a unique NK cell subset that is expanded and characterized by upregulation of CX3CR1, TBX21, MYOM2, DUSP1, and ZFP36L2, and negatively correlated with cognitive function in AD patients. Pseudo-temporal analysis revealed that this unique NK cell subset was at a late stage of NK cell development and enriched with transcription factors TBX21, NFATC2, and SMAD3. Together, our study identified a distinct NK cell subset and its potential involvement in AD.

TCL-306 Application of the Proposed Immunohistochemistry Algorithm for the Prognostication of GATA3 and TBX21 Subtypes of Peripheral T-Cell Lymphoma, Not Otherwise Specified.

Recent molecular studies have indicated the varied oncogenic pathways resulting in the subtypes of peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS), which affect prognosis and may have predictive value. To evaluate the immunohistochemistry (IHC) algorithm (Amador et al.) for the subtyping of PTCL, NOS and ascertain their relevance with correlation to the clinicopathological profile. This 7-year (2015-2021) ambispective study included 43 patients with diagnoses of PTCL, NOS from the archives of the Department of Pathology (AIIMS, New Delhi). IHC for transcription factors GATA3 (>50% immunopositivity considered positive) and TBX21 (>20%) and their target proteins CCR4 (>50%) and CXCR3 (>20%) was performed followed by subtyping by two pathologists. Detailed clinicopathological correlation was done. The association between the subtypes and clinical factors (demographics, performance status, organomegaly, IPI/PIT score, stage, marrow involvement), histologic features (monomorphous/polymorphous population), and survival (overall and progression-free survival) was analyzed. With the application of the algorithm by Amador et al., the cases were categorized into the subtypes GATA3 (18), TBX21 (13), and unclassifiable (7). There was no significant association observed with the clinical parameters of the subtypes. Though an increased proportion of TBX21-subgroup cases showed a polymorphous population compared to the GATA3 subgroup with monomorphous population, no significant P-value was found. Two of the Lennert lymphomas were categorized in the GATA3 subgroup. On multivariate analysis, no significant difference was present for overall survival (P-value=0.149) and progression-free survival (P-value=0.066) amongst the IHC-defined subtypes; the trends suggest that the GATA3 subgroup has worse overall and progression-free survival. The IHC-derived subtypes of GATA3 and TBX21 may have a role in prognostication and may guide further therapeutics aiding in better patient management of this aggressive group of lymphomas. The algorithm is reproducible, however some of the cases may be left unclassifiable and require further work up and gene expression profiling. The results trend toward the GATA3 subgroup having a monomorphous population with a worse overall prognosis, requiring a larger sample size for validation. The application of this algorithm in routine practice can help in risk stratification of the patients. Thanks to Science-Engineering-Research-Board and AIIMS-Research section (SERB-EEQ/2016/402, AIIMS-A-653).

Role of Diospyros peregrina fruit preparation in suppressing regulatory T (Treg) cells in the tumor microenvironment of breast and lung cancer

BackgroundThe most frequent cancers in the world are breast cancer and lung cancer. Regulatory T cells (Treg) are the subtype of T cells that promotes equilibrium between immunological surveillance and immune tolerance, and they are commonly found in high amounts within tumours. The presence of Treg cells in the tumour microenvironment results in immune suppression, due to which immune cells are not able to kill the tumour cells. Diospyros peregrina (Gaertn.) Gürke is an ethnomedicinal plant with hypoglycaemic, diuretic, and anti-cancer effects. This genus, Diospyros, is native to India but distributed throughout the tropics and is characterized by its ability to produce triterpenes of the lupine series and various other bioactive compounds. Diospyros peregrina fruit preparation (DFP) substantially increases tumour protective immunity by suppressing Treg cells. PurposeThis project aims to suppress the regulatory T cells in the tumour microenvironment by giving Diospyros peregrina fruit preparation (DFP) treatment to Treg cells, which will help in controlling and combating breast cancer and lung cancer. MethodFor this project's work, we isolated peripheral blood mononuclear cells from the blood, and the isolated peripheral blood mononuclear cells were used to isolate CD4+CD25+ Treg cells. Then, Diospyros peregrina fruit preparation (DFP) was prepared, which was used to treat CD4+CD25+ Treg cells. Lactate dehydrogenase (LDH) assays, enzyme-linked immunosorbent assays (ELISA), and RT-qPCR were used to determine the effect of DFP treatment on Treg cells. ResultDiospyros peregrina fruit preparation (DFP) treatment suppresses the expression of CD4+CD25+ Foxp3+Treg cells within tumours. DFP-treated CD4+CD25+Foxp3+ Treg cells increase the cytotoxicity within the tumours compared to non-treated Treg cells. DFP treatment help in reducing the release of (T helper type 2) TH2 cell cytokines (Interleukin-10) IL-10 as well as (Interleukin-4) IL-4 and escalating the release of (T helper type 1) TH1 cell cytokines (Interleukin-12) IL-12 and (Interferon-γ) IFN-γ, it also increases the transcriptional activity of T-box transcription factor TBX21 (TBX21) and reduces the expression of the transcription factor FOXP3 and GATA3. ConclusionDiospyros peregrina fruit preparation (DFP) treatment augments the expressions of TH1-specific cytokines and transcription factors, and DFP treatment diminishes the expressions of both TH2 and Treg-specific cytokines and transcription factors.

