Presence Of IL-2 Research Articles

JNK signaling during IL-3-mediated differentiation contributes to the c-kit-potentiated allergic inflammatory capacity of mast cells.

Mast cells are leukocytes that mediate various aspects of immunity and drive allergic hypersensitivity pathologies. Mast cells differentiate from hematopoietic progenitor cells in a manner that is largely IL-3 dependent. However, molecular mechanisms, including the signaling pathways that control this process, have yet to be thoroughly investigated. Here, we examine the role of the ubiquitous and critical mitogen-activated protein kinase signaling pathway due to its position downstream of the IL-3 receptor. Hematopoietic progenitor cells were harvested from the bone marrow of C57BL/6 mice and differentiated to bone marrow-derived mast cells in the presence of IL-3 and mitogen-activated protein kinase inhibitors. Inhibition of the JNK node of the mitogen-activated protein kinase pathway induced the most comprehensive changes to the mature mast cell phenotype. Bone marrow-derived mast cells differentiated during impaired JNK signaling expressed impaired c-kit levels on the mast cell surface, first detected at week 3 of differentiation. Following 1 wk of inhibitor withdrawal and subsequent stimulation of IgE-sensitized FcεRI receptors with allergen (TNP-BSA) and c-kit receptors with stem cell factor, JNK-inhibited bone marrow-derived mast cells exhibited impediments in early-phase mediator release through degranulation (80% of control), as well as late-phase secretion of CCL1, CCL2, CCL3, TNF, and IL-6. Experiments with dual stimulation conditions (TNP-BSA + stem cell factor or TNP-BSA alone) showed that impediments in mediator secretion were found to be mechanistically linked to reduced c-kit surface levels. This study is the first to implicate JNK activity in IL-3-mediated mast cell differentiation and also identifies development as a critical and functionally determinative period.

Clonally-Expanded, Thyrotoxic Autoimmune Mediator CD8 +T cells Driven by IL21 Contribute to Checkpoint Inhibitor Thyroiditis

Abstract Autoimmune toxicity occurs in up to 60% of patients treated with immune checkpoint inhibitor (ICI) cancer therapy and is an increasing clinical challenge with the expanding use of these treatments. Previous human studies into the cause of immune related adverse events (IRAEs) have often evaluated only circulating immune cells rather than affected tissues. Here, we directly sampled thyroid specimens from subjects with ICI-thyroiditis, one of the most common IRAEs, to determine driving mechanisms. We compared immune infiltrates in ICI-thyroiditis to those in subjects with spontaneous autoimmune Hashimoto’s thyroiditis (HT) or no thyroid disease. Single cell RNA sequencing revealed a dominant, clonally expanded population of thyroid-infiltrating cytotoxic CXCR6 +CD8 +T cells (“CD8 +autoimmune mediators”) present in ICI-thyroiditis, but not HT or healthy subjects. Furthermore, we identified a crucial role for interleukin 21, a cytokine secreted by intrathyroidal T follicular (Tfh) and T peripheral helper (Tph) cells, as a driver of these thyrotoxic CD8 +autoimmune mediators. In the presence of IL21, human CD8 +T cells acquired the autoimmune mediator phenotype with upregulation of cytotoxic molecules (IFNg, granzyme); the chemokine receptor CXCR6; and thyrotoxic capacity. We validated these findings in vivo using a novel mouse model of IRAEs, and further demonstrated that genetic inhibition of IL21 signaling protected ICI-treated mice from thyroid autoimmunity. Taken together, our data reveal novel mechanisms by which IL21 +Tfh/Tph cells drive thyrotoxic CD8 +autoimmune mediators for the development of IRAEs in humans. Furthermore, these studies highlight IL21 as a potential therapeutic target in patients to prevent IRAEs. Supported by grants from the NIH NIDDK (K08 DK129829), the American Thyroid Association, and the Aramont Charitable Foundation.

Mechanistic investigations of granzyme A adjuvant effects on γ 9δ 2T cells

Abstract γδ T cells are ideal effector cells against cancer and pathogens for numerous reasons: they are found in blood, mucosa, and lymphoid organs, develop memory responses independent of MHC restriction, present antigen, produce cytokines, and are cytolytic. An obstacle for the development of biomedical interventions targeting γδ T cells is the need for an adjuvant that promotes their expansion and is suitable for human use. The adjuvant routinely used, IL-2, has multiple drawbacks limiting its use for vaccination. Our lab has shown that memory γ 9δ 2T cells, induced by BCG vaccination, are expanded in vitro by M. tuberculosis, M.bovis(BCG), and our lab’s vaccine candidate 6-O-methylglucoselipopolysaccharide (mGLP), in the presence of IL-2. Additionally, the expanded γ 9δ 2T cells inhibit intracellular mycobacterial growth. This inhibition is dependent on granzyme A (GzmA) production by γ 9δ 2T cells, and GzmA alone can inhibit intracellular mycobacterial growth. We recently identified that GzmA can also promote expansion of γδ T cells, without exogenous IL-2, in response to mGLP and HMBPP, a phosphoantigen that stimulates all γ 9δ 2T cells. To identify γδ T cell adjuvants, in vitro expansion assays were performed using human peripheral blood mononuclear cells, dendritic cells, antigen, and adjuvant. The absolute numbers of expanded effector γδ T cells were calculated using flow cytometry after 7 days of culture. We identified that the ability of GzmA to promote γδ T cell expansion is dependent upon TLR4, CD14, and GzmA homodimerization, but independent of GzmA enzymatic activity. This new information has broad implications for vaccine design, promoting γδ T cell responses, and understanding the role of GzmA in infectious disease and cancer. Supported by grants from NIH (R01 AI048391) and Bill & Melinda Gates Foundation (OP1118659)

