Secretion Of TGF-beta Research Articles

Complement factor H is an ICOS ligand modulating Tregs in the glioma microenvironment.

The survival rate of glioma patients has not significantly increased in recent years despite aggressive treatment and advances in immunotherapy. The limited response to treatments is partially attributed to the immunosuppressive tumor microenvironment, where regulatory T cells (Tregs) play a pivotal role in immunological tolerance. In this study, we investigated the impact of complement factor H (FH) on Tregs within the glioma microenvironment and found that FH is an ICOS ligand. The binding of FH to this immune checkpoint molecule promoted the survival and function of Tregs and induced the secretion of TGF-beta (TGF-β) and IL-10, while also suppressing T-cell proliferation. We further demonstrated that cancer cells in human and mouse gliomas directly produce FH. Database investigations revealed that upregulation of FH expression was associated with the presence of Tregs and correlated with worse prognosis for glioma patients. We confirmed the effect of FH on glioma development in a mouse model, where FH knockdown was associated with decrease in number of ICOS+ Tregs and demonstrated a tendency of prolonged survival (p=0.064). Since the accumulation of Tregs represents a promising prognostic and therapeutic target, evaluating FH expression should be considered when assessing the effectiveness of and resistance to immunotherapies against glioma.

Abstract 5198: Humanized mice for preclinical efficacy evaluation of drugs targeting GARP

Abstract Introduction: GARP (Glycoprotein-A repetitions predominant) is a cell surface receptor which is coded by Lrrc32 (Leucine rich repeat containing protein 32) and involved in the activation of TGF-beta. The expression of GARP has been widely found in various cells including Treg, activated B cells, mesenchymal stromal cells and many types of cancer cells. In the tumor microenvironment (TME), GARP from Treg promotes the growth of tumor via secretion of immune suppressive TGF-beta. Result: We established GARP humanized mouse model (hGARP) on C57BL/6 background in which the signal peptide and extracellular domain of mouse GARP (mGARP) was replaced by human GARP (hGARP). Similar expression level of hGARP on Treg cells in splenocytes from C57BL/6-hGARP mice was observed compared with mGARP in wildtype mice. After treating with anti-hGARP antibody, tumor growth was inhibited in the cohort engrafted with MC38 colorectal cancer cell line. Flow cytometry analysis of the tumor infiltrating lymphocytes revealed an abundant accumulation of hGARP on the Treg cells suggesting that blocking of hGARP in Treg cells contributed to the therapeutic efficiency. In addition, hGARP expression was also found on platelet cells from C57BL/6-hGARP mice, which is similar to human. After treating with anti-hGARP, we also found a significant reduction of platelet cells in C57BL/6-hGARP mice. In vivo assay suggested that the reduction of platelet cells from C57BL/6-hGARP mice correlated well with the binding of anti-hGARP. Conclusion: C57BL/6-hGARP is a well-established mouse model for the pre-clinical evaluation of both therapeutic effect and toxicity of anti-hGARP therapy. Citation Format: Xing Liu, Meirong Wu, Huiyi Wang, Weiwei Yu, Jianming Xu, Hongyan Sun, Cunxiang Ju, Hongyu Wang, Santi Suryani Chen, Zhiying Li, Mark Wade Moore, Jing Zhao, Xiang Gao. Humanized mice for preclinical efficacy evaluation of drugs targeting GARP. [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 5198.

Immodulin peptides influence musculoskeletal homeostasis by linking extracellular cues to macrophage and myoblast nuclear receptors.

Immodulins are synthetic peptides derived from the C-terminal domains of insulin-like growth factor binding proteins (IGFBPs). Immodulins from the 3/5/6 (but not 1/2/4) IGFBP evolutionary clade transduce extracellular matrix (ECM) signals to RXR, NR4A1 and PPAR-alpha nuclear receptors (NRs) to stimulate novel macrophage lineages. The rationale of this study was to reconcile physical associations of immodulins with ECM and NRs, effects of siRNAs and chemical inhibitors in vivo, and immodulin-driven pro-differentiation effects in cell culture. When added to THP1D cells, immodulins stimulate CD169+ Clec9a+ and Clec12a+ macrophage lineages via a EP300/RXRγ/Nur77 transcriptional mechanism. This phenomenon is accompanied by the secretion of CCL22, IL-10 and TGFbeta and the ability to stimulate FoxP3+ T-cells in co-culture. ECM ligands of 3/5/6 immodulins include iron, zinc, glycosaminoglycans, transferrin and phosphatidylinositol-4,5,-biphosphate (PIP2), which can influence their pro-differentiation effects. Remarkably, immodulins also stimulate myogenesis in C2C12 myoblasts, thereby revealing a novel link between immune and musculoskeletal homeostasis. Distinct NR agonists stimulate these companion differentiation processes. Using solution NMR to guide design, immodulins with a tripeptide extension near the iron-binding pocket demonstrated higher iron-binding and improved pro-differentiation activities. Transferrin-bound immodulin shows binding preference for both high-molecular-weight hyaluronan (HMWHA) and HMWHA:CD44 complexes at endosomal pH, and interacts with PIP2 at normal physiological pH, offering intriguing mechanistic insights.