Transcriptional activity analysis of the immune response genes in the peripheral blood of patients with comorbid acute urticaria and lyme borreliosis

Acute urticaria (AU) and Lyme borreliosis (LB) are known to alter the transcriptional profile of blood cells. The nature of changes in the transcriptional activity of genes of the innate and adaptive immune system in the peripheral blood in patients with comorbid AU and LB is unknown. In our study, we applied a pathway-specific PCR array (Human Innate &amp; Adaptive Immune Responses RT2 Profiler PCR Array, QIAGEN, Germany) to detect and verify the innate and adaptive immune responses of pathway-focused genes expression in the blood of patients with a comorbid course of these pathologies. It was found that in patients with comorbid AU and Lyme disease, transcriptional induction of a number of genes of the innate immune system in PBMC was observed, in particular: TLR2, NOD2, NLRP3, APCS, complement component 3 (C3), CD14, CD86 compared with patients suffering from acute urticaria. These changes were accompanied by an increased transcriptional activity of systemic proinflammatory cytokines IL1B, IL6, IFNG and its receptor IFNGR1, TNF, ligands, and chemokine receptors CXCL10, CXCR3, CCR5, tyrosine kinase JAK2 and transcription factors STAT1 and TBX21. At the same time, the comorbid course of acute urticaria and Lyme disease led to the repression of the transcriptional activity of the CD80, IL4, and CXCL8 genes. The comorbid course of acute urticaria and Lyme borreliosis is accompanied by activation of the transcriptional activity of genes of the innate immune system and proinflammatory cytokines.

A44 PGC1-α INHIBITION WORSENS DSS-INDUCED COLITIS IN MICE

BackgroundInflammatory bowel diseases (IBD) represent a wide range of gastrointestinal diseases that are characterized by chronic inflammation of the gastrointestinal tract (GIT). Affecting more than 6 million individuals globally, the most prevalent forms of IBD are Ulcerative colitis (UC), characterized by inflammation and ulceration of the colon and rectum, and Crohn’s disease (CD), characterized by inflammation of several areas of the GIT. Due to the morbidity and occasionally mortality arising from IBD, as well as its increasing global prevalence, the search for novel treatments and cures for IBD presents great importance. Loss of immune tolerance significantly contributes to the inflammatory patterns observed in IBD patients. Dendritic Cells (DCs), key antigen-presenting cells, are important mediators of tolerance in the gut when polarized to tolerogenic DCs (tolDCs). The phenotype of tolDCs has been shown to directly depend on their intracellular metabolism, mostly by adopting mitochondrial oxidative phosphorylation. Peroxisome proliferator-activated receptor-gamma coactivator (PGC)1-α is a transcription coactivator that plays a crucial role in mediating cellular energy metabolism. Evidence shows that activation of PGC1-α stimulates mitochondrial biogenesis and promotes oxidative metabolism in a variety of biological processes.AimsThe aim of this project was to evaluate whether activation or inhibition of PGC1-α impacts inflammatory response in DSS-induced colitic in mice. We hypothesize that the PGC1-α pharmacological activator ZLN005 would ameliorate colitis as a result of tolDC polarization. On other hand, the PGC1-α pharmacological inhibitor SR18292 would increase inflammatory responses and decrease tolerance.MethodsC57/b6 mice received DSS in drinking water for 5 days, followed by 3 days of tap water. Mice were treated with vehicle, ZLN005 (10 mg/kg), or SR18292 treatment (25 mg/kg) for the 3 days of drinking tap water. Under necropsy, colitis was assessed by disease activity score (DAS), colon length, weight change, myeloperoxidase (MPO) activity assay, histopathology score, and cytokines by ELISA. Intestinal tolerance was accessed by evaluating T cells polarization to Treg, TH1, TH2, or TH17 by qPCR of the transcription factors Foxp3 (Treg), Tbx21 (TH1), Gata3 (TH2), and Rorγt (TH17) expressed in the colon.ResultsOur results demonstrated that ZLN005 did not alter the inflammatory response induced by DSS, gauged by MPO assay, DAS, and histopathology, suggesting that ZLN005 was ineffective in polarizing tolDCs. However, we observed a worsening of inflammatory conditions with treatment with SR18292 with a decrease in IL-10 levels and increased MPO values, potentially stemming from the loss of tolDCs and intestinal tolerance.ConclusionsPGC1-α inhibition worsens inflammatory response in DSS-induced colitis potentially by inhibiting tolDC polarization and loss of intestinal tolerance.Funding AgenciesNone

