Total Treg Research Articles

Inhibition of Immune Cell Subsets Is Differentially Affected By Dasatinib Dosage in Patients with Chronic Phase CML

Inhibition of Immune Cell Subsets Is Differentially Affected By Dasatinib Dosage in Patients with Chronic Phase CML

The Role of Regulatory T and B Cells in the Etiopathogenesis of Idiopathic Granulomatous Mastitis

ABSTRACT Background and objectives The aim of this study was to evaluate the role of T- and B-regulatory cells (Tregs and Bregs) in the pathogenesis of idiopathic granulomatous mastitis (IGM). Methods This study includes 47 patients with pathologically proven IGM (Group P) and 26 healthy subjects (Group C). The patients in Group P were divided into two groups according to whether their lesions were active (Group PA, n: 21) or in remission (Group PR, n: 26). By using flow-cytometry, the frequencies of CD3+CD4+CD45RA−Foxp3high activated Tregs (aTregs), CD3+CD4+CD45RA−Foxp3low non-suppressive Tregs, CD3+CD4+CD45RA+Foxp3low resting Tregs (rTregs), CD3+CD4+CD25+Foxp3− T-effector cells (Teff), total Tregs and Bregs were analyzed in all subjects. Results The frequency of the Teff cells was statistically higher in Group P when compared with Group C (p =.004). The Foxp3 expression of Treg cells and the frequency of non-suppressive Tregs in Group P were statistically lower than Group C (p =.032 and p =.02, respectively). In addition, Group PR’s Foxp3 expressions were statistically lower than Group C (p =.027); Group PR’s aTregs ratio was statistically lower than Group PA (p =.021); and the non-suppressive Tregs ratio of Group PR was lower than both Group PA and Group C (p =.006 and p <.0001). No significant differences were seen Bregs and B cell subsets. Conclusion Significant changes in Foxp3 expression and Treg subsets were seen in patients with active IGM lesion and in remission. This study shows an intrinsic defect of Tregs in patients with IGM.

Low-Dose IL-2 for Treating Moderate to Severe Alopecia Areata: A 52-Week Multicenter Prospective Placebo-Controlled Study Assessing its Impact on T Regulatory Cell and NK Cell Populations

Low-Dose IL-2 for Treating Moderate to Severe Alopecia Areata: A 52-Week Multicenter Prospective Placebo-Controlled Study Assessing its Impact on T Regulatory Cell and NK Cell Populations

Effect of Octreotide Long-Acting Release on Tregs and MDSC Cells in Neuroendocrine Tumour Patients: A Pivotal Prospective Study.

Octreotide long-acting repeatable (LAR) is largely used to treat functional and/or metastatic neuroendocrine neoplasms (NENs). Its effect in controlling carcinoid syndrome and partially reduce tumour burden is attributable to the ability of octreotide to bind somatostatin receptors (SSTRs) on the tumour and metastasis, regulating growth hormone secretion and cell growth. Notably, SSTRs are also expressed, at different levels, on Tregs. Tregs, together with myeloid-derived suppressor cells (MDSCs), are key components in the anti-tumour immunoregulation. This is the first prospective study aimed to explore the impact of Octreotide (OCT) LAR on the immune system, with a particular focus on Tregs and MDSC cells. Here, we show that circulating Tregs are elevated in NENs patients compared to healthy donors and that treatment with OCT LAR significantly decrease the level of total Tregs and of the three functional Tregs populations: nTregs, eTregs and non-Tregs. Furthermore, OCT LAR treatment induces a functional impairment of the remaining circulating Tregs, significantly decreasing the expression of PD1, CTLA4 and ENTPD1. A trend in circulating MDSC cells is reported in patients treated with OCT LAR. The results reported here suggest that the effect of OCT LAR on Tregs could tip the balance of the patients’ immune-system towards a durable anti-tumour immunosurveillance with consequent long-term control of the NENs disease.