How to Identify Patients for Treg DLI - Treg Subsets in Gvhd As Biomarker for Successful Treg DLI

Introduction:Regulatory T cells (Treg) play a major role in controlling GvHD. Therefore many clinical studies focus on prophylactic or therapeutic Treg approaches. We focus on Treg reconstitution and their function in GvHD. Our aim is to identify patients with altered Treg function which may benefit from Treg DLI.Methods:We analysed peripheral blood samples of 38 patients with acute and 11 patients with chronic GVHD as well as 18 patients without GVHD after Alemtuzumab based conditioning, 10 patients with ATG for TCD and 10 healthy individuals. To identify Treg we stained for CD3, CD4, CD25, CD127, FoxP3. As activation markers GARP, TGF-beta, HLA-DR, CD45RA, CD39, CD44, CD62L, Granzyme A, CCR5, ILT3, CTLA4, CXCR3 and GITR were used. To investigate the influence of the CD52-AB Alemtuzumab, we also stained for CD52.Treg function was evaluated in CSFE suppression assays and TGF beta secretion. To investigate patient derived natural occurring Treg we sorted Treg from patients with GVHD or healthy controls with FACS and performed a CFSE proliferation assay with healthy PBMCs as effector T cells. To rule out the influence of systemic immunosuppression we analysed the effect of corticosteroids and CSA A on Treg marker expression and activation in healthy Treg. Treg were activated in vitro with IL2 (100U/ml AIM V+ human serum 10%) and OKT3 (30ng/ml).Results:Like in other publications in our cohort there were differences in absolute and relative Treg numbers in patients with acute, chronic or no GVHD compared with healthy controls: patients with aGVHD (mean 2,2%) had less Treg in comparison to patients with chronic (mean 4,5% p=0.0076) or no GVHD (mean 4,4% p=0.0045).CSA A or corticosteroids ex vivo (and in vivo) did not influence Treg marker expression significantly.Especially in patients after Alemtuzumab CD52 expression is the most prominent marker: Frequency of CD52pos Treg differed highly significant between patients with aGVHD (mean 15,94%) in comparison to patients with chronic (mean 75,85%) or no GVHD (mean 92,09%; p<0,0001). For all patients with acute or chronic GVHD FACS sorted Treg showed reduced capacity to suppress T cell proliferation in the proliferation assay.To determine the suppressive capacity of CD52neg Treg in comparison with CD52pos Treg of the same patient we divided Treg from one patient by FACS-Sort in CD52pos/neg subpopulations, which were used in a CFSE-proliferation assay again.We could show that CD52neg Tregwere less able to suppress effector T cell proliferation in comparison to CD52pos Treg (tested in 3 patients). Independent of the conditioning regimen Treg from GVHD patients were less able to suppress effector T cells in comparison with Treg from patients without GVHD or healthy individuals.We could not show any difference in activation markers ex vivo except GARP/TGFbeta. CD52neg Treg showed a reduced GARP/TGFbeta expression (mean 4,6% vs 1,65% p=0,012). After stimulation in vitro GARP/TGFbeta healthy controls showed the highest GARP/TGF-ß expression compared to patients with any GVHD, patients without GVHD exhibited higher amounts of GARP/TGFbeta compared to patients with aGVHD.We also analyzed patients after Alemtuzmab weekly during the first 200 days after HSCT to monitor the reconstitution of CD52neg Treg as a predictive marker for the development of aGVHD (n= 10). Patients who reconstituted with more than 50% of CD52pos Treg before d50 never developed any aGVHD. Patients with aGVHD and persistence of high amounts of CD52neg Treg showed a steroid refractory aGVHD with high mortality.Conclusion:Patients with acute GVHD showed different marker expression and decreased Treg function. Especially CD52neg Treg derived from patients after Alemtuzumab showed a reduced suppressive capacity and lower expression of activation markers. Higher GARP/TGFbeta expression seems to be associated with less development of acute GVHD. CD52 and GARP expression/TGFbeta secretion could be helpful biomarkers to identify a patient cohort with functional altered Treg and high risk of severe acute GVHD. In particular this patient cohort may benefit from Treg-DLI. DisclosuresNo relevant conflicts of interest to declare.

Induction of CD4+CD25+ Regulatory T Cells from In Vitro Grown Human Mononuclear Cells by Sparteine Sulfate and Harpagoside.