T-Bet Deficiency Attenuates Bile Duct Injury in Experimental Biliary Atresia.

Biliary atresia (BA) is an obstructive neonatal cholangiopathy leading to liver cirrhosis and end stage liver disease. A Kasai portoenterostomy may restore biliary drainage, but most patients ultimately require liver transplantation for survival. At diagnosis, immune cells within the liver of patients with BA demonstrate a T-helper 1 (Th1) inflammatory profile similar to rhesus rotavirus (RRV)-infected mice livers developing BA. The transcription factor Tbx21 (T-bet) is essential for induction of a Th1 immune response in both the adaptive and innate immune system. Here we used animals with targeted deletion of the T-bet gene to determine its role in the progression of BA. Infection of newborn T-bet knockout (KO) pups with RRV resulted in a decreased Th1 inflammatory chemokine/cytokine profile when compared to infected wild-type mice. Analysis of the mononuclear cells profile from T-bet KO mice revealed both a significant decrease in the total number of CD3, CD4, and CD8 T cells and their effector molecules granzyme A, perforin, and FasL. Even though the percentage of T-bet KO mice displaying symptoms of an obstructive cholangiopathy and overall mortality rate was not different compared to wild-type mice, the extrahepatic bile ducts of T-bet KO mice remained patent.

Cytotoxic T lymphocyte antigen-4 regulates development of xenogenic graft versus host disease in mice via modulation of host immune responses induced by changes in human T cell engraftment and gene expression.

Graft versus host disease (GvHD) is a major clinical problem with a significant unmet medical need. We examined the role of cytotoxic T lymphocyte antigen-4 (CTLA-4) in a xenogenic GvHD (xeno-GvHD) model induced by injection of human peripheral mononuclear cells (hPBMC) into irradiated non-obese diabetic (NOD) SCID gamma (NSG) mice. Targeting the CTLA-4 pathway by treatment with CTLA-4 immunoglobulin (Ig) prevented xeno-GvHD, while anti-CTLA-4 antibody treatment exacerbated the lethality and morbidity associated with GvHD. Xeno-GvHD is associated with infiltration of hPBMCs into the lungs, spleen, stomach, liver and colon and an increase in human proinflammatory cytokines, including interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-5. Infiltration of donor cells and increases in cytokines were attenuated by treatment with CTLA-4 Ig, but remained either unaffected or enhanced by anti-CTLA-4 antibody. Further, splenic human T cell phenotyping showed that CTLA-4 Ig treatment prevented the engraftment of human CD45+ cells, while anti-CTLA-4 antibody enhanced donor T cell expansion, particularly CD4+ (CD45RO+ ) subsets, including T box transcription factor TBX21 (Tbet)+ CXCR3+ and CD25+ forkhead box protein 3 (FoxP3) cells. Comprehensive analysis of transcriptional profiling of human cells isolated from mouse spleen identified a set of 417 differentially expressed genes (DEGs) by CTLA-4 Ig treatment and 13 DEGs by anti-CTLA-4 antibody treatment. The CTLA-4 Ig regulated DEGs mapped to down-regulated apoptosis, inflammasome, T helper type 17 (Th17) and regulatory T cell (Treg ) pathways and enhanced Toll-like receptor (TLR) receptor signaling, TNF family signaling, complement system and epigenetic and transcriptional regulation, whereas anti-CTLA-4 antibody produced minimal to no impact on these gene pathways. Our results show an important role of co-inhibitory CTLA-4 signaling in xeno-GvHD and suggest the therapeutic utility of other immune checkpoint co-inhibitory pathways in the treatment of immune-mediated diseases driven by hyperactive T cells.