Regulatory T cell subsets in bullous pemphigoid and dipeptidyl peptidase-4 inhibitor-associated bullous pemphigoid

BackgroundRegulatory T (Treg) cells play an essential role in peripheral immune tolerance. Bullous pemphigoid (BP) is the most common blistering disease and is caused by autoantibodies to two BP antigens: type XVII collagen and BP230. Recently, we reported that Treg cell dysfunction may cause the production of autoantibodies to BP antigens. Several studies have suggested an association between Treg cells and BP pathogenesis. However, Treg cells are heterogeneous in humans, leading to inconsistent results in previous studies. ObjectiveTo assess functional Treg subsets in BP. MethodsWe examined three distinct Treg subsets in conventional BP (cBP) patients before versus after systemic corticosteroid treatment, dipeptidyl peptidase-4 inhibitor-associated BP (DPP-4i-BP) patients, younger controls and older controls. ResultsWe found that total Treg cells and all Treg cell subsets were increased in cBP patients before treatment and decreased by systemic corticosteroid treatment. In contrast, neither total Treg cells nor all Treg subsets were increased in DPP-4i-BP. Notably, CD45RA− Foxp3hi effector Treg cells positively correlated with disease severity in cBP, whereas CD45RA+Foxp3lo naïve Treg cells positively correlated with the disease severity in DPP-4i-BP. ConclusionThese findings suggest that Treg cells are differently involved in the pathogeneses of cBP and DPP-4i-BP.

Abstract 2913: Entinostat (KHK2375), class I histone deacetylase inhibitor, regulates human Treg function by decreasing Foxp3 expression and effector Treg population

Abstract Objective: Entinostat (KHK2375 also known as MS-275, SNDX-275 and ZN244894), a synthetic benzamide derivative, is a class I selective histone deacetylase inhibitor. Entinostat increases the acetylation of histone and non-histone proteins. Change of histone modification status leads to chromatin remodeling and alters gene expression resulting in the change of cellular phenotype. Also, it has been reported that entinostat decreased myeloid-derived suppressor cells (MDSCs) in both mice and humans, suggesting that entinostat may impact tumor immunology. In this study, we evaluated the effect of entinostat on CD4+ regulatory T cells (Treg) using human peripheral blood mononuclear cells (PBMCs). Materials/methods: Protein lysine acetylation (Ac-K) status and Treg population were evaluated by flow cytometric analysis. Expression of FoxP3, a master regulator of Treg development and function, was evaluated by both mRNA and protein levels. Treg function was also evaluated in terms of IFNγ production from CD4+ T cells (Tconv) and CD8+ T cells, and proliferation of these cells under a co-culture condition with Treg. Furthermore, the effect of entinostat on Treg population in PBMCs obtained in a phase 1 trial with Japanese breast cancer patients was evaluated. Results: Entinostat induced the accumulation of Ac-K signals in naive Treg (CD45RA+FoxP3low), effector Treg (CD45RA−FoxP3high) and non-Treg (CD45RA−FoxP3low). Interestingly, entinostat selectively reduced the proportion of effector Treg among CD4+ T cells in a dose dependent manner, while those of naive Treg and non-Treg, as well as total Treg defined by CD4+CD25+CD127low/− were not changed by entinostat. Entinostat showed the reduction of FoxP3 expression in total Treg in both mRNA and protein levels. Moreover, immuno-suppressive function of Treg as measured by T cell dependent IFNg production was cancelled by entinostat. Lastly, the decrease of effector Treg proportion was also observed in PBMCs on the day following the oral administration of entinostat in the phase 1 study (Cohort 1: 3 mg/day (N=3), Cohort 2: 5 mg/day (N=3), Cohort 3: 10 mg/day (N=2)). Conclusion: Entinostat (KHK2375) showed an inhibitory effect on immuno-suppressive function of human Treg by decreasing FoxP3 expression and effector Treg proportion. These results support a potential role for entinostat mediated immunomodulatory effects in providing clinical benefit to patients. Citation Format: Hiromichi Kosaka, Yozo Nishimura, Ayumi Kaneda, Norikazu Masuda, Kenji Tamura, Shigehira Saji, Hiroji Iwata, Kiyotoshi Mori. Entinostat (KHK2375), class I histone deacetylase inhibitor, regulates human Treg function by decreasing Foxp3 expression and effector Treg population [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2913.