Regulatory T cells (Tregs) are key players in the regulation of inflammatory responses. In this study, two natural molecules, namely, sparteine sulfate (SS) and harpagoside (Harp), were investigated for their ability to induce Tregs in human peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from healthy volunteers and grown in the presence or absence of ConA, with TGF-beta, SS or Harp. Expression of the mRNA of FoxP3, TGF-beta, IL-10 and GAPDH was assessed via q-PCR. The expression of Treg markers including CD4, CD25, CD127 and FoxP3 was measured via flow cytometry. The secretion of IL-10 and TGF-beta by cultured cells was assessed by ELISA. Furthermore, the suppressive role of SS and Harp on PBMCs in vitro was tested via allogeneic mixed lymphocyte reaction (MLR). Data obtained show that both compounds increased the expression of FoxP3, TGF-beta and IL-10 mRNA in resting PBMCs but to a lesser extent in activated cells. Moreover, they significantly increased the percent of CD4+CD25+FoxP3+CD127− Tregs in activated and naïve PBMCs. Functionally, both compounds caused a significant reduction in the stimulation index in allogeneic MLR. Together, our data demonstrate for the first time that SS and Harp can induce human Tregs in vitro and therefore have great potential as anti-inflammatory agents.

Controlling porous titanium/soft tissue interactions with an innovative surface chemical treatment: Responses of macrophages and fibroblasts.

In order to create a stable interface with the host tissue, porous implants are widely used to ensure the in-growth of the cells and the colonization of the implant. An ideal porous implant should have a 3D architecture that enables fast migration of incoming cells while not inducing a significant pro-inflammatory response by the immune cells. Moreover, in patients where the healing is impeded (patients with co-morbidities and metabolic diseases), porosity by itself is not enough for fast colonization, and the surface properties of the implant should also be controlled. In this study, we present a controlled oxidation-based surface treatment of microbead-based porous titanium implants which not only increases the colonization by connective tissue cells but also decreases the macrophage attachment. The treatment created a nanotextured surface on the implants with an acidic shift of isoelectric point (from 4.09 to 3.09) without endangering implant's mechanical integrity. The attachment and metabolic activity of activated macrophages were significantly lower on treated surfaces with an increase in the secretion of anti-inflammatory IL-1RA and a decrease in pro-fibrotic CCL-18. Human fibroblasts proliferated faster on the treated surfaces over 14days with near complete colonization of the whole thickness of the implant with an accompanying an increase in the secretion of TGF-beta. The surface treated samples demonstrated partial filling of the entire pores. We demonstrated that the use of nanoscale surface treatments that can be applied to the whole internal surface of porous titanium implants can significantly alter both the immune response and the colonization of the implants and can be used to fine-tune and personalize implant interfaces according to patient needs.

Clinical ALK5 Inhibitor, Vactosertib, Reverses TGFβ-1 Stimulated Smad-2 Driven Ineffective Hematopoiesis in MDS

Transforming growth factor beta is an important regulator of hematopoiesis and its overactivation has been shown to occur in myelodysplastic syndromes. Even though TGF-beta leads to activation of the transcription factor SMAD2, the exact gene expression program and chromatin changes stimulated in human hematopoiesis are not well studied. We generated primary erythroid progenitors from human CD34+ stem cells and assessed genome occupancy of SMAD2 upon TGF-beta stimulation by Chip-seq analysis. We observed widespread occupancy of important stem cell and differentiation regulators with approximately 7000 binding regions. Parallel RNA-seq identified gene expression changes that are associated with smad2 binding at respective promoters and enhancers. Genes involved in stem cell maintenance as well as erythroid differentiation were prominently altered by TGF-beta and contained prominent SMAD2 peaks in hematopoietic progenitors. Next, to determine the relative contribution of various TGF-beta ligands in bone marrow failure, we analyzed a large cohort of MDS serum samples and found TGF-beta 1 (TGF-b1) to be the predominantly elevated ligand in 30% samples. Exposure to MDS patient serum led to erythroid differentiation blocks in primary hematopoietic stem cells in clonogenic cultures. Finally, we analyzed the effect of constitutive secretion of TGF-b1 in a transgenic mouse containing TGF-b1 gene driven by an albumin promoter. These mice constitutively secrete TGF-b1 and become cytopenic and show marrow histological changes that mimic human MDS. We observed stem cell expansion and block at the stage of common myeloid progenitor (CMP) in these mice; findings that are similar to stem and progenitors alterations seen in human MDS patients. To inhibit TGF-b1 signaling we next used a novel ALK5 (TGF-beta receptor I kinase) inhibitor that has been shown to be safe for human use. Vactosertib is specific small molecule inhibitor of ALK5 with a IC50 between 1 x 10-8 M and 1 x 10-9 M and has been shown to have an acceptable safety profile in phase I trial in solid tumors (NCT02160106). Vactosertib was able to inhibit smad2 phosphorylation and activation in a variety of hematopoietic cell lines. Vactosertib treatment led to reversal of hematopoietic alterations upon exposure to MDS serum in clonogenic assays. Vactosertib dosing in vivo also led to increased blood counts in TGF/alb transgenic mice and resulted in normalization of stem and progenitor changes. In conclusion, we demonstrate increased TGF-b1 levels in subset of MDS samples. We also determined TGF-b1 stimulated smad2 binding in important regulatory regions of the genome in primary human erythroid progenitors. We also demonstrate the preclinical efficacy of novel TGF-beta receptor 1 kinase inhibitor in vitro and in vivo. These studies provide preclinical rationale for using this ALK5 inhibitor in trials. Disclosures Huh: MedPacto Inc: Employment. Hwang:MedPacto Inc: Employment. Seong-Jin:MedPacto Inc: Employment. Will:Novartis Pharmaceuticals: Research Funding. Steidl:BayerHealthcare: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; GlaxoSmithKline: Research Funding; Celgene: Consultancy; Aileron Therapeutics: Consultancy, Research Funding; Stelexis Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Scientific Co-Founder; Pieries Pharmaceuticals: Consultancy. Verma:Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria; Celgene: Honoraria; BMS: Research Funding; Janssen: Research Funding.