The role of interleukin-12-PRMT5 axis in generating suppressive Th1-like induced regulatory T cells

Abstract After activation, naïve CD4 T cells can differentiate into T helper cell subsets (Th1, Th2, Th17, and Follicular helper T cells) or into regulatory T cells (Tregs), depending on the type of antigen-presenting cells (APC) and cytokine milieu encountered during differentiation. Recent studies suggest that CD4+Foxp3+ Tregs can produce the pro-inflammatory cytokine IFNg when stimulated in a Th1 cytokine environment, such as in the presence of IL-12, and stimulation under these conditions makes iTregs sensors for inflammatory cytokines. The stability and suppressive functions of the iTregs depend on multiple factors. The goal of this study is to explore an epigenetic enzyme protein arginine methyltransferase 5 (PRMT5) and to understand the significance of this protein in the development of Th1-like iTregs. PRMT5 can either represses or activate transcription of target genes via symmetric di-methylation of arginine residues on histones. However, the role of PRMT5 in the differentiation of Th1-like iTregs has not been thoroughly investigated. We demonstrate that differentiating iTregs in a Th1 cytokine environment generated a specific population of Th1-like iTregs with increased suppressive function. In Th1-like iTregs, we observed symmetric arginine di-methylation on histone (H3R2me2s) mediated by PRMT5, while this epigenetic marker is absent in iTregs. We also demonstrate that si-RNA mediated knockdown of PRMT5 reduced the suppressive capacity of Th1-like iTregs. Therefore, we hypothesize that the IL12-PRMT5 axis is crucial for developing suppressive Th1-like iTregs. Goals of further research are to generate stable immunosuppressive iTregs for therapeutic use.

Severe asthma ILC2s demonstrate enhanced proliferation that is modified by biologics.

Type 2 (T2) innate lymphoid cells (ILC2s) contribute to airway inflammation and disease in asthma. We hypothesize that ILC2s isolated from people with severe allergic and eosinophilic asthma would exhibit an enhanced T2 inflammatory activity that would be altered following treatment with mepolizumab and omalizumab. We compare peripheral blood (PB) isolated ILC2's proliferative capacity, IL-5 and IL-13 secretion and phenotype between healthy without asthma (HC), non-asthma allergic (NAA), mild asthma (MA) and severe allergic and eosinophilic asthma (SA) subjects. We then determined the impact of 6 months treatment with either mepolizumab or omalizumab on ILC2s physiology of SA subjects. ILC2s were sorted and cultured in the presence of IL-2, IL-25, IL-33 and thymic stromal lymphopoietin (TSLP) for 14 days. ILC2s proliferation, phenotypes and functions were assessed using flowcytometry. The ILC2s response was then reassessed following clinically successful treatment of SA subjects with mepolizumab and omalizumab. SA ILC2s demonstrated increased proliferative capacity, TSLP receptor (TSLPR), GATA3 and NFATc1 protein expressions and increased IL-5 and IL-13 release. ILC2s were also capable of releasing IL-6 in response to stimulation. Mepolizumab treatment reduced ILC2s proliferative capacity and expression of TSLPR, GATA3 and NFATc1. Both mepolizumab and omalizumab were associated with reduced ILC2s release of IL-5 and IL-13, only mepolizumab reduced IL-6. ILC2s from severe allergic and eosinophilic asthma demonstrated an active phenotype typified by increased proliferation, TSLPR, GATA3 and NFATc1 expression and increased IL-5, IL-13 and IL-6 release. Mepolizumab reduced markers of ILC2s activation.

MCAM+CD161- Th17 Subset Expressing CD83 Enhances Tc17 Response in Psoriasis.