CCR4 and CCR6 Expression Characterizes Memory Tregs Subsets During HIV Infection and Correlates with Disease Progression

Abstract Chemokine receptor expression on Tregs has been associated with pathogenesis of several diseases such as cancer, as CCR4 promotes Treg recruitment into tumors. During HIV infection, continuous Treg stimulation due to chronic inflammation has negative effects such as tissue fibrosis and limited antiviral response. We studied CCR4, CCR6, CXCR3 and CXCR5 on Treg subsets to identify homing patterns during HIV disease. CD4+ T cells (Tc) from 33 chronic untreated (CHR) and 52 antiretroviral treated (ART) HIV donors were characterized by FACS using memory markers. Tregs were defined as CD127lowCD25hi. Analysis was performed on Cytobank and Prism 6 using non-parametric tests. Tregs represented 7.0% and 8.0% of total CD4+ Tc in CHR and ART, respectively. Average Treg memory subsets distribution in CHR vs. ART was: Tscm=17.2% vs. 15.3%, Tcm=30.7% vs. 36.3% (p=0.0008), Ttm=27.4% vs. 25.7%. Chemokine receptor expression on total Tregs was (CHR vs. ART): R4=46.9% vs. 55.2% (p=0.0261), R6=31.6% vs. 40.1% (p=0.0043), X3=20.8% vs. 20.9% and X5=7.6% vs. 10.3%. In CHR, R4 (p=0.0007 r=0.3161) and R6 (p=0.0054 r=0.2241) frequencies correlated with CD4+ Tc count. Both markers were preferentially found on memory Treg (Tcm: R4=70.5%, R6=51.2%; Ttm: R4=93.4%, R6=65.7%) and had a significantly lower expression on non-Treg subsets (Tcm: R4=43.2%, R6=46.9%; Ttm: R4=37.0%, R6=29.9%). A similar pattern was also observed in people on ART. We showed that Tregs are characterized by R4 and R6 expression during treated and untreated HIV infection. Both molecules are markedly found on memory phenotypes, suggesting a role in controlling homing of effector Tregs. Accumulation of R4+R6+ Tregs may reflect a response to control chronic activation that persists after ART.

The Endocannabinoid System: a new anti-inflammatory target in rheumatoid arthritis

Abstract Rheumatoid arthritis (RA) is characterised by the loss of regulatory T cell (Treg) mediated suppression of inflammatory T helper cells. RA patients with loss of immune tolerance display elevated levels of circulating pro-inflammatory cytokines. The endocannabinoid system (ES) present on immune cells may represent a novel target for development of anti-inflammatory treatments. We screened the therapeutic potential of a phytocannabinoid compound, GL1b, in an ex vivo, peripheral blood mononuclear cell (PBMC) inflammatory model. PBMCs were cultured with CD3/CD28 and IL-2 to activate and expand T cells. Inflammation was induced by phorbol 12-myristate-13-acetate (PMA)/ionomycin activation, over 4 hours. Inflamed PBMCs were treated with 0, 10, 20 and 50 μM GL1b for 24 hours, then harvested for flow cytometry analysis of Tregs. Cytokine mRNA expression and conditioned media concentration were analysed with qRT-PCR and ELISA, respectively. Results will be fed into in silico model. 50 μM GL1b significantly suppressed the total Treg population (p&amp;lt;0.05), and relative numbers of CD69+, FOXP3+ and CTLA4+ Treg subpopulations decreased in a dose-responsive manner. A trend toward increased mean fluorescent intensity (MFI) of FOXP3 and CTLA4 was observed at 10 μM, relative to vehicle. A significant (p&amp;lt;0.01) decrease was observed in IL-6 mRNA relative to vehicle with 50 μM GL1b, whereas at 10 – 20 μM concentrations, a trend toward lower IL-6, TNFα, IL-10 expression was evident. Conversely, secreted cytokine concentrations were increased at 10 – 20 μM GL1b, relative to vehicle. Although the phytocannabinoid GL1b reduces the overall Treg population, activated and suppressive Treg subpopulations are elevated and cytokine gene expression reduced.

Regulatory T-cell subset distribution in children with primary hypertension is associated with hypertension severity and hypertensive target organ damage.