Rapamycin-Based Expansion of Natural CD8+Tregs for Cell Therapy

Introduction We identified a subset of natural regulatory CD8+ T cells in peripheral blood of healthy volunteers characterized by the low expression of CD45RC surface molecule. These CD8+Tregs can inhibit allogeneic T cell proliferation in vitro through secretion of IFNg, IL-10, TGFbeta and IL-34. To date, there is no clinical study using CD8+Tregs as a cell-based therapy. Considering their potential for cellular therapy, we set up a process to expand these CD8+Tregs in a short term culture. Materials and Methods Tregs were expanded for 14 days with anti-CD3/CD28 mAbs or allogeneic APCs in presence of 1000U/ml IL-2, 10ng/ml IL-15, and with or without cyclosporine A (45ng/ml), rapamycin (45ng/ml), methylprednisolone (500pg/ml), tacrolimus (2ng/ml) or mycophenolate mofetil (1μg/ml). Suppressive function of CD8+Tregs was assessed in vitro on syngeneic CD4+CD25-T cells stimulated by alloAPCs, and in vivo into NSG mice infused with PBMCs and grafted or not with allogeneic human skin for allograft survival and xenogeneic GVHD studies. Results We demonstrated that natural CD8+Tregs can be efficiently expanded until 2000 fold in 14 days, when stimulated with either allogeneic APCs or anti-CD3 and anti-CD28 mAbs. High dose of IL-2 and IL-15 were required to maintain CD8+Tregs survival. After expansion, CD8+Tregs suppressive capacity was preserved and even increased in vitro, in accordance with their higher expression of immunoregulatory cytokines, while they displayed no cytotoxic activity. Furthermore, transfer of ex-vivo expanded CD8+Tregs allowed to delay GVHD development and allogeneic skin graft rejection in humanized mice models. As usually administered to patients after transplantation, we analyzed the impact of culture medium supplementation with immunosuppressive drugs during expansion. Interestingly, addition of rapamycin improved both CD8+Tregs expansion yield and suppressive activity by 16 and 1.6 fold respectively. Conclusions We demonstrated that natural CD8+Tregs can be greatly expanded while improved in their suppressive function particularly when cultured in presence of rapamycin, proving their relevance for cell therapy.

Modulation of TGFbeta 2 levels by lamin A in U2-OS osteoblast-like cells: understanding the osteolytic process triggered by altered lamins.

Transforming growth factor beta (TGFbeta) plays an essential role in bone homeostasis and deregulation of TGFbeta occurs in bone pathologies. Patients affected by Mandibuloacral Dysplasia (MADA), a progeroid disease linked to LMNA mutations, suffer from an osteolytic process. Our previous work showed that MADA osteoblasts secrete excess amount of TGFbeta 2, which in turn elicits differentiation of human blood precursors into osteoclasts. Here, we sought to determine how altered lamin A affects TGFbeta signaling. Our results show that wild-type lamin A negatively modulates TGFbeta 2 levels in osteoblast-like U2-OS cells, while the R527H mutated prelamin A as well as farnesylated prelamin A do not, ultimately leading to increased secretion of TGFbeta 2. TGFbeta 2 in turn, triggers the Akt/mTOR pathway and upregulates osteoprotegerin and cathepsin K. TGFbeta 2 neutralization rescues Akt/mTOR activation and the downstream transcriptional effects, an effect also obtained by statins or RAD001 treatment. Our results unravel an unexpected role of lamin A in TGFbeta 2 regulation and indicate rapamycin analogs and neutralizing antibodies to TGFbeta 2 as new potential therapeutic tools for MADA.

