Mesothelin CAR-T cells expressing tumor-targeted immunocytokine IL-12 yield durable efficacy and fewer side effects.

Chimeric antigen receptor (CAR)-modified T cell therapy has achieved remarkable efficacy in treating hematological malignancies, but it confronts many challenges in treating solid tumors, such as the immunosuppressive microenvironment of the solid tumors. These factors reduce the antitumor activity of CAR-T cells in clinical trials. Therefore, we used the immunocytokine interleukin-12 (IL-12) to enhance the efficacy of CAR-T cell therapy. In this study, we engineered CAR-IL12R54 T cells that targeted mesothelin (MSLN) and secreted a single-chain IL-12 fused to a scFv fragment R54 that recognized a different epitope on mesothelin. The evaluation of the anti-tumor activity of the CAR-IL12R54 T cells alone or in combination with anti-PD-1 antibody in vitro and in vivo was followed by the exploration of the functional mechanism by which the immunocytokine IL-12 enhanced the antitumor activity. CAR-IL12R54 T cells had potency to lyse mesothelin positive tumor cells in vitro. In vivo studies demonstrated that CAR-IL12R54 T cells were effective in controlling the growth of established tumors in a xenograft mouse model with fewer side effects than CAR-T cells that secreted naked IL-12. Furthermore, combination of PD-1 blockade antibody with CAR-IL12R54 T cells elicited durable anti-tumor responses. Mechanistic studies showed that IL12R54 enhanced Interferon-γ (IFN-γ) production and dampened the activity of regulatory T cells (Tregs). IL12R54 also upregulated CXCR6 expression in the T cells through the NF-κB pathway, which facilitated T cell infiltration and persistence in the tumor tissues. In summary, the studies provide a good therapeutic option for the clinical treatment of solid tumors.

IL-12 regulates type 3 immunity through interfollicular keratinocytes in psoriasiform inflammation.

Psoriasis is a chronic inflammatory skin disorder underpinned by dysregulated cytokine signaling. Drugs neutralizing the common p40 subunit of interleukin-12 (IL-12) and IL-23 represented a therapeutic breakthrough; however, new drugs that block the IL-23p19 subunit and spare IL-12 are more effective, suggesting a regulatory function of IL-12. To pinpoint the cell type and underlying mechanism of IL-12­mediated immune regulation in psoriasis, we generated a conditional Il12rb2-knockout (KO)/reporter mouse strain. We detected Il12rb2 expression in T cells and a specific subset of interfollicular (IF) keratinocytes. Analysis of single-cell RNA-sequencing (scRNAseq) data from patients with psoriasis confirmed a similar expression pattern in the human skin. Deletion of Il12rb2 across the hematopoietic compartment did not alter the development of Aldara-induced psoriasiform inflammation. However, depletion of Il12rb2 in keratinocytes exacerbated disease development, phenocopying the Il12rb2 germline knockout. Protective IL-12 signaling blocked the hyperproliferation of keratinocytes, maintained skin barrier integrity, and diminished disease-driving IL-23/type 3 immune circuits. In line, specific IL-23p19 blockade led to a more profound reduction of psoriatic keratinocyte expression signatures in the skin of patients with psoriasis than combined IL-12/IL-23 inhibition. Collectively, we provide a potential explanation for the superior efficacy of IL-23p19 inhibitors in psoriasis and describe an unperceived role of IL-12 in maintaining skin epithelial cell homeostasis.

Clinical and Molecular Findings in Mendelian Susceptibility to Mycobacterial Diseases: Experience From India.