Candida tropicalis Systemic Infection Redirects Leukocyte Infiltration to the Kidneys Attenuating Encephalomyelitis.

Environmental factors, including infections, are strongly associated with the pathogenesis of multiple sclerosis (MS), which is an autoimmune and demyelinating disease of the central nervous system (CNS). Although classically associated with bacterial and viral agents, fungal species have also been suspected to affect the course of the disease. Candida tropicalis is an opportunistic fungus that affects immunocompromised individuals and is also able to spread to vital organs. As C. tropicalis has been increasingly isolated from systemic infections, we aimed to evaluate the effect of this fungus on experimental autoimmune encephalomyelitis (EAE), a murine model to study MS. For this, EAE was induced in female C57BL/6 mice 3 days after infection with 106 viable C. tropicalis yeasts. The infection decreased EAE prevalence and severity, confirmed by the less inflammatory infiltrate and less demyelization in the lumbar spinal cord. Despite this, C. tropicalis infection associated with EAE results in the death of some animals and increased urea and creatinine serum levels. The kidneys of EAE-infected mice showed higher fungal load associated with increased leukocyte infiltration (CD45+ cells) and higher expression of T-box transcription factor (Tbx21) and forkhead box P3 (Foxp3). Altogether, our results demonstrate that although C. tropicalis infection reduces the prevalence and severity of EAE, partially due to the sequestration of leukocytes by the inflamed renal tissue, this effect is associated with a poor disease outcome.

Fine particulate matter induces airway inflammation bydisturbing the balance between Th1/Th2 and regulation of GATA3 and Runx3 expression in BALB/c mice.

The present study aimed to examine the effects of 2.5 µm particulate matter (PM2.5) on airway inflammation and to investigate the possible underlying mechanism. Specifically, the focus was on the imbalance of T helper (Th)1/Th2 cells and the dysregulated expression of transcription factors, including trans-acting T cell-specific transcription factor 3 (GATA3), runt-related transcription factor 3 (Runx3) and T-box transcription factor TBX21 (T-bet). In this study, ambient PM2.5 was collected and analyzed, male BALB/c mice were sensitized and treated with PBS, ovalbumin (OVA), PM2.5 or OVA + PM2.5. The effects of PM2.5 alone or PM2.5 + OVA on immunopathological changes, the expression of transcription factors GATA3, Runx3 and T-bet, and the imbalance of Th1/Th2 were investigated. It was found that PM2.5 + OVA co-exposure significantly enhanced inflammatory cell infiltration, increased higher tracheal secretions in lung tissue and upregulated respiratory resistance response to acetylcholine compared with PM2.5 or OVA single exposure and control groups. In addition, higher protein and mRNA expression levels of Th2 inflammatory mediators interleukin (IL)-4, IL-5 and IL-13 in bronchoalveolar lavage fluid were observed in PM2.5 + OVA treated mice, whereas the expression levels of GATA3 and STAT6 were exhibited in mice exposed to OVA + PM2.5 compared with the OVA and PM2.5 groups. By contrast, PM2.5 exposure decreased the protein and mRNA expression levels of Th1 cytokine interferon-γ and transcription factors Runx3 and T-bet, especially among asthmatic mice, different from OVA group, PM2.5 exposure only failed to influence the expression of T-bet. To conclude, PM2.5 exposure evoked the allergic airway inflammation response, especially in the asthmatic mouse model and led to Th1/Th2 imbalance. These effects worked mainly by upregulating GATA3 and downregulating Runx3. These data suggested that Runx3 may play an important role in PM2.5-aggravated asthma in BALB/c mice.

Exhausted T cells in systemic lupus erythematosus patients in long-standing remission.

