Treg Cell Population Research Articles

Restoring Ovarian Function With Human Placenta-Derived Mesenchymal Stem Cells in Autoimmune-Induced Premature Ovarian Failure Mice Mediated by Treg Cells and Associated Cytokines.

Regulatory T (Treg) cells play a key role in the regulation of autoimmunity and transplantation. Human placenta-derived mesenchymal stem cell (hPMSC) transplantation has a potential to restore ovarian dysfunction associated with premature ovarian failure (POF), while the exact function of the Treg cells in the transplantation still needs to be further investigated. In this study, hPMSCs were intravenously injected into POF mice following zona pellucida glycoprotein 3 (pZP3) treatment. Ovarian function was measured by analyzing estrous cycle, folliculogenesis, and hormone secretion, also, with the detection of apoptotic granular cells (GCs) in ovarian tissues. To determine whether immune response is involved in the regulation of ovarian function change, the population of Treg cell populations and expression of associated cytokines, for example, transforming growth factor β (TGF-β) and interferon γ (IFN-γ) were measured. After hPMSCs transplantation, the injured ovarian function is significantly improved. Also, the pZP3-treatment-induced apoptotic GCs were significantly decreased as compared with the POF mice. The transplantation of hPMSCs significantly increased the population of Treg cells which was inhibited by pZP3 treatment. The decrease in TGF-β and increase in IFN-γ in serum caused by pZP3 treatment have been reversed following hPMSCs transplantation. These findings strongly suggest that the recovery of ovarian function in POF mice is mediated via the regulation of Treg cells and production of associated cytokines following hPMSCs transplantation.

Abstract 1754: Smac mimetics APG-1387 synergizes with immune checkpoint inhibitors in preclinical models

Abstract APG-1387 is a novel, SMAC-mimetic, small molecule inhibitor for inhibitor of apoptosis proteins (IAPs). IAPs are a class of proteins which negatively regulate caspases and apoptosis. The IAP proteins are frequently dysregulated in many cancers and have been suggested as contributing to apoptosis-resistance of cancer cells to a variety of anticancer drugs. DNA amplification of the cellular IAP-1 (cIAP-1) and cellular IAP-2 (cIAP-2) genes (BIRC2 and BIRC3, respectively) has been observed in various human cancer types, including lung cancer, pancreatic cancer, and liver carcinoma. Dysregulation of IAP proteins has also been frequently observed at the protein level in multiple cancer cell lines and tumor samples. IAPs contribute to tumor cell survival, chemo-resistance, disease progression, and poor prognosis. In addition, IAPs play an important role in immune regulation. For example, IAPs regulate ubiquitin (Ub)-dependent pathways that modulate innate immune signaling via activation of nuclear factor κB (NF-κB). Due to their significant biological functions in cell death and immune responses, IAPs have been emerging as drug targets in a wide range of malignancies. Several IAP inhibitors have been developed and their clinical benefits are currently in investigation in multiple clinical trials in both single agent and combination regimen with immune checkpoint inhibitors. APG-1387 has been developed as a novel drug candidate targeting IAPs. Mechanistically, in both cancer cells and xenograft tumors, APG-1387 induces degradation of cIAP-1 and XIAP proteins, as well as caspase-3 activation and PARP cleavage, which leads to apoptosis. Anti-proliferative and anti-tumor activities of APG-1387 have been well demonstrated in a panel of cancer cell lines and xenograft tumor models. In this study, we explored the synergistic effect of APG-1387 in combination therapy with immune checkpoint inhibitors. We found that APG-1387 enhanced human PBMC co-stimulation of anti-CD3 and anti-CD28, and T cell proliferation in vitro. In syngeneic tumor tissues, the combination treatment with APG-1387 and anti-PD-1 antibody led to an increase in CD3+ and CD8+ T cell populations and a decrease in Treg cell population. Synergistic effect of the combination was further demonstrated in syngeneic tumor models. Taken together, our results suggest that the treatment with APG-1387 may enhance the effect of anti-PD-1 antibody therapy. Consequently, APG-1387 has been approved by FDA for phase I clinical trials as single agent and in combination with anti-PD-1 antibody in solid tumors. Citation Format: Wentao Pan, Douglas D. Fang, Guangfeng Wang, Miaoyi Wu, Yingfeng Li, Keji Lian, Dingxiong Cheng, Yanhui Kong, Guoqin Zhai, Ming Guo, Shaomeng Wang, Dajun Yang, Yifan Zhai. Smac mimetics APG-1387 synergizes with immune checkpoint inhibitors in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1754.