Mendelian Susceptibility to Mycobacterial diseases (MSMD) are a group of innate immune defects with more than 17 genes and 32 clinical phenotypes identified. Defects in the IFN-γ mediated immunity lead to an increased susceptibility to intracellular pathogens like mycobacteria including attenuated Mycobacterium bovis-Bacillus Calmette-Guérin (BCG) vaccine strains and non-tuberculous environmental mycobacteria (NTM), Salmonella, fungi, parasites like Leishmania and some viruses, in otherwise healthy individuals. Mutations in the IL12RB1 gene are the commonest genetic defects identified. This retrospective study reports the clinical, immunological, and molecular characteristics of a cohort of 55 MSMD patients from 10 centers across India. Mycobacterial infection was confirmed by GeneXpert, Histopathology, and acid fast bacilli staining. Immunological workup included lymphocyte subset analysis, Nitro blue tetrazolium (NBT) test, immunoglobulin levels, and flow-cytometric evaluation of the IFN-γ mediated immunity. Genetic analysis was done by next generation sequencing (NGS). Disseminated BCG-osis was the commonest presenting manifestation (82%) with a median age of presentation of 6 months due to the practice of BCG vaccination at birth. This was followed by infection with Salmonella and non-typhi Salmonella (13%), Cytomegalovirus (CMV) (11%), Candida (7%), NTM (4%), and Histoplasma (2%). Thirty-six percent of patients in cohort were infected by more than one organism. This study is the largest cohort of MSMD patients reported from India to the best of our knowledge and we highlight the importance of work up for IL-12/IL-23/ISG15/IFN-γ circuit in all patients with BCG-osis and suspected MSMD irrespective of age.

Interleukin 35 Delays Hindlimb Ischemia-Induced Angiogenesis Through Regulating ROS-Extracellular Matrix but Spares Later Regenerative Angiogenesis.

Interleukin (IL) 35 is a novel immunosuppressive heterodimeric cytokine in IL-12 family. Whether and how IL-35 regulates ischemia-induced angiogenesis in peripheral artery diseases are unrevealed. To fill this important knowledge gap, we used loss-of-function, gain-of-function, omics data analysis, RNA-Seq, in vivo and in vitro experiments, and we have made the following significant findings: i) IL-35 and its receptor subunit IL-12RB2, but not IL-6ST, are induced in the muscle after hindlimb ischemia (HLI); ii) HLI-induced angiogenesis is improved in Il12rb2-/- mice, in ApoE-/-/Il12rb2-/- mice compared to WT and ApoE-/- controls, respectively, where hyperlipidemia inhibits angiogenesis in vivo and in vitro; iii) IL-35 cytokine injection as a gain-of-function approach delays blood perfusion recovery at day 14 after HLI; iv) IL-35 spares regenerative angiogenesis at the late phase of HLI recovery after day 14 of HLI; v) Transcriptome analysis of endothelial cells (ECs) at 14 days post-HLI reveals a disturbed extracellular matrix re-organization in IL-35-injected mice; vi) IL-35 downregulates three reactive oxygen species (ROS) promoters and upregulates one ROS attenuator, which may functionally mediate IL-35 upregulation of anti-angiogenic extracellular matrix proteins in ECs; and vii) IL-35 inhibits human microvascular EC migration and tube formation in vitro mainly through upregulating anti-angiogenic extracellular matrix-remodeling proteins. These findings provide a novel insight on the future therapeutic potential of IL-35 in suppressing ischemia/inflammation-triggered inflammatory angiogenesis at early phase but sparing regenerative angiogenesis at late phase.

IL-12 p40 monomer is different from other IL-12 family members to selectively inhibit IL-12Rß1 internalization and suppress EAE.

Multiple sclerosis (MS) is the most common human demyelinating disease of the central nervous system. The IL-12 family of cytokines has four members, which are IL-12 (p40:p35), IL-23 (p40:p19), the p40 monomer (p40), and the p40 homodimer (p402). Since all four members contain p40 in different forms, it is important to use a specific monoclonal antibody (mAb) to characterize these molecules. Here, by using such mAbs, we describe selective loss of p40 in serum of MS patients as compared to healthy controls. Similarly, we also observed decrease in p40 and increase in IL-12, IL-23, and p402 in serum of mice with experimental autoimmune encephalomyelitis (EAE), an animal model of MS, as compared to control mice. Interestingly, weekly supplementation of mouse and human recombinant p40 ameliorated clinical symptoms and disease progression of EAE. On the other hand, IL-12, IL-23, and p402 did not exhibit such inhibitory effect. In addition to EAE, p40 also suppressed collagen-induced arthritis in mice. Using IL-12Rß1-/-, IL-12Rß2-/-, and IL-12Rß1+/-/IL-12Rß2-/- mice, we observed that p40 required IL-12Rß1, but not IL-12Rß2, to suppress EAE. Interestingly, p40 arrested IL-12-, IL-23-, or p402-mediated internalization of IL-12Rß1, but neither IL-12Rß2 nor IL-23R, protected regulatory T cells, and suppressed Th1 and Th17 biasness. These studies identify p40 as an anti-autoimmune cytokine with a biological role different from IL-12, IL-23, and p402 in which it attenuates autoimmune signaling via suppression of IL-12Rß1 internalization, which may be beneficial in patients with MS and other autoimmune disorders.