The mechanisms that drive systemic lupus erythematosus (SLE) patients to achieve remission are unknown; one possible explanation might be T cell exhaustion. The aim of the present study was to measure CD4+ and CD8+ T cell exhaustion in SLE patients in prolonged remission (PR-SLE) and compared them with patients with active SLE (Act-SLE) and healthy subjects. We included 15PR-SLE patients, 15Act-SLE and 29healthy subjects. T cell exhaustion was determined by flow cytometry according to the expression of programmed cell death 1 (PD)-1, T cell immunoglobulin and mucin 3 (Tim-3), natural killer cell receptor (2B4), eomesodermin (EOMES) and T-box transcription factor TBX21 (T-bet) in CD4+ and CD8+ T cells. Dimensionality reduction using the T-distributed stochastic neighbor-embedding algorithm and clustering analysis was used for the identification of relevant populations. Percentages of CD3+ , CD4+ and CD8+ T cells were similar among groups. We identified five subpopulations of CD8+ and seven of CD4+ cells. The CD4+ T-bet+ CD45RO+ cells identified in the unsupervised analysis were significantly increased in PR-SLE versus Act-SLE [median=0·20, interquartile range (IQR)=1·74-30·50 versus 1·68, IQR=0·4-2·83; P<0·01]. CD4+ EOMES+ cells were also increased in PR-SLE versus Act-SLE (5·24, IQR=3·38-14·70 versus 1·39, IQR=0·48-2·87; P<0·001). CD8+ EOMES+ cells were increased in PR-SLE versus Act-SLE (37·6, IQR=24·9-53·2 versus 8·13, IQR=2·33-20·5; P<0·001). Exhausted and activated T cells presented an increased frequency of PD-1, CD57 and EOMES in SLE patients versus healthy subjects. Some subpopulations of T cells expressing markers associated with exhaustion are increased in patients in remission, supporting T cell exhaustion as a tolerance mechanism in SLE. Exhaustion of specific populations of T cells might represent a potential therapeutic tool that will contribute to the goal of achieving sustained remission in these patients.

Immunology of IL-12: An update on functional activities and implications for disease.

As its first identified member, Interleukin-12 (IL-12) named a whole family of cytokines. In response to pathogens, the heterodimeric protein, consisting of the two subunits p35 and p40, is secreted by phagocytic cells. Binding of IL-12 to the IL-12 receptor (IL-12R) on T and natural killer (NK) cells leads to signaling via signal transducer and activator of transcription 4 (STAT4) and subsequent interferon gamma (IFN-γ) production and secretion. Signaling downstream of IFN-γ includes activation of T-box transcription factor TBX21 (Tbet) and induces pro-inflammatory functions of T helper 1 (TH1) cells, thereby linking innate and adaptive immune responses. Initial views on the role of IL-12 and clinical efforts to translate them into therapeutic approaches had to be re-interpreted following the discovery of other members of the IL-12 family, such as IL-23, sharing a subunit with IL-12. However, the importance of IL-12 with regard to immune processes in the context of infection and (auto-) inflammation is still beyond doubt. In this review, we will provide an update on functional activities of IL-12 and their implications for disease. We will begin with a summary on structure and function of the cytokine itself as well as its receptor and outline the signal transduction and the transcriptional regulation of IL-12 secretion. In the second part of the review, we will depict the involvement of IL-12 in immune-mediated diseases and relevant experimental disease models, while also providing an outlook on potential translational approaches.

Peptidoglycan-treated tumor antigen-pulsed dendritic cells impart complete resistance against tumor rechallenge.

Solid tumors elicit suppressive T cell responses which impair antigen-presenting cell (APC) functions. Such immune suppression results in uncontrolled tumor growth and mortality. Addressing APC dysfunction, dendritic cell (DC)-mediated anti-tumor vaccination was extensively investigated in both mice and humans. These studies never achieved full resistance to tumor relapse. Herein, we describe a repetitive RM-1 murine tumor rechallenge model for recurrence in humans. Using this newly developed model, we show that priming with tumor antigen-pulsed, Toll-like receptor (TLR)2 ligand-activated DCs elicits a host-protective anti-tumor immune response in C57BL/6 mice. Upon stimulation with the TLR2 ligand peptidoglycan (PGN), the tumor antigen-pulsed DCs induce complete resistance to repetitive tumor challenges. Intra-tumoral injection of PGN reduces tumor growth. The tumor resistance is accompanied by increased expression of interleukin (IL)-27, T-box transcription factor TBX21 (T-bet), IL-12, tumor necrosis factor (TNF)-α and interferon (IFN)-γ, along with heightened cytotoxic T lymphocyte (CTL) functions. Mice primed four times with PGN-stimulated tumor antigen-pulsed DCs remain entirely resistant to repeat challenges with RM-1 tumor cells, suggesting complete prevention of relapse and recurrence of tumor. Adoptive transfer of T cells from these mice, which were fully protected from RM-1 rechallenge, confers anti-tumor immunity to syngeneic naive recipient mice upon RM-1 challenge. These observations indicate that PGN-activated DCs induce robust host-protective anti-tumor T cells that completely resist tumor growth and recurrence.