The relationship between circulating regulatory T-cell (Tregs) subset distribution and hypertension severity in children with primary hypertension is not known. We aimed to find out if target organ damage (TOD) in children with primary hypertension is related to defects in Tregs distribution reflected by their phenotype characteristics. The study constituted 33 nontreated hypertensive children and 35 sex-matched and age-matched controls. Using multicolor flow cytometry technique, we assessed a distribution of the total Tregs (CD4CD25CD127) and their subsets (CD45RA-naive Tregs, CD45RA memory/activated Tregs, CD45RACD31 recent thymic emigrants Tregs and mature naive CD45RACD31 Tregs) in the whole blood. Hypertensive children showed decreased percentage of the total Tregs, the CD45RA-naive Tregs, the total CD31 Tregs and the recent thymic emigrants Tregs but elevation of the CD45RA memory/activated Treg and mature naive CD45RACD31 Tregs. Decreased frequency of the total Tregs, naive Tregs and CD31-bearing Treg cell subsets (CD31 total Tregs, CD45RACD31 recent thymic emigrants Tregs) negatively correlated to TOD markers, arterial stiffness and blood pressure elevation. In contrast, increased percentage of memory Tregs and CD31 Tregs subsets positively correlated to organ damage markers, arterial stiffness and blood pressure values. These changes were independent of BMI, age, sex and hsCRP. Both diagnosis of hypertension, TOD and arterial stiffness in hypertensive children were associated with decreased population of total CD4 Tregs, limited output of recent thymic emigrants Tregs, and increased pool of activated/memory Tregs. Hypertension was an independent predictor of the circulating Treg subsets distribution irrespective of hsCRP.

Reduced CD4+ CD25++ CD45RA− Foxp3hi activated regulatory T cells and its association with acute rejection in patients with kidney transplantation

BackgroundIt was found that regulatory T cells (Tregs) importantly affect the maintenance of the kidney graft. However, Tregs are a heterogeneous population with less to more suppressive activity. The aim of this study was to determine the effects of different subsets of Tregs, as well as their ratio to effector T cells (Teff), on kidney transplantation outcomes. MethodsA total of 58 participants were enrolled in this study and divided into four groups: (i) first kidney transplant recipients (stable 1); (ii) second kidney transplant recipients (stable 2); (iii) transplant recipients with acute rejection (AR); and (iv) healthy control subjects. By using flow cytometer, the frequencies of CD4+ CD25++ CD45RA− Foxp3hi activated Tregs (aTregs), CD4+ CD25+ CD45RA+ Foxp3lo resting Tregs (rTregs), CD4+ CD25+ CD45RA− Foxp3lo non-suppressive T cells, CD4+ CD25+ Foxp3− cells Teff, and total Tregs were analyzed in all subjects. ResultsThe frequency of aTregs (as well as the ratio of aTregs/Tregs) was significantly lower in the AR patients than the other three groups. In contrast to AR patients, stables 1 and 2 had a higher aTreg/Treg ratio than those in the control group. Although patients with AR had a significantly lower total Tregs than the other three groups, the balance of total Tregs and Teff was similar between patients with and without AR. ConclusionPatients with AR had poorer immunoregulatory properties than those with normal graft functioning, as well as those in the control group. These reduced immunoregulatory properties in patients with AR could lead to graft rejection.

Regulatory T cells and IL-17A levels in noninfectious uveitis.

Regulatory T cells (Tregs) have been intensively studied in a myriad of autoimmune diseases. As for noninfectious uveitis (NIU), results have been contradictory, and studies have failed to demonstrate a consistent reduction in Treg cell frequency in patients with active disease. The present study aims to characterize T lymphocyte subsets, including naïve and memory Tregs as well as their respective CD39 expression, in the peripheral blood of NIU patients. Inflammatory as well as suppressive cytokine profiles were also evaluated. T cell subpopulations were evaluated by multiparametric flow cytometry using anti-CD3, anti-CD4, anti-CD45, anti-CD45RA, anti-CD197, anti-CD25, anti-CD127, and anti-CD39. Treg cells were defined as CD3 + CD4+CD25hiCD127low. A multiplex bead-based immunoassay was used to determine TNF-α, IFN-ɣ, IL-17A, IL-10, and TGF-β levels. Twenty-nine patients with active NIU were included as well as 15 sex- and age-matched controls. There were no significant differences in T lymphocyte subsets, including Tregs, between patients and controls. However, patients with a lower grade of anterior chamber or vitreous inflammatory cellular reaction showed higher memory Treg counts than controls, with no respective increase in CD39+ expression, and a tendency for higher IL-17A levels (p = 0.06). This IL-17A elevation was present in the total NIU group (p = 0.08) as well as a positive correlation between IL-17A levels and the absolute counts of memory Tregs (p = 0.013; R = 0.465). Patients with higher IL-17A levels also showed higher serum concentrations of memory (p = 0.001) and naïve (p = 0.003) Tregs as well as elevated TNF-α (p < 0.0001) and IFN-ɣ (p = 0.016) levels. Negative correlations were observed between IL-10 and TGF-β levels and the percentages of memory (p = 0.030; R = - 0.411) and total CD39+ Tregs (p = 0.051; R = - 0.373) in the peripheral blood of NIU patients. Our results showed that total Treg levels were not reduced in patients with NIU. Further characterization of Treg subsets, including memory Tregs and respective CD39 expression, may provide additional insight on the role of Treg cells in NIU. Consistent high levels of circulating IL-17A in NIU patients are in accordance with previous studies and reinforce this cytokine's vital role in uveitis pathogenesis and its possible use as a therapeutic target.