Production of IL-17A at Innate Immune Phase Leads to Decreased Th1 Immune Response and Attenuated Host Defense against Infection with Cryptococcus deneoformans.

IL-17A is a proinflammatory cytokine produced by many types of innate immune cells and Th17 cells and is involved in the elimination of extracellularly growing microorganisms, yet the role of this cytokine in the host defense against intracellularly growing microorganisms is not well known. Cryptococcus deneoformans is an opportunistic intracellular growth fungal pathogen that frequently causes fatal meningoencephalitis in patients with impaired immune responses. In the current study, we analyzed the role of IL-17A in the host defense against C. deneoformans infection. IL-17A was quickly produced by γδT cells at an innate immune phase in infected lungs. In IL-17A gene-disrupted mice, clearance of this fungal pathogen and the host immune response mediated by Th1 cells were significantly accelerated in infected lungs compared with wild-type mice. Similarly, killing of this fungus and production of inducible NO synthase and TNF-α were significantly enhanced in IL-17A gene-disrupted mice. In addition, elimination of this fungal pathogen, Th1 response, and expression of IL-12Rß2 and IFN-γ in NK and NKT cells were significantly suppressed by treatment with rIL-17A. The production of IL-12p40 and TNF-α from bone marrow-derived dendritic cells stimulated with C. deneoformans was significantly suppressed by rIL-17A. In addition, rIL-17A attenuated Th1 cell differentiation in splenocytes from transgenic mice highly expressing TCR for mannoprotein 98, a cryptococcal Ag, upon stimulation with recombinant mannoprotein 98. These data suggest that IL-17A may be involved in the negative regulation of the local host defense against C. deneoformans infection through suppression of the Th1 response.

Increased Interleukin-35 suppresses peripheral CD14+ monocytes function in patients with Kawasaki disease.

BACKGROUND: Interleukin-35 (IL-35) is a newly identified IL-12 cytokine family member, which regulates the activity of immune cells in infectious diseases and autoimmune disorders. However, the regulatory function of IL-35 in Kawasaki disease is not well elucidated. METHODS: Thirty-three patients with Kawasaki disease and seventeen healthy controls were studied. Peripheral IL-35 concentration was measured by enzyme linked immunosorbent assay. CD14+ monocytes were purified, and mRNA expression of IL-35 receptor (IL-12Rß2 and gp130) was semi-quantified by real-time polymerase chain reaction. CD14+ monocytes were stimulated with recombinant IL-35. The modulatory role of IL-35 treated CD14+ monocytes to naïve CD4+ T cell activation was investigated by flow cytometry. The influence of IL-35 to cytotoxicity of CD14+ monocytes was assessed by measuring target cell death, cytokine and granzyme secretion. RESULTS: Plasma IL-35 concentration was elevated in patients with Kawasaki disease. There was no significant differences of either IL-12Rß2 or gp130 mRNA expression in CD14+ monocytes between Kawasaki disease patients and controls. IL-35 suppressed CD14+ monocytes induced naïve CD4+ T cell activation in Kawasaki disease, and this process required direct cell-to-cell contact. IL-35 also inhibited tumor necrosis factor-α and granzyme B secretion by CD14+ monocytes from patients with Kawasaki disease, however, only granzyme B was responsible for the cytotoxicity of CD14+ monocytes. CONCLUSIONS: IL-35 played an important immunosuppressive role to CD14+ monocytes function in Kawasaki disease.