Abstract 2644: Relative T regulatory cell populations are indicative of HCC in patients with underlying liver disease

Abstract Hepatocellular carcinoma continues to be an increasing burden to patients and the health care system despite recent advances in treatment of viral hepatitis. Since the vast majority of cancers develop in patients with underlying liver disease, we would expect that efforts to cull out the patients at highest risk for cancer emergence would be possible, yet we have struggled to do so. The difficulty lies in the complex microenvironment of the diseased liver that leads to striking aberrancies, many of which are related to inflammation and can overlap with biomarkers felt to be related to cancer. Our goal with lymphocyte profiling is to stratify patients with underlying liver disease into those with and without cancer. Improved surveillance modalities for patients at highest risk of developing HCC are required. Moreover, there is an increasing push to include immunology in cancer detection and treatment. To address these issues, we performed lymphocyte profiling in patients with underlying liver disease with and without HCC and living related renal donors (LRD) by selective cell marker staining and flow cytometry, in order to determine if lymphocytes, which are key to cancer immunology, are modulated concordant with HCC. Specifically, whole blood was stained in order to quantitate T helper (Th), T cytotoxic (Tctx), B cells, natural killer (NK), and T regulatory cells (Treg) by flow cytometry. Th, Tctx and B cells were significantly reduced and NK cells were significantly increased in patients with underlying liver disease. These changes were independent of HCC diagnosis. In contrast, Treg cell levels were dependent on HCC diagnosis. In patients with underlying liver disease without HCC, Treg levels, when compared to LRD (30±6cells/mm3), were significantly reduced (15±3), whereas in patients with underlying liver disease and HCC, Treg levels were increased when compared to patients without HCC and unchanged when compared to LRD (28±6). To correct for the observed lower T-cell levels associated with underlying liver disease, lymphocyte populations were expressed as a ratio when compared to total T-cell numbers. This conversion identified proportional Treg cells population as a marker of HCC. In patients without HCC the Treg/T cell ratio was unchanged when compared to living related donors (0.02±0.002, 0.02±0.002, P=0.3). In contrast, Treg/Tcell ratio was significantly increased in patients with HCC (0.08±0.02, P=0.001). Consequently, given that the primary function of Treg cells is to inhibit T cytotoxic cells, a proportional increase in Treg cells would be associated with immunologic escape for HCC cells. Therefore, surveillance of lymphocyte populations, in particular T cells and Treg, would evaluate changes in immunologic capacity and increased Treg/Tcell ratio would be indicative of an increased risk for HCC in patients with underlying liver disease. Citation Format: Nicholas J. Skill, Mary A. Maluccio. Relative T regulatory cell populations are indicative of HCC in patients with underlying liver disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2644.

Abstract LB-118: A conserved checkpoint molecule signature offers novel targets for tumor specific regulatory T cell manipulation

Abstract Regulatory T (Treg) cells play an important role in modulating the tumor microenvironment, leading to the suppression of anti-tumor immune responses. Tumor Tregs could be targeted by taking advantage of the differential expression of immune checkpoints on these cells as compared to other T cell subsets. While the elevated expression of checkpoints such as CTLA-4 on Tregs compared to conventional T cells is well-characterized, less is known about the collective landscape of Treg specific checkpoint expression specific in the tumor microenvironment. We identified a network of Treg-associated checkpoint markers including those previously associated with Treg function (CTLA4, ICOS) as well as several additional checkpoints (BTLA, LAG3, TIGIT, TNFRSF9) by characterizing the expression of 28 checkpoint molecules across 7,608 bulk tumor samples (31 tumor types) from TCGA. We then utilized whole transcriptome RNA sequencing of purified peripheral and tumor Treg cell populations across 4 different tumor types (glioblastoma multiforme, prostatic adenocarcinoma, urothelial transitional cell carcinoma, renal clear cell carcinoma) to characterize the Treg specific expression of checkpoint molecules. Peripheral and tumor Tregs demonstrate differential checkpoint expression landscapes, with tumor Tregs uniquely discriminated by collective expression of TNFRSF9 (4-1BB), HAVCR-2 (TIM-3), and LAG-3. This discriminatory signature was conserved across the 4 profiled tumor types, was associated with changes in Treg biology, and was confirmed in published data from three additional cancer cohorts (breast, colorectal, and non-small-cell-lung cancers). Furthermore, normalized TNFRSF9 expression in TCGA was also associated with a worse survival outcome, suggesting that elevated Treg TNFRSF9 expression may be a poor prognostic factor across a wide range of tumors. Taken together, these data highlight the potential of targeting TNFRSF9 expressing Tregs in the tumor as a novel immunotherapeutic approach for several types of cancer. Citation Format: Zachary T. Freeman, Daniel H. Hovleson, Alexander T. Pearson, Jingyi Zhai, Ali Ghasemzadeh, Thomas R. Nirschl, Hui Jiang, Scott A. Tomlins, Charles G. Drake. A conserved checkpoint molecule signature offers novel targets for tumor specific regulatory T cell manipulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-118.