GAD-alum immunotherapy in type 1 diabetes expands bifunctional Th1/Th2 autoreactive CD4 T cells

Aims/hypothesisAntigen-specific therapy aims to modify inflammatory T cell responses in type 1 diabetes and restore immune tolerance. One strategy employs GAD65 conjugated to aluminium hydroxide (GAD-alum) to take advantage of the T helper (Th)2-biasing adjuvant properties of alum and thereby regulate pathological Th1 autoimmunity. We explored the cellular and molecular mechanism of GAD-alum action in the setting of a previously reported randomised placebo-controlled clinical trial conducted by Type 1 Diabetes TrialNet.MethodsIn the clinical trial conducted by Type 1 Diabetes TrialNet, participants were immunised with 20 μg GAD-alum (twice or three times) or alum alone and peripheral blood mononuclear cell samples were banked at baseline and post treatment. In the present study, GAD-specific T cell responses were measured in these samples and GAD-specific T cell lines and clones were generated, which were then further characterised.ResultsAt day 91 post immunisation, we detected GAD-specific IL-13+ CD4 T cell responses significantly more frequently in participants immunised with GAD-alum (71% and 94% treated twice or three times, respectively) compared with those immunised with alum alone (38%; p = 0.003 and p = 0.0002, respectively) accompanied by high secreted levels of IL-13, IL-4 and IL-5, confirming a GAD-specific, GAD-alum-induced Th2 response. Of note, GAD-specific, IL-13+ CD4 T cells observed after immunisation co-secreted IFN-γ, displaying a bifunctional Th1/Th2 phenotype. Single-cell transcriptome analysis identified IL13 and IFNG expression in concert with the canonical Th2 and Th1 transcription factor genes GATA3 and TBX21, respectively. T cell receptor β-chain (TCRB) CDR3 regions of GAD-specific bifunctional T cells were identified in circulating naive and central memory CD4 T cell pools of non-immunised participants with new-onset type 1 diabetes and healthy individuals, suggesting the potential for bifunctional responses to be generated de novo by GAD-alum immunisation or via expansion from an existing public repertoire.Conclusions/interpretationGAD-alum immunisation activates and propagates GAD-specific CD4 T cells with a distinctive bifunctional phenotype, the functional analysis of which might be important in understanding therapeutic responses.

Dysregulated Transcription Factor TFAP2A After Peripheral Nerve Injury Modulated Schwann Cell Phenotype.

Transcription factors regulate the transcriptions and expressions of numerous target genes and direct a variety of physiological and pathological activities. To obtain a better understanding of the involvement of transcription factors during peripheral nerve repair and regeneration, significantly differentially expressed genes coding for transcription factors in rat sciatic nerves after sciatic nerve crush injury were identified. A total of 9 transcription factor genes, including GBX2, HIF3A, IRF8, LRRC63, SNAI3, SPIB, TBX21, TFAP2A, and ZBTB16 were identified to be commonly differentially expressed at 1, 4, 7, and 14 days after nerve injury. TFAP2A, a gene encoding transcription factor activating enhancer binding protein 2 alpha, was found to be critical in the regulatory network. PCR validation and immunohistochemistry staining of injured rat sciatic nerves showed that TFAP2A expression was significantly up-regulated in the Schwann cells after nerve injury for at least 2 weeks. Schwann cells transfected with TFAP2A-siRNA exhibited elevated proliferation rate and migration ability, suggesting that TFAP2A suppressed Schwann cell proliferation and migration. Collectively, our study provided a global overview of the dynamic changes of transcription factors after sciatic nerve injury, discovered key transcription factors for the regeneration process, and deepened the understanding of the molecular mechanisms underlying peripheral nerve repair and regeneration.