The monogenic basis of human tuberculosis.

The pathogenesis of tuberculosis (TB) remains poorly understood, as no more than 5-10% of individuals infected with Mycobacterium tuberculosis go on developing clinical disease. The contribution of human genetics to TB pathogenesis has been amply documented by means of classic genetics since the turn of the twentieth century. Over the last 20 years, following-up on the study of Mendelian susceptibility to mycobacterial disease (MSMD), monogenic disorders have been found to underlie TB in some patients. Rare inborn errors of immunity, such as autosomal recessive, complete IL-12Rß1 and TYK2 deficiencies, impairing the IL-12- and IL-23-dependent induction of IFN-γ, were initially identified in a few patients. More recently, homozygosity for a common variant of TYK2 (P1104A) that selectively disrupts cellular responses to IL-23 was found in two cohorts of TB patients. It shows high penetrance in areas endemic for TB and appears to be responsible for about 1% of TB cases in populations of European descent. Both rare and common genetic etiologies of TB affect IFN-γ immunity, providing a rationale for novel preventive and therapeutic approaches for TB control, including the use of recombinant IFN-γ.

Novel nonsense IL-12Rß1 mutation associated with recurrent tuberculosis.

The interleukin (IL)-12/interferon(IFN)γ axis plays an important role in the control of mycobacterial diseases as demonstrated by the increased susceptibility to mycobacterial species in patients with an inborn error of the IL-12-dependent IFNγ immunity. Here, we report a novel mutation in the IL-12Rß1 gene in a female Pakistani patient who was born in a consanguineous marriage and developed severe bacille Calmette-Guérin (BCG) infection and recurrent tuberculosis. After reviewing the patient's clinical records, she was investigated for IL-12/IFNγ defects using enzyme-linked immunosorbent assay (ELISA), flow cytometry, and DNA genetic Sanger sequencing. Quantification of secretory cytokines from the patient's peripheral blood mononuclear cells (PBMCs) revealed significantly reduced IFNγ production. Flow cytometric analysis revealed no surface expression of IL-12Rß1 on PHA-activated T lymphocytes. In addition, IL-12-induced impaired STAT4 phosphorylation in the patient's lymphocytes when compared with those from five healthy controls. The genetic analysis of IL-12Rß1 gene identified a novel nonsense mutation c.199G>T/p.E67* within exon 3, which encodes part of the cytokine-binding region (CBR). In silico analysis indicates that this novel nonsense mutation generates a truncated protein with an apparent inactivating effect. Our data expand the genetic spectrum of IL-12Rß1 deficiency. Moreover, our findings highlight the need for developing newborn screening for patients with primary immunodeficiency associated with mycobacterial infections in areas where BCG vaccination is mandatory in order to improve the treatment of patients, and consequently their quality of life.

Three novel compound heterozygous IL12RB1 mutations in Chinese patients with Mendelian susceptibility to mycobacterial disease.

Mendelian Susceptibility to Mycobacterial Diseases (MSMD) is a primary immunodeficiency disease (PID) characterized by variable susceptibility to weakly virulent mycobacteria (Bacille Calmette-Guerin, BCG) and various intramacrophagic bacteria, fungi, parasites. Mycobacterial disease generally begins in childhood, more rarely during adolescence and adulthood. The pathogenesis of MSMD is the inherited impaired production of interferon gamma (IFN-γ) or inadequate response to it. Autosomal recessive IL12RB1 deficiency is the most common genetic etiology of MSMD. Here we identified three novel compound heterozygous mutations in IL12RB1 gene (c.635G>A, c.765delG; c.632G>C, c.847C>T; c.64G>A, c.1673insGAGCTTCCTGAG) in three Chinese families with MSMD.

Interleukin-12 receptor ß2 from grass carp: Molecular characterization and its involvement in Aeromonas hydrophila-induced intestinal inflammation.