Vitamin D, autoimmunity and recurrent pregnancy loss: More than an association.

Recurrent pregnancy loss (RPL) affects close to 1% of couples; however, the etiology is known in only about 50% of the cases. Recent studies show that autoimmune dysregulation is a probable cause of RPL, which in some cases may be overlooked. In order for a pregnancy to proceed to term, early modulation of immunologic response is required to induce tolerance to the semi-allogenic fetus. Certain subsets of both the innate and adaptive immune responses play a role in the induction of fetomaternal tolerance. A relatively predominant T-cell helper (Th) 2 and T regulatory (Treg) cell population seem to favor a better pregnancy outcome, whereas Th1 and Th17 cell populations appear to have an opposite effect. Lately, the role of vitamin D in the modulation of immune response was established. Vitamin D has been shown to promote a more favorable environment for pregnancy through various mechanisms, such as enhancement of the shift toward Th2 cells and regulation of immune cell differentiation and cytokine secretion. Therefore, it seems that vitamin D deficiency sways the balance toward a worse outcome and may play a part in recurrent pregnancy loss. This review sheds light on the immunologic changes, which occur in early pregnancy and the regulatory role vitamin D has in the maintenance of this delicate balance.

Challenges to Reshape the Future of Type 1 Diabetes Research.

Immunotherapy trials to prevent type 1 diabetes have been unsuccessful for >15 years. Understanding pitfalls and knowledge gaps in the immunology of type 1 diabetes should lead us in new directions that will yield better trial outcomes. A proposal is made for precision medicine trial design in future type 1 diabetes studies. High-quality peer-reviewed basic science and clinical research trials for type 1 diabetes were used in this Perspective article. Type 1 diabetes publications were reviewed from 2000 to 2018 by using Google Scholar and PubMed reference databases. Personalized medicine for type 1 diabetes should recognize that each individual has phenotypic and genotypic quirks that distinguish them from other study participants. A uniform protocol for antigen-specific immunotherapy has consistently failed to prevent disease. An alternative approach using molecular tools to personalize the preventive treatment strategy might be a road forward for type 1 diabetes research. Assumptions or lack of knowledge about disease stratification (not all type 1 diabetes is the same disease), individualized antigen-specific T cells, regulatory T-cell populations, and T-cell receptor rearrangement are just a few aspects of immunology that require integration with clinical trial design. The type 1 diabetes research community continues to bring forward novel immunotherapy trials to prevent disease, but this approach is unlikely to succeed until several fundamental aspects of clinical immunology are recognized and addressed. Here, we identify several knowledge gaps that could rectify type 1 diabetes trial design and lead to future success.

Anti-pruritic and anti-inflammatory effects of oxymatrine in a mouse model of allergic contact dermatitis

BackgroundAllergic contact dermatitis (ACD) is a highly prevalent inflammatory disease of the skin. As a result of the complex etiology in ACD, therapeutic compounds targeting refractory pruritus in ACD lack efficacy and lead to numerous side effects. ObjectiveIn this study, we investigated the anti-pruritic effects of oxymatrine (OMT) and explored its mechanism of action in a mouse model of ACD. Method72 male SPF C57BL/6 mice were randomly divided into control group, ACD model group, dexamethasone positive control group (0.08 mg kg−1) and 3 OMT groups (80, 40, 20 mg kg−1). OMT was administrated by intraperitoneal injection 1 h before video recording on day 10, 24 h after 2nd challenge with SADBE. Cheek skin fold thickness was measured before treatment and after recording. H&E staining was used for pathological observation. RT-qPCR, Immunohistochemistry and LEGENDplexTM assay were used to detect cytokines levels. The population of Treg cells in peripheral blood were detected via flow cytometry. ResultsOMT treatment significantly decreases the skin inflammation and scratching bouts. It rescues defects in epidermal keratinization and inflammatory cell infiltration in ACD mice. Administration of OMT significantly reduced levels of IFN-γ, IL-13, IL-17A, TNF-α, IL-22 and mRNA expression of TNF-α and IL-1β. Furthermore, it increased the percentage of Treg cells in peripheral blood of ACD mice. ConclusionWe have demonstrated that OMT exhibits anti-pruritic and anti-inflammatory effects in ACD mice by regulating inflammatory mediators. OMT might emerge as a potential drug for the treatment of pruritus and skin inflammation in the setting of ACD.