Recent studies have highlighted the pathogenic roles of IL-17-producing CD8+ T cells (T-cytotoxic 17 [Tc17]) in psoriasis. However, the underlying mechanisms of Tc17 induction remain unclear. In this study, we focused on the pathogenic subsets of Th17 and their mechanism of promotion of Tc17 responses. We determined that the pathogenic Th17-enriched fraction expressed melanoma cell adhesion molecule (MCAM) and CCR6, but not CD161, because this subset produced IL-17A abundantly and the presence of these cells in the peripheral blood of patients has been correlated with the severity of psoriasis. Intriguingly, the serial analysis of gene expression revealed that CCR6+MCAM+CD161-CD4+ T cells displayed the gene profile for adaptive immune responses, including CD83, which is an activator for CD8+ T cells. Coculture assay with or without intercellular contact between CD4+ and CD8+ T cells showed that CCR6+MCAM+CD161-CD4+ T cells induced the proliferation of CD8+ T cells in a CD83-dependent manner. However, the production of IL-17A by CD8+ T cells required exogenous IL-17A, suggesting that intercellular contact via CD83 and the production of IL-17A from activated CD4+ T cells elicit Tc17 responses. Intriguingly, the CD83 expression was enhanced in the presence of IL-15, and CD83+ cells stimulated with IL-1β, IL-23, IL-15, and IL-15Rα did not express FOXP3. Furthermore, CCR6+MCAM+CD161-CD4+ T cells expressing CD83 were increased in the peripheral blood of patients, and the CD83+ Th17-type cells accumulated in the lesional skin of psoriasis. In conclusion, pathogenic MCAM+CD161- Th17 cells may be involved in the Tc17 responses via IL-17A and CD83 in psoriasis.

Dual IL-6 and CTLA-4 blockade regresses pancreatic tumors in a T cell- and CXCR3-dependent manner.

This study aimed to enhance antitumor immune responses to pancreatic cancer via Ab-based blockade of IL-6 and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Mice bearing s.c. or orthotopic pancreatic tumors were treated with blocking Abs to IL‑6 and/or CTLA-4. In both tumor models, dual IL-6 and CTLA-4 blockade significantly inhibited tumor growth. Additional investigations revealed that dual therapy induced an overwhelming infiltration of T cells into the tumor as well as changes in CD4+ T cell subsets. Dual blockade therapy elicited CD4+ T cells to secrete increased IFN-γ in vitro. Likewise, in vitro stimulation of pancreatic tumor cells with IFN-γ profoundly increased tumor cell production of CXCR3-specific chemokines, even in the presence of IL-6. In vivo blockade of CXCR3 prevented orthotopic tumor regression in the presence of the combination treatment, demonstrating a dependence on the CXCR3 axis for antitumor efficacy. Both CD4+ and CD8+ T cells were required for the antitumor activity of this combination therapy, as their in vivo depletion via Abs impaired outcomes. These data represent the first report to our knowledge of IL-6 and CTLA‑4 blockade as a means to regress pancreatic tumors with defined operative mechanisms of efficacy.

Abstract 3200: Non-engineered T cells with specific anticancer effects in KRAS-mutated colorectal cancer

Abstract Cytokine-induced killer (CIK) cell therapy is known as a useful treatment for cancer immunotherapy due to its anticancer activity and safety. However, its clinical efficacy is limited due to its lack of antigen specificity and low in vivo persistence. KRAS mutations play an important role in promoting tumorigenesis in cancer. But, its clinical application as a biomarker for cancer treatment is still difficult. Non-engineered T cell therapy approaches could be a promising strategy for treating KRAS-mutated cancer. Herein we developed non-engineered T cells empowered with specific anticancer effects in KRAS-mutated colorectal cancer (CRC), and investigated its properties and anticancer effects compared to CIK cells. A recombinant overlapping peptide was designed to overlap the KRAS protein by 15 amino acids linked by a cathepsin S cleavage site (LRMK). It contained KRASG12D, KRASG12V, KRASG13D, and KRASWT epitope peptides and other KRAS protein regions. Peripheral blood mononuclear cells (PBMCs) from CRC patients with KRAS mutations were stimulated with the recombinant overlapping peptides in vitro for two days in the presence of interleukin (IL)-4, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF)-α, IL-1β, and prostaglandin 2, and expanded using the 12-day CIK cell expansion method in the presence of IL-2 (200 IU/mL). To verify the enrichment of KRAS mutant-specific T cells, the expanded T cells were restimulated by pulsed monocyte-derived dendritic cells with KRAS mutant antigen for two days and the frequency of KRAS mutation-specific T cells was determined using the interferon (INF)-γ capture assay. Red fluorescent protein (RFP)-expressing HLA-matched cell lines and cell line-derived spheroids T3M10 (KRASG12D) and MCF-7 (KRASWT) were used for the Incucyte immune cell killing assay. T-cell phenotypes were determined by flow cytometry. The expanded non-engineered T cells stimulated with KRAS mutant antigen showed skewed CD4 T cell differentiation and increased central memory T cells compared to CIK cells, suggesting that they may have more long-term immunity and functionality than CIK cells. The KRASG12D-specific T cells were enriched 12-13% (9.4-fold) by in vitro stimulation compared to 1.3% in CIK cells. Anticancer activity in a 2D-culture model was significantly higher in KRASG12D-specific enriched T cells (82.5%) than in CIK cells (64.9%) co-cultured with T3M10 cells for 48 h. In a 3D spheroid-culture model, increased cytotoxicity was also detectable in the KRASG12D-specific enriched T cells (13.2% at 72 h and 14.1% at 142 h) compared to CIK cells. These results demonstrated that non-engineered T cells with KRAS mutated-specific anticancer activity are an ideal candidate for KRAS mutant cancer-targeted immunotherapy. Further studies are necessary to verify the anticancer effects in vivo. Citation Format: Sunki Lim, Yujeong Gho, Seoyoung Kim, Min Ki Kim, Jong Seob Park, Wang Jun Lee, Young-Kwan Lee, Hyoun Jong Moon, Excelsisbio.Inc, Goyang, Korea. Non-engineered T cells with specific anticancer effects in KRAS-mutated colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3200.