Interleukin-12 receptor ß2 (IL-12Rß2) is a signaling subunit of heterodimeric receptors for IL-12 and IL-35. It plays important regulatory functions in the development of Th1 cells and in the expression of inflammatory cytokines in mammals and other higher vertebrates. However, little is known about IL-12Rß2 in teleost fish. In this work, we have cloned and characterized IL-12Rß2 from grass carp (Ctenopharyngodon idella). The full-length cDNA of grass carp IL-12Rß2 is 2875 bp, which encodes a mature protein with 741 amino acids. This mature protein contains three fibronectin type III domains, a transmembrane helix, and CXW and WSXWS-like motifs that are characteristic of the type I cytokine receptor family. Phylogenetic analysis revealed that cyprinid fish IL-12Rß2 formed a single branch, clearly separated from those of other vertebrates. We expressed and purified a recombinant grass carp IL-12Rß2 protein containing major antigenic regions, which was used to raise a polyclonal antibody. The specificity of the antibody was assessed by Western blotting analysis of whole cell lysates from Escherichia coli cells expressing the recombinant IL-12Rß2, grass carp intestinal intraepithelial lymphocytes, and cultured C. idella kidney cells. To explore the potential regulatory role of IL-12Rß2 in inflammation, we generated an intestinal inflammation model by anal intubation of fish with Aeromonas hydrophila. Immunohistochemical staining of the inflamed intestines revealed that IL-12Rß2 expression is consistent with inflammatory cell recruitment during intestinal inflammation. Real-time quantitative PCR revealed that IL-12Rß2 is widely expressed in normal tissues and is up-regulated in most tissues after infecting with A. hydrophila. We found that IL-12Rß2, IL-12p35, and interferon-γ were expressed in similar patterns in the intestines during inflammation. Taken together, our results suggest that IL-12Rß2 is involved in the regulation of intestinal inflammation.

Behçet's disease: An immunogenetic perspective.

Behçet's disease (BD) is a chronic and rare multisystemic disorder defined by autoimmunity and inflammatory characteristics, manifested by ocular lesions, recurrent genital and oral ulcers, skin symptoms and arthritis as well as neurological, intestinal, and vascular involvement. Despite the unknown cause of BD, there is some strong documentation for immunological, genetic, environmental, and infectious factors playing a role in the pathogenesis of BD. While the nature of the genetic variants remains unidentified, many genetic risk factors are considered to contribute to BD susceptibility. Along with human leukocyte antigen gene encoding B*51 (HLA-B*51) and areas including the major histocompatibility complex class I, genome-wide association studies have recognized numerous other BD susceptibility genes including those encoding interleukin (IL)-10, IL-12 receptor ß 2 (IL-12RB2), IL-23 receptor (IL-23R), C-C chemokine receptor 1 gene, signal transducer and activator of transcription 4 (STAT4), endoplasmic reticulum aminopeptidase (ERAP1), and genes encoding killer cell lectin-like receptor family members (KLRC4-KLRK1). It is believed that BD could be considered as a disorder lying in between autoimmune and autoinflammatory syndromes. The positive responses to classical immunosuppressive agents like azathioprine and cyclosporine and involvement of autoantigens in the initiation of the disorder are the main BD features that reflect the autoimmune nature of the disorder. In this review, we address recent findings on the role of common cytokines, antibodies and immunogenetic factors in BD.

JNK2 modulates the CD1d-dependent and -independent activation of iNKT cells.

Invariant natural killer T (iNKT) cells play critical roles in autoimmune, anti-tumor, and anti-microbial immune responses, and are activated by glycolipids presented by the MHC class I-like molecule, CD1d. How the activation of signaling pathways impacts antigen (Ag)-dependent iNKT cell activation is not well-known. In the current study, we found that the MAPK JNK2 not only negatively regulates CD1d-mediated Ag presentation in APCs, but also contributes to CD1d-independent iNKT cell activation. A deficiency in the JNK2 (but not JNK1) isoform enhanced Ag presentation by CD1d. Using a vaccinia virus (VV) infection model known to cause a loss in iNKT cells in a CD1d-independent, but IL-12-dependent manner, we found the virus-induced loss of iNKT cells in JNK2 KO mice was substantially lower than that observed in JNK1 KO or wild-type (WT) mice. Importantly, compared to WT mice, JNK2 KO mouse iNKT cells were found to express less surface IL-12 receptors. As with a VV infection, an IL-12 injection also resulted in a smaller decrease in JNK2 KO iNKT cells as compared to WT mice. Overall, our work strongly suggests JNK2 is a negative regulator of CD1d-mediated Ag presentation and contributes to IL-12-induced iNKT cell activation and loss during viral infections.