Glucocorticosteroids administration is associated with increased regulatory T cells in equine asthmatic lungs

Recurrent inflammation in severe equine asthma causes a remodeling of the airways leading to incompletely reversible airway obstruction. Despite the improvement of clinical signs and lung function with glucocorticoids (GC), inflammation, translated by an increased percentage of neutrophils, persists in the airways. Regulatory T cells (Treg) have been shown to have anti-inflammatory properties and play an important role in balancing the immune response by suppressing effector lymphocyte activity. However, interactions between Treg, neutrophils and glucocorticosteroids in vivo are unclear, particularly in asthma. Furthermore, the effects of GC on Treg in the airway of asthmatic horses have not been investigated. We hypothesized that horses with severe asthma display a decreased population of pulmonary Treg when compared to heathy controls, and that treatment with GC lead to an increased pulmonary Treg cell population only in affected horses. Using lung function measurements and flow cytometry with surface antigens CD4 and FoxP3, we investigated Treg in airway luminal cells obtained by bronchoalveolar lavage fluid (BALF) from 6 asthmatic horses in exacerbation of the disease and 6 aged-match controls, kept in the same environment, before and following a 2-week treatment with dexamethasone. Results showed that the number of Treg increases only in the lungs of asthmatic horses following GC therapy, despite continued presence of increased numbers of neutrophils. Our results support the complexity of the interaction between Treg, neutrophils and GC.

Interaction Between CCR6+ Th17 Cells and CD34+ Fibrocytes Promotes Inflammation: Implications in Graves' Orbitopathy in Chinese Population.

Recent reports suggest that Th17 immunity and bone marrow-derived CD34+ fibrocytes contribute to the pathogenesis of Graves' orbitopathy (GO). This study investigated interactions between Th17 cells and fibrocytes in GO inflammation in Chinese subjects. Th17 cells and fibrocytes were derived from blood samples from Chinese GO patients and healthy controls. Proportions and phenotypes of Th17 cells, regulatory T cells (Tregs), and fibrocytes were examined by flow cytometry. Exogenous IL-17A was used to study inflammatory activity of fibrocytes from GO patients and control subjects. Coculture, quantitative RT-PCR, Luminex, and transwell assays were performed to investigate the relationship between Th17 cells and fibrocytes. CC-chemokine receptor 6 (CCR6+) Th17 cells were increased in both active (P < 0.001) and inactive (P < 0.05) GO patients, compared with healthy controls. There was a positive correlation between number of CCR6+ Th17 cells and GO clinical activity score (P < 0.0001, r = 0.8176). Further, CD34+ fibrocytes were increased in GO patients, with increased expression of IL-17RA (P < 0.05), CD80 (P < 0.05), and CD86 (P < 0.05). A decreased population of effector Treg cells (P < 0.01) and increased CTLA-4 expression on naïve Treg cells (P < 0.05) were observed in GO patients. IL-17A stimulated cytokine production in fibrocytes; GO fibrocytes exhibited more robust production than normal fibrocytes. Autologous Th17 cells promoted inflammatory and antigen-presenting functions of GO fibrocytes; conversely, fibrocytes enhanced Th17 cell-function and recruited Th17 cells in a macrophage inflammatory protein 3 (MIP-3)/CCR6-dependent manner. The crosstalk between CCR6+ Th17 cells and fibrocytes plays a role in the pathogenesis of GO. Suppressing these interactions may be a candidate molecular target for therapeutic approaches of GO.

Transplantation of adipose tissue-derived stem cells ameliorates autoimmune pathogenesis in MRL/lpr mice : Modulation of the balance between Th17 and Treg.