Abstract 4076: 4-1bb selection augments DC/AML fusion vaccine-educated T cells for adoptive cell therapy

Abstract Introduction: Our group has pioneered a novel vaccine by fusion of patient-derived tumor with autologous dendritic cells (DCs) that presents an array of tumor antigens generating a polyclonal immune response. DC/AML vaccination led to expansion of leukemia-specific T cells with survival benefit in a phase II clinical trial. We postulated that ex vivo generation of vaccine-educated T cells would provide a powerful platform for adoptive immunotherapy with opportunity to augment T-cell functional potency prior to infusion. We report on an ex vivo system in which vaccine-educated T cells are further enriched for activated antigen-specific effector cells via an agonistic 4-1bb antibody. We report phenotypic and functional characteristics of 4-1bb-enriched vaccine-educated T cells. Methods: DC/AML fusion vaccines were generated from C57BL/6J mice DCs and syngeneic C1498 mCh/luc+ AML cells. Splenic T cells were co-cultured with autologous irradiated DC/AML fusions in presence of IL-2/7/15. Selection with biotinylated agonistic 4-1bb (3H3) was performed on vaccine-educated T cells followed by expansion with anti-CD3/CD28 activation beads. T cells were phenotyped for activation (CD25/CD69), immune checkpoints (PD1/LAG3/TIM3), memory (CD44+CD62L-) and enrichment (anti-rat H&L). Cytotoxicity was evaluated by luminescence. Mice were inoculated with C1498 and injected with T cells 7 days later. BLI imaging was performed and 100-day survival measured. Results: Vaccine-educated T cells demonstrated evidence of immune activation and memory phenotype compared to unstimulated naïve T-cell controls (TN) (7.24-fold, CD4+CD25+CD69+; 1.7-fold, CD3+CD44+CD62L-). Vaccine-educated T cells selected based on 4-1bb expression showed enhanced markers of activation (15.3-fold, CD4+CD25+CD69+) and memory phenotype (5-fold, CD3+CD44+CD62L-) compared to TN. Selection enriched for 4-1bb+ vaccine-educated T cells resulting in enhanced antigen-specific recognition as measured by tumor lysate induction of IFNg expression. Tumor specificity and activation was maintained following CD3/CD28-mediated expansion. The 4-1bb+ vaccine-educated T cells showed enhanced cytotoxicity (1.9-fold increase/TN at 10:1 E/T, P<0.0001). Phenotypic and functional analysis support 3-5 days as the optimal duration of time for T-cell vaccine education. In vivo, 60% of mice treated with 4-1bb+ vaccine-educated cells were alive at 60 days vs. 20% treated with unselected vaccine-educated cells. Conclusion: We demonstrate that vaccine-educated T cells subject to selection via an agonist 4-1bb antibody confer enhanced tumor selectivity and potency. Optimal duration for T-cell education was 3-5 days. T-cell stimulation and enrichment by agonistic 4-1bb selection enhanced cytotoxicity and memory phenotype. Thus, 4-1bb selection is a novel approach for antigen-specific T-cell enrichment for superior adoptive immunotherapy in AML. Citation Format: Kathrine S. Rallis*, Jessica Liegel*, Giulia Cheloni, Dina Stroopinsky, Poorva Bindal, Kenel Dufort, Daniela Torres, Isabella Saldarriaga, Samuel Herzlinger, Abigael Morin, Raphael Kesselman, Jeremy Rosenbaum, Georges Chedid, Sophia Adamia, Donald Kufe, Jacalyn Rosenblatt, David Avigan. 4-1bb selection augments DC/AML fusion vaccine-educated T cells for adoptive cell therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4076.

Can montelukast sodium be an alternative treatment in the treatment of interstitial cystitis?