Morphological and Functional Characterization of IL-12Rß2 Chain on Intestinal Epithelial Cells: Implications for Local and Systemic Immunoregulation.

Interaction between intestinal epithelial cells (IECs) and the underlying immune systems is critical for maintaining intestinal immune homeostasis and mounting appropriate immune responses. We have previously showed that the T helper type 1 (TH1) cytokine IL-12 plays a key role in the delicate immunological balance in the gut and the lack of appropriate levels of IL-12 had important consequences for health and disease, particularly with regard to food allergy. Here, we sought to understand the role of IL-12 in the regulation of lymphoepithelial cross talk and how this interaction affects immune responses locally and systemically. Using a combination of microscopy and flow cytometry techniques we observed that freshly isolated IECs expressed an incomplete, yet functional IL-12 receptor (IL-12R) formed solely by the IL-12Rß2 chain that albeit the lack of the complementary IL-12ß1 chain responded to ex vivo challenge with IL-12. Furthermore, the expression of IL-12Rß2 on IECs is strategically located at the interface between epithelial and immune cells of the lamina propria and using in vitro coculture models and primary intestinal organoids we showed that immune-derived signals were required for the expression of IL-12Rß2 on IECs. The biological relevance of the IEC-associated IL-12Rß2 was assessed in vivo in a mouse model of food allergy characterized by allergy-associated diminished intestinal levels of IL-12 and in chimeric mice that lack the IL-12Rß2 chain on IECs. These experimental models enabled us to show that the antiallergic properties of orally delivered recombinant Lactococcus lactis secreting bioactive IL-12 (rLc-IL12) were reduced in mice lacking the IL-12ß2 chain on IECs. Finally, we observed that the oral delivery of IL-12 was accompanied by the downregulation of the production of the IEC-derived proallergic cytokine thymic stromal lymphopoietin (TSLP). However, further analysis of intestinal levels of TSLP in IL-12Rß2-/- mice suggested that this event was not directly linked to the IEC-associated IL-12Rß2 chain. We interpreted these data as showing that IEC-associated IL12Rß2 is a component of the cytokine network operating at the interface between the intestinal epithelium and immune system that plays a role in immune regulation.

Susceptibility to mycobacterial disease due to mutations in IL-12Rß1 in three Iranian patients.

In the last decade, autosomal recessive interleukin-12 receptor ß1 (IL-12Rß1) deficiency, the most common cause of Mendelian susceptibility to mycobacterial disease (MSMD), has been diagnosed in a few children and adults with severe tuberculosis in Iran. Here, we report three cases referred to the Immunology, Asthma and Allergy ward at the National Research Institute of Tuberculosis and Lung Diseases (NRITLD) at Masih Daneshvari Hospital from 2012 to 2017 with Mycobacterium tuberculosis and non-tuberculous mycobacteria infections due to defects in IL-12Rß1 but with different clinical manifestations. All three were homozygous for either an IL-12Rß1 missense or nonsense mutation that caused the IL-12Rß1 protein not to be expressed on the cell membrane and completely abolished the cellular response to recombinant IL-12. Our findings suggest that the presence of IL-12Rß1 deficiency should be determined in children with mycobacterial infections at least in countries with a high prevalence of parental consanguinity and in areas endemic for TB like Iran.

A high ratio of IL-12Rß2-positive tumor-infiltrating lymphocytes indicates favorable prognosis in laryngeal cancer.