Immune homeostasis maintenance from decidualization to implantation: macrophages, dendritic cells and Treg network

Immune homeostasis maintenance from decidualization to implantation: macrophages, dendritic cells and Treg network

PLGA nanoparticle-entrapped inactivated porcine reproductive and respiratory syndrome virus vaccine coadministered intranasally with a potent adjuvant elicits cross-protective immunity in the pig respiratory system (VAC7P.981)

Abstract Porcine reproductive and respiratory syndrome (PRRS) is caused by a highly mutagenic RNA virus, PRRS virus (PRRSV). Currently used vaccines have failed to prevent PRRS outbreaks induced losses of estimated one billion dollar annually to the US pig farmers. The PRRSV infects primarily the lung macrophages. Because of safety advantage, we attempted to strengthen the immunogenicity of killed PRRSV vaccine antigens (KAg) in the pig respiratory system. We entrapped the KAg in biodegradable PLGA nanoparticles (NP-KAg) and coadministered the NP-KAg with a potent mucosal adjuvant, whole cell lysate of Mycobacterium tuberculosis (M. tb WCL), twice intranasally to growing pigs and challenged with a heterologous virus. Our results indicated that formulation of NP-KAg and unentrapped soluble M. tb WCL significantly cleared detectable replicating infective PRRSV with a 10-fold reduction in viral RNA load in the lungs, associated with substantially reduced microscopic lung pathology. Immunologically, enhanced virus neutralizing antibody titers by high avidity antibodies and augmented populations of IFN-γ secreting CD4+ and CD8+ lymphocytes, and reduced secretion of TGFbeta was detected in the lungs. Currently, we are in the process of identifying PRRSV epitope-specific T-cell response and frequency of apoptotic/necrotic immune cells in the lungs of vaccinated pigs. In conclusion, our vaccine formulation elicited broadly cross-protective anti-PRRSV immunity in the pig respiratory system.

Eosinophil–Epithelial Cell Interactions Stimulate the Production of MUC5AC Mucin and Profibrotic Cytokines Involved in Airway Tissue Remodeling

Predominant eosinophil infiltration and tissue remodeling are common characteristics of chronic airway inflammation such as nasal polyposis and bronchial asthma. This study was designed to elucidate the role of eosinophils in tissue remodeling of chronic airway inflammation; eosinophil-epithelial interactions were examined by the coculture of airway epithelial cell line NCI-H292 with the eosinophilic cell line EoL-1 or with human blood eosinophils. The coculture-induced production of MUC5AC mucin, platelet-derived growth factor AB (PDGF-AB), vascular endothelial growth factor (VEGF), transforming growth factor (TGF) beta1, and interleukin-8 (IL-8) were evaluated by enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction. Eosinophil-epithelial interactions significantly stimulated the secretion of MUC5AC, PDGF-AB, VEGF, TGF-beta1, and IL-8 in culture supernatants. The epidermal growth factor receptor tyrosine kinase inhibitor AG1478 inhibited the coculture-induced secretion of MUC5AC, PDGF-AB, VEGF, and IL-8. Neutralizing antibodies directed against TGF-alpha or amphiregulin and pan-metalloproteinase inhibitor GM6001 inhibited the coculture-induced secretion of MUC5AC and amphiregulin from the cocultured NCI-H292 cells. Coculture of NCI-H292 cells with peripheral blood eosinophils also significantly stimulated MUC5AC production. The results of this study indicate that eosinophil-epithelial cell interactions are important in the pathogenesis of tissue remodeling of eosinophil-predominant airway inflammation such as occurs in nasal polyposis and bronchial asthma.

Alteration of Th17 and Treg cells in patients with unexplained recurrent spontaneous abortion before and after lymphocyte immunization therapy.