The leukotriene D4 receptors have been detected in human bladder detrusor myocytes, and they can play the role of interstitial cystitis etiology. Our study aims to explain the role of mast cells histologically and immunohistochemically in the pathogenesis and the effectiveness of montelukast that leukotriene D4 receptor antagonist in the treatment of interstitial cystitis. Twenty-four Wistar albino adult female rats were used. Group 1 (n = 8): control (sham) group, Group 2 (n = 8): interstitial cystitis group, and Group 3 (n = 8): treatment group. Groups 2 and 3 rats were administered 75 mg/kg cyclophosphamide four times every three days intraperitoneally. The rats in the treatment group were started on montelukast sodium as 10 mg/kg, 1 × 1/day per orally after the last administration of cyclophosphamide and were given for 14 days. Mast cells in the bladder tissues were examined histologically, and the presence of IL-6, 8, VEGF, and TNF alpha was examined immunohistochemically. Thin transitional epithelium, loose connective tissue, weak smooth muscle bundles, and signs of chronic inflammation were observed in the interstitial cystitis group. Regenerated transitional epithelium, intact basement membrane, compact lamina propia, thick smooth muscle bundles, and rare inflammatory cells were observed after the treatment with the montelukast. Mast cells were decreased in bladder tissue after treatment. IL-6, IL-8, VEGF, and TNF alpha levels were significantly decreased after treatment. We found that inflammatory mediators were significantly reduced after treatment with montelukast in the interstitial cystitis group. Montelukast can be used as an effective drug in the treatment of interstitial cystitis.

IL-12 and IL-27 Promote CD39 Expression on CD8+ T Cells and Differentially Regulate the CD39+CD8+ T Cell Phenotype.

Tumor-specific CD8+ T cells are critical components of antitumor immunity; however, factors that modulate their phenotype and function have not been completely elucidated. Cytokines IL-12 and IL-27 have recognized roles in promoting CD8+ T cells' effector function and mediated antitumor responses. Tumor-specific CD8+ tumor-infiltrating lymphocytes (TILs) can be identified based on surface expression of CD39, whereas bystander CD8+ TILs do not express this enzyme. It is currently unclear how and why tumor-specific CD8+ T cells uniquely express CD39. Given the important roles of IL-12 and IL-27 in promoting CD8+ T cell functionality, we investigated whether these cytokines could modulate CD39 expression on these cells. Using invitro stimulation assays, we identified that murine splenic CD8+ T cells differentially upregulate CD39 in the presence of IL-12 and IL-27. Subsequently, we assessed the exhaustion profile of IL-12- and IL-27-induced CD39+CD8+ T cells. Despite the greatest frequency of exhausted CD39+CD8+ T cells after activation with IL-12, as demonstrated by the coexpression of TIM-3+PD-1+LAG-3+ and reduced degranulation capacity, these cells retained the ability to produce IFN-γ. IL-27-induced CD39+CD8+ T cells expressed PD-1 but did not exhibit a terminally exhausted phenotype. IL-27 was able to attenuate IL-12-mediated inhibitory receptor expression on CD39+CD8+ T cells but did not rescue degranulation ability. Using an immunogenic neuro-2a mouse model, inhibiting IL-12 activity reduced CD39+CD8+ TIL frequency compared with controls without changing the overall CD8+ TIL frequency. These results provide insight into immune regulators of CD39 expression on CD8+ T cells and further highlight the differential impact of CD39-inducing factors on the phenotype and effector functions of CD8+ T cells.

Natural killer cells suppress human cutaneous squamous cell carcinoma cancer cell survival and tumor growth.

Cutaneous squamous cell carcinoma (CSCC), which develops in response to ultraviolet irradiation exposure, is among the most common cancers. CSCC lesions can be removed by surgical excision, but 4.5% of these cancers reappear as aggressive and therapy-resistant tumors. CSCC tumors display a high mutation burden, and tumor frequency is dramatically increased in immune-suppressed patients, indicating a vital role for the immune system in controlling cancer development. Natural killer cells (NK cells) play a key role in cancer immune surveillance, and recent studies suggest that NK cells from healthy donors can be expanded from peripheral blood for use in therapy. In the present study, we test the ability of ex vivo expanded human NK cells to suppress the CSCC cell cancer phenotype and reduce tumor growth. We expanded human NK cells from multiple healthy donors, in the presence of IL-2, and tested their ability to suppress the CSCC cell cancer phenotype. NK cell treatment produced a dose-dependent reduction in SCC-13 and HaCaT cell spheroid growth and matrigel invasion and induced SCC-13 and HaCaT cell apoptosis as evidenced by increased procaspase 9, procaspase 3, and PARP cleavage. Moreover, two important CSCC cell pro-cancer signaling pathways, YAP1/TAZ/TEAD and MEK1/2-ERK1/2, were markedly reduced. Furthermore, tail-vein injection of NK cells markedly suppressed the growth of SCC-13 xenograft tumors in NSG mice, which was also associated with a reduction in YAP1 and MEK1/2-P levels and enhanced apoptosis. These findings show that NK cell treatment suppresses CSCC cell spheroid formation, invasion, viability, and tumor growth, suggesting NK cell treatment may be a candidate therapy for CSCC.