Mesenchymal stem cells (MSCs) are multipotent cells characterized by immunomodulatory properties and are therefore considered apromising tool for the treatment of autoimmune diseases. In this study, we aimed to investigate whether transplantation of adipose tissue-derived stem cells (ADSCs) affects the autoimmune pathogenesis in MRL/lpr mice. Fifteen12-week-oldMRL/lprmice were randomly divided into three groups: ADSC, cyclophosphamide (CTX), and control groups, with five mice in each group. ADSC and control groups were injected with 1 × 106ADSCs orPBS, respectively, via the tail vein, once aweek for 8weeks.The CTX group was injected with CTX at adose of 15 mg/kg body weight, once aweek for 2weeks, and this was repeated after 2weeks rest. Proteinuria, anti-double-stranded DNA (anti-dsDNA) antibody, and serum creatinine levels were then measured. The populations of Th17 and Treg cells in the spleen were detected by flow cytometry. All statistical analyses were performed using least square difference. Eight weeks after treatment, the 24 hproteinuria, anti-dsDNA antibody levels, and serum creatinine were decreased significantly with transplantation of mouse ADSCs. ADSCs markedly reduced the number of TH17 cells, increased Treg cells, and improved renal pathology. Our results indicate that transplantation of ADSCs could significantly inhibit autoimmune progression in MRL/lpr mice and the efficacy of ADSCs was comparable to that of CTX.

Helios expression defines a phenotypically distinct population of Treg cells

Abstract The transcription factor Helios is expressed by 65–75% of Treg and we have proposed that it is a marker of thymic derived Treg (tTreg). In order to study the differences between Helios+ and Helios− Treg, we have generated a Helios-GFP/Foxp3-RFP double reporter mouse. Helios+ Treg expressed a more activated phenotype and had higher suppressive capability in vitro. Both subsets were equivalent in their ability to suppress IBD in vivo, but Helios+ Treg were more protective against disease induced by the transfer of splenocytes from scurfy mice. Both subsets expressed a highly demethylated Foxp3 TSDR, with slightly higher demethylation in the Helios+ Treg subset. Upon transfer to normal mice, both Helios+ and Helios− Treg cells maintained equal Foxp3 stability. However, upon transfer to lymphopenic mice, the percentage of cells from the Helios− subset that maintained Foxp3 expression was significantly lower than that of the Helios+ subset. Upon transfer to both normal and lymphopenic mice, Foxp3+Helios+ Treg maintained stable expression of Helios, while 30% of the Helios− Treg acquired low levels of Helios expression upon transfer to normal mice and 70–80% of the Helios− Treg acquired Helios expression when transferred to lymphopenic mice. Gene expression profiling of the two populations demonstrated 1000 differentially expressed genes. Additionally, TCR repertoire deep sequencing analysis revealed little to no overlap of the two populations. Taken together, our data indicate that Helios expression can differentiate two distinct populations of Treg with overlapping functions, most likely representing tTreg (Helios+) and stable peripherally-induced pTreg (Helios−). Supported by the Intramural Research Program of the NIH, NIAID.

IL-1 and IL-33 differentially regulate the functional specialization of mucosal Foxp3+ regulatory T cells.

Abstract CD4+ regulatory T (TREG) cells are critical mediators of peripheral immune tolerance and homeostasis, and express the forkhead box p3 (Foxp3) transcription factor, the master-regulator driving the programming of the TREG cell suppressive phenotype. TREG cells are abundant at mucosal surfaces, where they acquire tissue-specific adaptations. The biological consequences of these adaptations on the stability of thymic-derived TREG (tTreg) cells remain largely unknown. To determine the signals that drive the fate of TREG cells, we isolated and compared stable from unstable TREG cells using a TREG transfer model where we previously observed the functional reprogramming of Foxp3+ TREG cells into Th1/Th17 effector T cells. We identified the expression of the IL-33 receptor (IL-33R, ST2) on stable tTREG cells and the IL-1 receptor (IL1R1) on unstable exTREG cells undergoing functional reprogramming. We show that both TREG cell populations represent competing subsets in inflammatory conditions. This is further underlined by the fact that the absence of IL1R1 expression (IL1R1−/−) leads to the accumulation of ST2+ TREG cells at mucosal sites in vivo. In two distinct lung infection models, we demonstrate that ST2-expressing TREG cells express GATA3 and resist production of inflammatory cytokines, whereas IL1R1-expressing TREG cells express RORγT and lose Foxp3 expression in vivo. While IL-1 signaling impairs TREG cell suppressive function, ST2 is required for the maintenance of the lineage identity and suppressive function of TREG cells. These observations demonstrate that IL-1 and IL-33 produced during immune challenge exert distinct roles on the functional adaptation of Foxp3+ tTREG cells at mucosal surfaces.