BackgroundSeveral types of T cells have been associated with the pathogenesis of unexplained recurrent spontaneous abortion (URSA), including Th1/Th2/Th17/Tregs cell. It has been appreciated that immunotherapy with paternal or third party lymphocytes is an effective method of treatment for URSA patients. The balance of Th1/Th2 cells could be maintained and an increase of Treg cells would be beneficial after immunotherapy; however, the mechanism by which the Th17/Treg balance affects URSA has not yet been fully elucidated.MethodsHere, we used flow cytometry, liquid chip technology and quantitative real-time PCR (qPCR) methods to characterize Th17/Treg cell populations after immunotherapy. We found that after immunotherapy in URSA patients, the percentage of Th17 cells decreased and the percentage of Treg cells in peripheral blood mononuclear cells (PBMC) increased, as detected by flow cytometry.ResultsImmunotherapy may induce a decrease in the Th17/Treg ratio and the Treg bias, which may be beneficial for the maintenance of pregnancy. The expression level of ROR gamma t, a transcription factor found in Th17 cells, decreased and the expression of the Treg-specific transcription factor Foxp3 increased in peripheral blood as detected by qPCR. Immunotherapy may induce a decrease in the ratio of ROR gamma t to Foxp3 and a Treg cell bias, which would be beneficial for pregnancy maintenance. The secretion of the Treg-associated cytokine TGF-beta, as well as Th2 cytokines, was increased in serum, while the secretion of Th17-associated cytokine IL-17A and Th1 cytokine production was decreased. The Th1/Th2 cytokine ratio significantly decreased. Similarly, the Th17/Treg ratio significantly decreased in the total patient after immunotherapy.ConclusionsThese results indicate that in patients with URSA, immunotherapy with mononuclear cells derived from the baby’s father could affect both Th1/Th2 and Th17/Treg balance, and we found that the Th2 and Treg bias would be beneficial for pregnancy, which may lead to a balancing of the Th17/Treg ratio in URSA patients after immunotherapy.

Foxp3+ Regulatory T Cell Subsets Are Induced In Alloreactive Microenvironment and Associated With Chronic Graft-Versus-Host Disease Severity After Allogeneic Hematopoietic Stem Cell Transplantation

BackgroundRegulatory T cells (Tregs), in particular CD4+ Foxp3+ T cells (CD4+Tregs), have been shown to play important roles in the maintenance of tolerance after allogeneic hematopoietic stem cell transplantation (Allo-HSCT). CD8+ Foxp3+ T cells (CD8+Treg) have also been shown to control alloimmune responses in preclinical and clinical models (Renee J. Robb et al, Blood, 2012;119(24):5898-5908). In our previous study, we found that Foxp3+ gamma delta TCR+ Treg cells (gamma delta Tregs) played important regulatory roles in alloimmunity and in xenogeneic GVHD protection (Hu Yong-xian, et al. Leukemia, 2013;27(7):1580-5). Whether these Treg subsets are simultaneously present or have synergetic functional roles in allo-HSCT or, more specifically, GVHD biology is not known. To address this, we analyzed the frequency and possible roles of these Treg cell subsets in patients after allo-HSCT. MethodsPatients who underwent allo-HSCT in our single center from January 2000 to July 2012 were selected. They were divided into 3 groups according to cGVHD criteria including non-cGVHD group, limited cGVHD group and extensive cGVHD group. We also selected healthy volunteers as healthy group. 10ml peripheral blood was drawn from all the selected patients and volunteers for CD4+Tregs, CD8+Tregs and gamma delta Tregs percentage and CD4/CD8 ratio analysis by flow cytometry. Serum cytokine levels of tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (INF-gamma) and transforming growth factor-beta1 (TGF-beta1) were evaluated by ELISA. Data were processed and analyzed using statistical software (SPSS 17.0). Statistical significance for the difference in different groups was assessed by two-sample t-test and one-way analysis of variance (ANOVA). Spearman's correlation was used to test the correlation between two continuous variables. Findings21, 18 and 23 patients were selected in non-cGVHD group, limited cGVHD group and extensive cGVHD group respectively. 15 healthy volunteers were also selected in healthy group. As shown in Figure 1A, the percentages of CD4+Tregs, CD8+Tregs and gamma delta Tregs percentage were all significantly increased in non-cGVHD group compared to healthy group, limited cGVHD group and extensive cGVHD group while these 3 types of Tregs were increased in limited cGVHD group compared with extensive cGVHD group (p<0.05 respectively). TNF-alpha, INF-gamma, and CD4/CD8 have been reported to be associated with cGVHD severity. Our results got similar results (Figure 1B and 1D). Foxp3+ Tregs play their immunosuppressive roles partly via TGF-beta1 secretion. As shown in Figure 1C, the levels of TGF-beta1 increased dramatically in non-cGVHD group compared to healthy controls and cGVHD patients (P < 0.05). Spearman's correlation analysis revealed that the increased level of TGF-beta1 was positively associated with increased regulatory T cell subsets but was negatively associated with cGVHD severity (p<0.05). We also found that all the 3 types of Treg cell percentages were positively correlated with CD4/CD8 ratio in cGVHD cohort. [Display omitted] ConclusionsFoxp3+ regulatory T cell subsets (CD4+ Tregs, CD8+ Tregs and gamma delta Tregs) can be simultaneously induced in alloreactive microenvironment and may be associated with cGVHD severity after allo-HSCT. They may play synergetic functional roles in controlling cGVHD. Our findings support the development of new strategies to increase the number of 3 types of Treg cells including CD4+ Tregs, CD8+ Tregs and gamma delta Tregs following allo-HSCT to prevent or correct cGVHD. Disclosures:No relevant conflicts of interest to declare.