B. abortus Infection Promotes an Imbalance in the Adipocyte-Osteoblast Crosstalk Favoring Bone Resorption.

Osteoarticular injury is the most common presentation of active brucellosis in humans. Osteoblasts and adipocytes originate from mesenchymal stem cells (MSC). Since those osteoblasts are bone-forming cells, the predilection of MSC to differentiate into adipocytes or osteoblasts is a potential factor involved in bone loss. In addition, osteoblasts and adipocytes can be converted into each other according to the surrounding microenvironment. Here, we study the incumbency of B. abortus infection in the crosstalk between adipocytes and osteoblasts during differentiation from its precursors. Our results indicate that soluble mediators present in culture supernatants from B. abotus-infected adipocytes inhibit osteoblast mineral matrix deposition in a mechanism dependent on the presence of IL-6 with the concomitant reduction of Runt-related transcription factor 2 (RUNX-2) transcription, but without altering organic matrix deposition and inducing nuclear receptor activator ligand kβ (RANKL) expression. Secondly, B. abortus-infected osteoblasts stimulate adipocyte differentiation with the induction of peroxisome proliferator-activated receptor γ (PPAR-γ) and CCAAT enhancer binding protein β (C/EBP-β). We conclude that adipocyte-osteoblast crosstalk during B. abortus infection could modulate mutual differentiation from its precursor cells, contributing to bone resorption.

STAT6 controls the stability and suppressive function of regulatory T cells.

Signal transducer and activator of transcription 6 (STAT6) promotes tumorigenesis by decreasing the Forkhead box P3+ (Foxp3+) cell frequency allowing for the infiltration of inflammatory cells during the early stages of colitis-associated cancer (CAC). In this study, we dissected the role of STAT6 in the generation of inducible in vitro regulatory T cells (iTregs) and peripheral in vivo Tregs (pTregs) under inflammatory conditions. In in vitro assays, when STAT6 was lacking, iTregs preserved a stable phenotype and expressed high levels of Foxp3 and CD25 during long expansion periods, even in the presence of IL-6. This effect was associated with increased in vitro suppressive ability, over-expression of programmed death-1 (PD-1), CTLA-4, and Foxp3, and decreased IFN-γ expression. Furthermore, iTregs developed during STAT6 deficiency showed a higher demethylation status for the FOXP3 Treg-specific demethylated region (TSDR), coupled with lower DNA methyltransferase 1 (DNMT1) mRNA expression, suggesting that STAT6 may lead to Foxp3 silencing. Using a mouse model of CAC, the STAT6-/- pTregs expressed a more activated phenotype at the intestine, had higher suppressive capacity, and expressed more significant levels of PD-1 and latency-associated peptide of TGF-β (LAP) associated with their ability to attenuate tumor development. These data suggest that STAT6 signaling impairs the induction, stability, and suppressive capacity of Tregs developed in vitro or in vivo during gut inflammation.

Estimation of Sensitization Status in Renal Transplant Recipients by Assessing Indirect Pathway CD4 + T Cell Response to Donor Cell-pulsed Dendritic Cell.

Generation of donor-specific human leukocyte antigen antibody (DSA) via indirect allorecognition is detrimental to long-term survival of transplant organs. The detection of such immune responses would make it possible to define patients with high risk of sensitization. In this study, we established a novel method for evaluating indirect allorecognition to assess sensitization in kidney transplant recipients. Recipient CD14 + monocytes were mixed with donor peripheral blood mononuclear cells; cultured in the presence of IL-4, GM-CSF, IL-1β, and TNFα; and used as pulsed dendritic cells (DCs). Cell proliferation and cytokine production were evaluated by carboxyfluorescein diacetate succinimidyl ester-based T cell proliferation assay and Enzyme-Linked ImmunoSpot assay, respectively. CD4 + T cell proliferation was strongly observed in following coculture with allogeneic antigen-pulsed DC leading to interferon-γ and IL-21 production. About 1% of CD4 + T cells exhibited Tfh-like phenotype (PD-1 high CXCR5 + ICOS + CD40L + ). Recipient DC pulsed with donor peripheral blood mononuclear cells was cocultured with recipient CD45RA+CD4+ and CD45RA-CD4+ (generally defined as naive and memory in humans, respectively) T cells. Irrespective of preformed or de novo DSA status, CD45RA + CD4 + T cells constantly produced IL-21. In contrast, IL-21-produced CD45RA - CD4 + T cells were significantly higher in preformed DSA-positive patients than those in negative patients (80.8 ± 51.2 versus 14.8 ± 20.4, P < 0.001). In de novo DSA-positive patients, IL-21-produced CD45RA - CD4 + T cells were significantly increased after transplantation compared with before transplantation (9.23 ± 9.08 versus 43.9 ± 29.1, P < 0.001). Assessment of indirect pathway CD4 + T cell response could provide new insights into the underlying mechanism of de novo DSA production, leading to the development of effective strategies against antibody-mediated rejection.