Helios But Not CD226, TIGIT and Foxp3 is a Potential Marker for CD4 Treg Cells in Patients with Rheumatoid Arthritis

Background: Rheumatoid arthritis (RA) is a progressive, chronic, even disabling systemic autoimmune disease. Imbalance between pathogenic immune cells and immunosuppressive cells is associated with the pathogenesis and development of RA and other autoimmune diseases. As Foxp3 is also expressed on activated CD4 cells in the presence of inflammation, the identification of Treg cells in patients with RA remains a challenge. Methods: Comprehensive analyses were carried out by Flow cytometry. Expression of Helios, CD226, T cell immunoreceptor with Ig and ITIM domains (TIGIT) was analyzed by clinical samples of rheumatoid arthritis patients and healthy controls. Findings: we have systemically examined three potential markers, Helios, CD226 and TIGIT that are possibly related to Treg identification, and found that Helios expression on CD4 Foxp3 cells was decreased and negatively correlated with the disease activity of RA patients, while CD226 and TIGIT both showed elevated expression levels in CD4 Foxp3 cells in RA patients and they were not associated with disease activity of RA patients. Interpretation: Our findings indicate that CD4 CD25hiCD127low/-Foxp3 Helios may represent the real Treg cell population in patients with RA. Funding: This work was supported in part by grants from the National Key R&D Program of China (2017YFA0105800), General Program of National Natural Science Foundation of China (81671611) and the Zhujiang Innovative and Entrepreneurial Talent Team Award of Guangdong Province (2016 ZT 06S 252) and NIH R01 AR 059103. Declaration of Interest: All authors have no conflict of interest. Ethical Approval: All human studies have been approved by the Research Ethical Committee of the Third Affiliated Hospital at Sun Yat-sen University. Before study, written informed consents were received from all participants.

The role of Treg population in pathogenesis of Crimean Congo hemorrhagic fever

Crimean-Congo hemorrhagic fever (CCHF) is a severe human infection caused by CCHF virus (CCHFV). Today, although the literature on CCHF pathogenesis is still limited, it is thought to be associated with immunosuppression in the early phase of infection followed by pro-inflammatory immune response that may lead to fatal outcomes. The aim of this study is to investigate the role of regulatory T-cells (Treg cells) in the pathogenesis of CCHFV.Peripheral blood mononuclear cell samples collected from 14 acute CCHF patients with mild disease course and 13 healthy subjects were included in this study. Treg expression and functional levels were analyzed by flow cytometry. Treg cells were identified as CD4+CD25 + CD127dim cells, and their functional levels were compared by measuring their ability to suppress CD69 and CD154 expression by activated T-cells.The flow cytometry analysis revealed that total T-cell and helper T-cell levels did not vary between the two groups. In contrast, CCHF patients displayed higher Treg cell levels but lower Treg suppressive activities when compared with control subjects.This is the first study on the involvement of Treg cells in CCHF pathogenesis. Our results indicate that even though Treg cell levels are elevated during acute phase of CCHF infection, not all generated Treg cells has immunosuppressive capacity, and therefore may not represent ‘true’ Treg cell population. Future studies on the intrinsic mechanisms responsible for the reduced Treg inhibitory activities are required for further enlightening the CCHF pathogenesis, especially in the acute phase of the disease.

Gene Therapy With Regulatory T Cells: A Beneficial Alliance.