The beneficial effect of leflunomide on systemic lupus erythematosus: the role of Tregs repopulation?

We read with great interest the recent article by Wu et al. [1]. They reported the beneficial effects of leflunomide treatment systemic lupus erythematosus (SLE) and speculated that it might suppresses the production of proinflammatory cytokines such as tumor necrosis factor (TNF)alpha and interleukin (IL)-17, blocks TNF-mediated NF-jB activation and induces inhibition of T cell activation [1]. However, we would like to add another possible mechanism on the beneficial effect of leflunomide on SLE. It has been reported that regulatory T cells (Treg) play important roles in the induction of peripheral tolerance to selfand foreign antigens [2] and naturally occurring CD4?CD25? Treg, which express the transcription factor forkhead box protein P3 (Foxp3), have been widely studied as deficiency of Tregs abrogates self-tolerance and causes autoimmune disease, such as SLE [2]. Recently, there have been some reports showing that leflunomide repopulate Tregs [3, 4]. Wang et al. [3] showed that by leflunomide inhibited arthritis by upregulating CD4?CD25? Tregs, and the active metabolite of leflunomide promoted the differentiation of spleen lymphocytes into CD4?CD25? Tregs and increased anti-inflammatory cytokine (TGF-beta1) secretion. Also, Baban et al. [4] demonstrated that leflunomide increased FoxP3 Tregs and IL-10-positive cells but reduced IL-17and IL-23expressing cells in both the peripheral blood and kidney cells in the ischemic-reperfused kidney model. Therefore, there is a possibility that leflunomide might treat SLE by repopulating Tregs in addition to the suppressing inflammatory cytokines. Further studies are necessary to evaluate serial changes of Tregs after leflunomide therapy in SLE and the exact molecular role of Tregs in the pathogenesis in SLE development should be further elucidated in the future.

Homozygosity for killer immunoglobin-like receptor haplotype A predicts complete molecular response to treatment with tyrosine kinase inhibitors in chronic myeloid leukemia patients

Several recent reports suggest a possible role for killer immunoglobulin-like receptors (KIR) in the onset of chronic myeloid leukemia (CML) and response to therapy with tyrosine kinase inhibitors (TKIs). To explore this hypothesis, we studied KIRs and their human leukocyte antigen class I ligands in 59 consecutive patients with chronic-phase CML (mean age, 53 years; range, 23-81 years) and a group of 121 healthy control participants belonging to the same ethnic group as the patients. The 2-year cumulative incidence of complete molecular response, obtained after a median of 27 months (range, 4-52 months), was 51.2%. An increased frequency of the activating receptor KIR2DS1 (pm = 0.05) and a reduced frequency of the KIR-ligand combination KIR2DS2/2DL2 absent/C1 present (pm = 0.001) were significantly associated with CML. Moreover, KIR repertoires in patients appeared to influence response to TKI therapy. Homozygosity for KIR haplotype A (pm = 0.01), a decreased frequency of the inhibitory KIR gene KIR2DL2 (pm = 0.02), and low numbers of inhibitory KIR genes (pm = 0.05) were all significantly associated with achievement of complete molecular remission. These data suggest that a decrease in properly stimulated and activated NK cells might contribute to the occurrence of CML and indicate homozygosity for KIR haplotype A as a promising immunogenetic marker of complete molecular response that could help clinicians decide whether to withdraw treatment in patients with CML.