BACKGROUND: The purpose of this study was to elucidate IL-12Rß2's roles as a tumor-associated immunological molecule, delineate the complex roles of IL-12Rß2+ tumor-infiltrating lymphocytes (TILs) and tumor cell IL-12Rß2 expression in the tumor microenvironment, and determine the correlation of IL-12Rß2+ TILs and tumor cell IL-12Rß2 expression with clinical prognosis. METHODS: We assessed mRNA and protein levels in matched laryngeal cancer tissues (LTs) and adjacent normal mucous membrane tissues (ANMMTs) from 3 laryngeal cancer (LC) patients and ratios of IL-12Rß2+ TILs in matched LTs and ANMMTs from 61 LC patients. We used the Kaplan-Meier log-rank test and Cox regression hazard ratios to analyze survival. RESULTS: Comparative proteomic and transcriptomic assays revealed that matched LTs and ANMMTs from the 3 patients had significantly different IL-12Rß2 and IFN-γ expression; the ratio of IL-12Rß2+ TILs decreased with lower degrees of tumor differentiation. Among all 61 LC patients, the IL-12Rß2+ TIL ratio in ANMMTs (38.5% ±â€¯22.8%) was significantly higher than that in LTs (29.7% ±â€¯19%; p<.001). Kaplan-Meier analysis revealed that patients with an IL-12Rß2+ TIL ratio ≥35% had significantly better survival than those with an IL-12Rß2+ TIL ratio <35% (log rank p=0.041). Multivariable analysis showed a significant association between a high IL-12Rß2+ TIL ratio and overall survival (hazard ratio, 0.14; 95% confidence interval, 0.03-0.77). CONCLUSION: Tumor cell differentiation is associated with TILs' expression of IL-12Rß2, and an IL-12Rß2+ TIL ratio ≥35%) indicates favorable prognosis in LC.

H3K27 Methylation Dynamics during CD4 T Cell Activation: Regulation of JAK/STAT and IL12RB2 Expression by JMJD3.

The changes to the epigenetic landscape in response to Ag during CD4 T cell activation have not been well characterized. Although CD4 T cell subsets have been mapped globally for numerous epigenetic marks, little has been done to study their dynamics early after activation. We have studied changes to promoter H3K27me3 during activation of human naive and memory CD4 T cells. Our results show that these changes occur relatively early (1 d) after activation of naive and memory cells and that demethylation is the predominant change to H3K27me3 at this time point, reinforcing high expression of target genes. Additionally, inhibition of the H3K27 demethylase JMJD3 in naive CD4 T cells demonstrates how critically important molecules required for T cell differentiation, such as JAK2 and IL12RB2, are regulated by H3K27me3. Our results show that H3K27me3 is a dynamic and important epigenetic modification during CD4 T cell activation and that JMJD3-driven H3K27 demethylation is critical for CD4 T cell function.

Gain-of-function STAT1 mutations are associated with intracranial aneurysms.

Chronic mucocutaneous candidiasis, characterized by persistent or recurrent fungal infections, represents the clinical hallmark in gain-of-function (GOF) signal transducer and activator of transcription 1 (STAT1) mutation carriers. Several cases of intracranial aneurysms have been reported in patients with GOF STAT1 mutation but the paucity of reported cases likely suggested this association still as serendipity. In order to endorse this association, we link the development of intracranial aneurysms with STAT1 GOF mutation by presenting the two different cases of a patient and her mother, and demonstrate upregulated phosphorylated STAT4 and IL-12 receptor ß1 upon stimulation in patient's blood cells. We also detected increased transforming growth factor (TGF)-ß type 2 receptor expression, particularly in CD14+ cells, and a slightly higher phosphorylation rate of SMAD3. In addition, the mother of the patient developed disseminated bacille Calmette-Guérin disease after vaccination, speculating that GOF STAT1 mutations may confer a predisposition to weakly virulent mycobacteria.

Evaluation of IL-12RB1, IL-12B, CXCR-3 and IL-17a expression in cases affected by a non-healing form of cutaneous leishmaniasis: an observational study design.