Gene therapy aims to replace a defective or a deficient protein at therapeutic or curative levels. Improved vector designs have enhanced safety, efficacy, and delivery, with potential for lasting treatment. However, innate and adaptive immune responses to the viral vector and transgene product remain obstacles to the establishment of therapeutic efficacy. It is widely accepted that endogenous regulatory T cells (Tregs) are critical for tolerance induction to the transgene product and in some cases the viral vector. There are two basic strategies to harness the suppressive ability of Tregs: in vivo induction of adaptive Tregs specific to the introduced gene product and concurrent administration of autologous, ex vivo expanded Tregs. The latter may be polyclonal or engineered to direct specificity to the therapeutic antigen. Recent clinical trials have advanced adoptive immunotherapy with Tregs for the treatment of autoimmune disease and in patients receiving cell transplants. Here, we highlight the potential benefit of combining gene therapy with Treg adoptive transfer to achieve a sustained transgene expression. Furthermore, techniques to engineer antigen-specific Treg cell populations, either through reprogramming conventional CD4+ T cells or transferring T cell receptors with known specificity into polyclonal Tregs, are promising in preclinical studies. Thus, based upon these observations and the successful use of chimeric (IgG-based) antigen receptors (CARs) in antigen-specific effector T cells, different types of CAR-Tregs could be added to the repertoire of inhibitory modalities to suppress immune responses to therapeutic cargos of gene therapy vectors. The diverse approaches to harness the ability of Tregs to suppress unwanted immune responses to gene therapy and their perspectives are reviewed in this article.

Complete B Cell Deficiency Reduces Allograft Inflammation and Intragraft Macrophages in a Rat Kidney Transplant Model.

Increasingly, it is being appreciated that B cells have broad roles beyond the humoral response and are able to contribute to and regulate inflammation. The specific role of B cells in the pathogenesis of early allograft inflammation remains unclear. To address this question, we generated B cell-deficient (B) Lewis rats via clustered regularly interspaced short palindromic repeats (CRISPR) technology. In a full mismatch transplant model, kidneys from Brown Norway donors were transplanted into B Lewis recipients or wild type Lewis recipients. T cell-mediated rejection was attenuated with cyclosporine. Renal inflammation was reduced at 1 week after transplant (Banff scores for interstitial inflammation, microvascular inflammation, glomerulitis, and C4d) in allografts from B recipients. The reduction in interstitial inflammation was predominantly due to a decline in graft infiltrating macrophages. Intragraft T-cell numbers remained unchanged. In addition, B-cell deficiency was associated with increased T regulatory cells and reduced splenic T follicular helper cells at baseline; and significantly increased intragraft and splenic IL-10 mRNA levels after transplant. In vitro, B and wild type splenic T cells produced similar levels of IFN-γ in response to T cell-specific activation. B-cell deficiency in this model produced an anti-inflammatory phenotype with a shift toward regulatory T-cell populations, production of anti-inflammatory cytokines (IL-10), and a reduction in allograft inflammation. These findings define a role for B cells to influence the cell populations and mediators involved in the pathogenesis of early allograft inflammation.

Investigating the roles of regulatory T cells, mast cells and interleukin-9 in the control of skin inflammation by vitamin D.

Topical application of biologically active vitamin D [1,25-dihydroxyvitamin D (1,25(OH)2D)], or low-calcemic analogues, curb skin inflammation through mechanisms that involve migratory dendritic cells (DCs) and regulatory T (TReg) cells. 1,25(OH)2D also promotes immunoregulation by mast cells, and inhibits the development of T helper type-9 (Th9) cells that secrete interleukin-9 (IL-9). Here, we investigated the ability of topical 1,25(OH)2D to suppress contact dermatitis through an IL-9-dependent process, examining mast cells and IL-9-secreting T cells. Contact dermatitis was modelled in adult BALB/c female mice by initiating a "biphasic ear swelling response" following a single application of 2,4-dinitrofluorobenzene (DNFB). Topical 1,25(OH)2D (125ng) applied to ear pinnae prior to (but not after) DNFB sensitisation suppressed the efferent phase of the ear swelling response. This dose of 1,25(OH)2D did not cause hypercalcemia. At the peak of the efferent ear swelling response, proportions of TReg (CD3 + Foxp3+) cells and numbers of mast cells were increased in ear skin of 1,25(OH)2D-treated mice. Topical 1,25(OH)2D increased the proportion of Foxp3 + IL-9 + TReg cells and the capacity of TReg cells to secrete IL-9 ex vivo. However, the proportion of the IL-9 + cells of the total TReg cell population was small (< 1%), and the amount of IL-9 secreted by TReg cells from mice treated with IL-9 was low (< 50pg/ml). Furthermore, injection of anti-IL-9 neutralising antibody (100µg, intraperitoneally) prior to sensitisation did not significantly reverse the suppressive effects of 1,25(OH)2D. In conclusion, topically applied 1,25(OH)2D suppressed the efferent phase of a biphasic cutaneous ear swelling response through mechanism(s) that may be dependent on mast cells and TReg cells; however, the role of IL-9 in mediating these responses is uncertain. More studies are needed to further characterise the mechanisms by which topical 1,25(OH)2D modulates cell-mediated immune responses central to its suppressive effects upon contact dermatitis.