In situ expression of Th17 immunologic mediators in American cutaneous leishmaniasis caused by Leishmania (V.) braziliensis and Leishmania (L.) amazonensis in the Brazilian Amazon

American cutaneous leishmaniasis (ACL) presents a wide spectrum of clinical and immunopathological manifestations. In Brazil, Leishmania (L.) amazonensis[La] and Leishmania(V.)braziliensis[Lb] show the highest pathogenic potential for humans causing different clinical forms: localized cutaneous leishmaniasis (LCL : Lb/La), anergic diffuse cutaneous leishmaniasis (ADCL : La) and mucocutaneous leishmaniasis (MCL : Lb). ADCL and MCL are the most severe forms and infection leads to a cellular immune response at the hyposensitivity and hypersensitivity poles. Th17-cells are involved in the ACL pathogenesis, are derived from naïve TCD4+ cells regulated by RORγt, differentiate in presence of IL-6, TGF-β, IL- 1β, IL-23 and express IL-17. Aim of this study was to characterize the cellular immune response mediated by Th17-profile cells through in situ determination of the expression of RORγt, IL-17, IL-6, TGF-β, IL-1β, and IL-23 in the ACL clinical-immunopathological spectrum caused by L.(L.)amazonensis and L.(V.)braziliensis. Biopsies of skin and mucosal lesions from forty patients including ADCL(n=8), LCL[La](n=17), LCL[Lb](n=9) and MCL(n=6), were examined by immunohistochemistry. The immunostained cells density (cells/mm2) was determined in image analysis system using AxionVision 4.8 software (Zeiss). As the disease evolution time (DET) was different among ACL patients, the effect of DET on the expression of immunological markers was evaluated in different clinical forms and histopathological changes, using ANCOVA. Our results showed significantly increased expression of RORγt, IL-17, IL-6, IL-1β and IL-23 in patients with ACL polar forms (ADCL and MCL); higher TGF-β expression was found in ADCL. DET influenced the expression of RORγt and IL-6 in: clinical forms of ACL and in categories of parasitism. DET also affected the production of RORγt, IL-17, IL-6, TGF-β and IL-1β in types of inflammatory infiltrate, evidencing that DET had effect on the expression of Th17 profile cytokines in ACL. Together, the expression of immunological mediators of Th17 profile in the ACL spectrum, as well as the DET effect, demonstrate the participation of this cell lineage in the immunopathogenesis of ACL, mainly in the polar and more severe forms of ACL spectrum. The dubious role played by Th17-cells may favors immune response suppression and parasitic persistence in ADCL, while in MCL it contributes to an exacerbated immune response and parasite scarcity.

Pan-HDAC inhibitors augment IL2-induced proliferation of NK cells via the JAK2-STAT5B signaling pathway

BackgroundNatural killer (NK) cells are a subtype of lymphocytes with the ability to quickly and efficiently identify and eliminate tumor cells. In the presence of IL2, NK cells can divide rapidly but in limited numbers. According to previous studies, in vivo treatment with histone deacetylase (HDAC) inhibitors did not impair NK-cell function. This study aimed to investigate the effect of HDAC inhibitors on NK-cell proliferation and the underlying regulatory mechanism. MethodsNK92 cells, primary NK (pNK) cells, and CD19-CAR-NK92 cells were treated with low concentrations of pan-HDACi Dacinostat (Dac) and Panobinostat (Pan) in the presence of IL2, and Cell Counting Kit-8 (CCK8), 5-ethynyl-2′-deoxyuridine (EdU), and flow cytometry assays were used to assess cell proliferation and apoptosis. The expression of granzyme B was detected by immunofluorescence, and the expression of CD107a and NKG2D was determined by flow cytometry. The downstream regulatory genes were identified by RNA-seq, and the “JAK-STAT signaling pathway”- and “Cell cycle signaling pathway”-related genes were detected by real-time quantitative polymerase chain reaction (RT–qPCR) and Western blot analysis. The JAK2V617F mouse model was constructed to simulate the upregulation of the JAK2 signaling pathway in vivo, and the NK proliferation was evaluated by flow cytometry. A tumor-bearing nude mouse model was constructed to determine the anti-tumor efficacy of NK92 cells following Dac treatment. ResultsIn the presence of IL2, the proliferation rate of NK92 cells, pNK cells, and CD19-CAR-NK92 cells treated with pan-HDACi Dac and Pan at low nanomolar doses was significantly increased, although cell function was unaffected. Low doses of Dac upregulated the JAK-STAT signaling pathway and enhance the cell cycle via that pathway. In addition, the in vivo experiment in nude mice showed that the capacity of Dac treated NK92 cells to eliminate tumor cells was unaffected. ConclusionLow nanomolar doses of Pan-HDACi enhanced IL2-induced NK cell proliferation without compromising the functioning of NK cells.