INTRODUCTION: Seldom cutaneous leishmaniasis (CL) may present as a lasting and active lesion(s), known as a non-healing form of CL (NHCL). Non-functional type 1 T helper (Th1) cells are assumed the most important factor in the outcome of the disease. The present study aims to assess some molecular defects that potentially contribute to Th1 impairment in NHCL. METHODS AND ANALYSIS: This prospective observational study will be implemented among five groups. The first and second groups comprise patients afflicted with non-healing and healing forms of CL, respectively. The third group consists of those recovered participants who have scars as a result of CL. Those participants who have never lived or travelled to endemic areas of leishmaniasis will comprise the fourth group. The fifth group comprises participants living in hyperendemic areas for leishmaniasis, although none of them have been afflicted by CL. The aim is to recruit 10 NHCL cases and 30 participants in each of the other groups. A leishmanin skin test (LST) will be performed to assess in vivo immunity against the Leishmania infection. The cytokine profile (interleukin (IL)-12p70, interferon (IFN)-γ, C-X-C motif chemokine ligand (CXCL)-11 and IL-17a) of the isolated peripheral blood mononuclear cells (PBMCs) will be evaluated through ELISA. Real-time PCR will determine the C-X-C motif chemokine receptor (CXCR)-3 and IL-17a gene expression and expression of IL-12Rß1 will be assessed by flow cytometry. Furthermore, IL-12B and IL-12RB1 mutation analysis will be performed. DISCUSSION: It is anticipated that the outcome of the current study will identify IL-12B and IL-12RB1 mutations, which lead to persistent lesions of CL. Furthermore, our expected results will reveal an association between NHCL and pro-inflammatory cytokines (IL-12p70, IFN-γ IL-17a and CXCL-11), as well as CXCR-3 expression. ETHICS AND DISSEMINATION: This study has been approved by a local ethical committee. The final results will be disseminated through peer-reviewed journals and scientific conferences.

Differential Expression of Long Noncoding RNAs During Cardiac Allograft Rejection.

BACKGROUND: Organ transplantation is the most effective treatment for end-stage diseases. Although transplant rejection is the major restriction in successful long-lasting graft survival, induction of sustainable immune tolerance against transplant is one of the major goals in transplantation to enable long-term graft survival. Although various mechanisms have been suggested that induce immune tolerance during transplantation, the roles of long noncoding RNAs (lncRNAs), which modulate gene expression and regulate innate and adaptive immune responses, are not clearly understood in transplantation. Here, we described the role of 2 essential lncRNAs, lncRNA-A930015D03Rik and mouselincRNA1055, in regulating T helper 1 (Th1) response in graft rejection. METHODS: To understand the gene expression and lncRNA profile during transplantation, we performed microarray to profile lncRNA and messenger (m)RNA of the heart graft and graft-infiltrating lymphocytes (GILs) in allogeneic and syngeneic mouse heart transplantation model. We screened the differentially expressed lncRNAs and mRNAs, and generated the network of lncRNA-mRNA coexpression and computationally predicted their association in transplantation. We further validated the selected T cell related lncRNAs by qPCR, which we identified in gene set enrichment analysis. The functional validation of these lncRNAs in the regulation of Th1 response in transplantation was performed by short hairpin RNA-mediated inhibition. RESULTS: We established a profile of lncRNA and mRNA, which are differentially expressed during transplant rejection in mouse model of heart transplant. Consistent with the microarray results, we have confirmed and validated the expression of 7 lncRNA by qPCR. The lncRNA-A930015D03Rik and mouselincRNA1055 were highly expressed in allogeneic heart graft and GILs. We further identified that expression of IL-12Rß1 is strongly correlated with lncRNA-A930015D03Rik and mouselincRNA1055 in GILs. Further analysis revealed the association of lncRNA-A930015D03Rik and mouselincRNA1055 with Th1 cells in graft rejection. The functions of lncRNA-A930015D03Rik and mouselincRNA1055 were validated in differentiation of Th1 cells by knocking down their expression. Inhibition of lncRNA-A930015D03Rik and mouselincRNA1055 substantially suppressed the expression of IL-12Rß1 and IFN-γ induction in Th1 cells. CONCLUSIONS: Our results provide a detailed profile of lncRNAs that may regulate immune response and graft outcomes. Our data not only suggest the involvement of lncRNA-A930015D03Rik and mouselincRNA1055 in the regulation of Th1 cells response during graft rejection but also identify them as novel biomarkers for subclinical graft rejection.