The analysis of the level of Treg lymphocytes in the blood of patients with endometrial cancer before and after the surgery — preliminary study

Introduction. The progression of cancer is a complex process involving host-tumour interactions taking place in cancer and in the cancer microenvironment. The tumour remodels the microenvironment into the suppressive profile by various mechanisms. One of the most important elements of this mechanism is the inducing of the infiltration of Treg lymphocytes into cancer and its microenvironment. The aim of the present study was to evaluate the alterations of the Treg cell population in the peripheral blood of patients before and after the surgical treatment for endometrial cancer. Material and methods. For the present study 24 patients with endometrial cancer were recruited. All the patients were treated surgically. The peripheral blood samples were collected from the endometrial cancer patients before operation and three days after the surgical procedure and evaluated using flow cytometry method. Results. CD25+ CD4+ FOXP3+ T cells were found in all the examined peripheral blood samples derived from the endometrial cancer patients in the days before and following applied surgery. We observed differences before and after the applied surgical procedure in patients treated for uterine cancer. The highest number of Treg cells in the peripheral blood was demonstrated before the surgical procedure; it diminished statistically significantly following the surgery. Conclusions. The decrease of the percentage of Treg cells in blood sera in patients following radical surgical treatment might be useful in measuring the radicalism of the treatment. The monitoring of the level of selective immune system suppression related to Treg cell blood serum levels during cancer therapy might support a decision to supplement the standard therapy with immunotherapy or to increase the degree of radicalism of the applied therapy.

Natural Course of Human T-Cell Leukemia Virus Type 1 Proviral DNA Levels in Carriers During Pregnancy.

The measurement of human T-cell leukemia virus type 1 (HTLV-1) proviral DNA levels by using polymerase chain reaction has been beneficial for confirming HTLV-1 infection during pregnancy. However, the influence of pregnancy on HTLV-1 infection and proviral DNA levels among pregnant women with HTLV-1 has not been clarified. We prospectively gathered blood samples from 36 pregnant women in whom HTLV-1 carriage was previously diagnosed and sequentially measured their proviral DNA levels. The HTLV-1 proviral DNA levels remained at a plateau during pregnancy but were elevated after delivery. Moreover, flow cytometry and serological analyses revealed that the regulatory T-cell population and soluble interleukin 2 receptor levels were similarly elevated after birth in comparison with those in control pregnant women. This study is the first to provide data on sequential changes in HTLV-1 proviral DNA levels during and after pregnancy. These findings will guide the establishment of a better program to prevent mother-to-child transmission of HTLV-1.

An open label study of a novel immunosuppression intervention for the treatment of amyotrophic lateral sclerosis

Neuroinflammation is increasingly tied to disease progression in amyotrophic lateral sclerosis (ALS). Participants in the first-in-human trial of intra-spinal allogeneic stem cell therapy for ALS received immunosuppression, and one participant saw dramatic improvement across multiple outcome measures. The primary objective of this study (NCT01884571) was to assess the rate of clinical response to the same immunosuppressive regimen using basiliximab, tacrolimus, mycophenolate, and prednisone in people with ALS. A clinical response was defined as an improvement on the revised ALS Functional Rating Scale (ALSFRS-R) by six points over a 6-month period. Thirty-one participants were enrolled in this 15-month open label study and received an identical immunosuppression regimen. Clinical outcome measures and biospecimens were collected before, during, and after the treatment regimen. No patients met the pre-defined responder criteria. No difference in mean ALSFRS-R slope was seen in the treatment period compared to the pretreatment period (p = 0.200). The regimen was generally safe in an ALS population, although only 18 out of 31 patients completed the full 6 months of immunosuppression. Analyses of immune markers showed no change in peripheral regulatory T-cell populations during treatment compared to pretreatment (p = 0.200). Analysis of cerebrospinal fluid (CSF) cytokine levels showed an increase in IL-2 levels with immunosuppression (p = 0.004) followed by decrease during post-treatment follow-up (p = 0.031). Further studies are needed to understand how manipulation of the immune system may affect disease progression in ALS.