Tregs Specific for Minor Histocompatibility Antigens

Abstract 1891 Background:The risk of acute GVHD after HSCT is increased in male recipients of female grafts. Disparities for the male-associated H-Y and other minor histocompatibility antigens (mHAs) have the capacity to sensitize alloreactive donor T cells and cause GVHD in HLA-matched recipients. These mHAs are polymorphic proteins that differ between donor and recipient and are presented as peptides by HLA molecules on recipient or donor antigen-presenting cells to donor immune cells. Currently, there is no evidence that minor histocompatibility antigen specific Tregs exist. Earlier in our laboratory, we have measured the frequency, growth requirements, and function of human blood Tregs specific for allo-MHC. In the present study, we sought to detect the frequency, expansion kinetics and characteristics of the minor antigens specific Tregs in the blood of HLA-matched sibling pair. Methods:CD4+CD25+CD127− Tregs were isolated by immunoabsorption from sibling donors, and cultured with HLA-matched sibling recipient antigen-presenting cells in the presence of IL-2, IL-15 and rapamycin. We detected 30–50 fold increase in H-TdR uptake at 6 days in Treg cultures stimulated by HLA-identical sibling compared to self DC. The precursor frequency of mHA-specific Tregs are between 7 and 43 (median - 13) cells per one million blood Tregs. The frequency of mHA-specific conventional CD4 T cells among total blood CD4 T cells is similar in HLA-matched sibling donors. Ex vivo expanded mHA-specific Tregs maintained higher levels of Foxp3 expression, retained the lymphoid homing receptor CD62L and a chemokine receptor, CCR7, suggesting that they are functional and are able to migrate to lymphoid tissue in vivo. Split well assay on day 12 demonstrated the mHA specificity, since Treg responded to restimulation with DC from the original HLA-identical sibling, but not self DC. The mHA-specific Tregs expanded to more than 100 fold in vitro, and exhibited antigen specific suppression. When Tregs were cultured at limiting dilution, we obtained 6 mHA-specific Treg clones that retained TGF-beta secretion in response to the sibling's mHA-disparate DC but not self DC. Conclusion:We demonstrated for the first time that it is possible to detect and expand mHA specific Tregs from HLA-matched sibling pairs, immunotherapy with mHA-specific Tregs may prevent GVHD. Disclosures:No relevant conflicts of interest to declare.

Regulatory T cells in children undergoing rush venom immunotherapy

Venom immunotherapy (VIT) induces immune tolerance to Hymenoptera venom but the underlying mechanisms are not clarified. Regulatory T cells are thought to play an important role in tolerance induction during specific immunotherapy. Our objective was to determine the effects of rush VIT on the percentage of regulatory T cells and immunosuppressive cytokines interleukin (IL)-10 and transforming growth factor beta (TGF-beta) in children. Blood samples were collected from 18 children with a previous systemic allergic reaction to a Hymenoptera sting, with a positive skin test and positive specific IgE, before rush VIT, after 6 weeks and 6 months of rush VIT. Ten children with no history of venom allergy were studied as controls. Isolated peripheral blood mononuclear cells were stained with specific markers for regulatory T cells and analyzed by flow cytometry. The percentage of regulatory T cells did not change during rush VIT in children. No change was noticed in the percentage of IL-10 and TGF-beta secreting cells after 6 weeks or 6 months of VIT. No difference in expression of cytotoxic T-lymphocyte antigen 4 on CD4(+)CD25(+high) was found. Rush VIT is a safe and effective treatment for patients allergic to Hymenoptera venom. Although regulatory T cells are considered to be responsible for this effect; no significant changes in the percentage of these cells or immunosuppressive cytokines were noticed during rush VIT in children. Additional investigations are needed to clarify the role of regulatory T cells in the induction of tolerance during rush VIT in children.

Phosphatidylserine Exposure by Toxoplasma gondii Is Fundamental to Balance the Immune Response Granting Survival of the Parasite and of the Host

Phosphatidylserine (PS) exposure on the cell surface indicates apoptosis, but has also been related to evasion mechanisms of parasites, a concept known as apoptotic mimicry. Toxoplasma gondii mimics apoptotic cells by exposing PS, inducing secretion of TGF-beta1 by infected activated macrophages leading to degradation of inducible nitric oxide (NO) synthase, NO production inhibition and consequently persisting in these cells. Here PS+ and PS− subpopulation of tachyzoites were separated and the entrance mechanism, growth and NO inhibition in murine macrophages, and mice survival and pathology were analyzed. Infection index in resident macrophages was similar for both PS subpopulations but lower when compared to the total T. gondii population. Growth in resident macrophages was higher for the total T. gondii population, intermediate for the PS+ and lower for the PS− subpopulation. Production of NO by activated macrophages was inhibited after infection with the PS+ subpopulation and the total populations of tachyzoites. However, the PS− subpopulation was not able to inhibit NO production. PS+ subpopulation invaded macrophages by active penetration as indicated by tight-fitting vacuoles, but the PS− subpopulation entered macrophages by phagocytosis as suggested by loose-fitting vacuoles containing these tachyzoites. The entrance mechanism of both subpopulations was confirmed in a non-professional phagocytic cell line where only the PS+ tachyzoites were found inside these cells in tight-fitting vacuoles. Both subpopulations of T. gondii killed mice faster than the total population. Clear signs of inflammation and no tachyzoites were seen in the peritoneal cavity of mice infected with the PS− subpopulation. Moreover, mice infected with the PS+ subpopulation had no sign of inflammation and the parasite burden was intense. These results show that PS+ and PS− subpopulations of T. gondii are necessary for a successful toxoplasma infection indicating that both subpopulations are required to maintain the balance between inflammation and parasite growth.

The effects of different concentration of opium on the secretion of TGF-β in Jurkat cells

The effects of different concentration of opium on the secretion of TGF-beta in Jurkat cells