TRPA1 as a potential factor and drug target in scleroderma: dermal fibrosis and alternative macrophage activation are attenuated in TRPA1-deficient mice in bleomycin-induced experimental model of scleroderma

BackgroundSystemic sclerosis is a rheumatoid disease best known for its fibrotic skin manifestations called scleroderma. Alternatively activated (M2-type) macrophages are normally involved in the resolution of inflammation and wound healing but also in fibrosing diseases such as scleroderma. TRPA1 is a non-selective cation channel, activation of which causes pain and neurogenic inflammation. In the present study, we investigated the role of TRPA1 in bleomycin-induced skin fibrosis mimicking scleroderma.MethodsWild type and TRPA1-deficient mice were challenged with intradermal bleomycin injections to induce a scleroderma-mimicking disease. Macrophages were investigated in vitro to evaluate the underlying mechanisms.ResultsBleomycin induced dermal thickening and collagen accumulation in wild type mice and that was significantly attenuated in TRPA1-deficient animals. Accordingly, the expression of collagens 1A1, 1A2, and 3A1 as well as pro-fibrotic factors TGF-beta, CTGF, fibronectin-1 and YKL-40, and M2 macrophage markers Arg1 and MRC1 were lower in TRPA1-deficient than wild type mice. Furthermore, bleomycin was discovered to significantly enhance M2-marker expression particularly in the presence of IL-4 in wild type macrophages in vitro, but not in macrophages harvested from TRPA1-deficient mice. IL-4-induced PPARγ-expression in macrophages was increased by bleomycin, providing a possible mechanism behind the phenomenon.ConclusionsIn conclusion, the results indicate that interfering TRPA1 attenuates fibrotic and inflammatory responses in bleomycin-induced scleroderma. Therefore, TRPA1-blocking treatment could potentially alleviate M2 macrophage driven diseases like systemic sclerosis and scleroderma.

Treg-based immunotherapy for antigen-specific immune suppression and stable tolerance induction: a perspective.

FoxP3-expressing regulatory T cells (Tregs), whether naturally generated in the immune system or unnaturally induced from conventional T cells (Tconvs) in the laboratory, have much therapeutic value in treating immunological diseases and establishing transplantation tolerance. Natural Tregs (nTregs) can be selectively expanded in vivo by administration of low-dose IL-2 or IL-2 muteins for immune suppression. For adoptive Treg cell therapy, nTregs can be expanded in vitro by strong antigenic stimulation in the presence of IL-2. Synthetic receptors such as CAR can be expressed in nTregs to equip them with a particular target specificity for suppression. In addition, antigen-specific Tconvs can be converted in vitro to functionally stable Treg-like cells by a combination of antigenic stimulation, FoxP3 induction, and establishment of the Treg-type epigenome. This review discusses current and prospective strategies for Treg-based immune suppression and the issues to be resolved for achieving stable antigen-specific immune suppression and tolerance induction in the clinic by targeting Tregs.

Regulation of Cytotoxic Immune Effector Function by AJ3 Probiotic Bacteria in Amyotrophic Lateral Sclerosis (ALS).

Our recent studies indicated that amyotrophic lateral sclerosis (ALS) patients suffer from significantly elevated levels of interferon-gamma (IFN-γ) secretion by natural killer (NK) and CD8+ T cells, which may be responsible for the immune-pathologies seen in central nervous system and in peripheral organs of the patients. In order to counter such elevated induction of IFN-γ in patients we designed a treatment strategy to increase anti-inflammatory cytokine interleukin-10 (IL-10) by the use of probiotic strains which significantly increase the levels of IL-10. Therefore, in this paper we demonstrate disease specific functions of Al-Pro (AJ3) formulated for the adjunct treatment of auto-immune diseases including ALS, and compared the function with CA/I-Pro (AJ4) for the treatment of cancer and viral diseases, and NK-CLK (AJ2) for maintenance of immune balance and promotion of disease prevention. The three different formulations of probiotic bacteria have distinct profiles of activation of peripheral blood mononuclear cells (PBMCs), NK, and CD8+ T cells, and their induced activation is different from those mediated by either IL-2 or IL-2 + anti-CD16 monoclonal antibodies (mAbs) or IL-2 + anti-CD3/CD28 mAbs. IL-2 + anti-CD16 mAb activation of PBMCs and NK cells had the highest IFN-γ/IL-10 ratio, whereas IL-2 combination with sAJ4 had the next highest followed by IL-2 + sAJ2 and the lowest was seen with IL-2 + sAJ3. Accordingly, the highest secretion of IFN-γ was seen when the PBMCs and NK cells were treated with IL-2 + sAJ4, intermediate for IL-2 + sAJ2 and the lowest with IL-2 + sAJ3. The levels of IFN-γ induction and the ratio of IFN-γ to IL-10 induced by different probiotic bacteria formulation in the absence of IL-2 treatment remained much lower when compared to those treated in the presence of IL-2. Of note is the difference between NK cells and CD8+ T cells in which synergistic induction of IFN-y by IL-2 + sAJ4 was significantly higher in NK cells than those seen by CD8+ T cells. Based on these results, sAJ3 should be effective in alleviating auto-immunity seen in ALS since it will greatly regulate the levels and function of IFN-γ negatively, decreasing overactivation of cytotoxic immune effectors and prevention of death in motor neurons.