HIV-1 elite controllers present a high frequency of activated regulatory T and Th17 cells.

HIV-1 infection is characterized by generalized deregulation of the immune system, resulting in increased chronic immune activation. However, some individuals called HIV controllers (HICs) present spontaneous control of viral replication and have a more preserved immune system. Among HICs, discordant results have been observed regarding immune activation and the frequency of different T cell subsets, including Treg and Th17 cells. We evaluated T cell immune activation, differentiation and regulatory profiles in two groups of HICs—elite controllers (ECs) and viremic controllers (VCs)—and compared them to those of cART-treated individuals (cART) and HIV-1-negative (HIV-neg) individuals. ECs demonstrated similar levels of activated CD4+ and CD8+ T cells in comparison to HIV-neg, while cART and VCs showed elevated T cell activation. CD4+ T cell subset analyses showed differences only for transitional memory T cell frequency between the EC and HIV-neg groups. However, VC individuals showed higher frequencies of terminally differentiated, naïve, and stem cell memory T cells and lower frequencies of transitional memory and central memory T cells compared to the HIV-neg group. Among CD8+ T cell subsets, ECs presented higher frequencies of stem cell memory T cells, while VCs presented higher frequencies of terminally differentiated T cells compared to the HIV-neg group. HICs showed lower frequencies of total Treg cells compared to the HIV-neg and cART groups. ECs also presented higher frequencies of activated and a lower frequency of resting Treg cells than the HIV-neg and cART groups. Furthermore, we observed a high frequency of Th17 cells in ECs and high Th17/Treg ratios in both HIC groups. Our data showed that ECs had low levels of activated T cells and a high frequency of activated Treg and Th17 cells, which could restrict chronic immune activation and be indicative of a preserved mucosal response in these individuals.

Preventing Post-Transplant Diabetes Mellitus Via Orchestration of the IL-33/ST2 Axis in Pancreatic Islets

Post-transplant diabetes mellitus (PTDM) occurs after allogeneic hematopoietic cell transplantation (HCT) in over 50% of HCT recipients and will increase 3-fold their risk of death. Further, levels of soluble Stimulation-2 (ST2) are predictive of PTDM in adults and children. However, molecular pathology of PTDM remains poorly understood, and therapeutic strategies for PTDM are limited. PTDM is initiated by hyperinsulinemia and hyperglycemia, and the alarmin IL-33 has been found to contribute to the secretion of insulin in islets by activating innate lymphoid cells type 2 (ILC2s) via Stimulation2 (ST2), the IL-33 receptor (<i>Dalmas E. et al, Immunity 2017</i>). This ST2/IL-33 regulatory axis is also critical for regulating inflammation and the balance between cytopathic T effector cells (Teffs) and regulatory cells (CD4⁺FOXP3⁺ Tregs and ILC2s). Therefore, we explored the role of ST2/IL-33 in pancreatic islets post-HCT. First, neither fasting blood glucose nor glucose tolerance (time and area under the curve) was significantly different between syngeneic and allogeneic wild-type (WT) or ST2^−/− recipients of minor mismatch HCT (B6→C3H.SW) (Fig. 1A, 1B). Total infiltrating CD90.2⁺ T cells in the pancreas were increased in the allogeneic groups but not different between WT or ST2^−/− recipients (Fig. 1C). However, pancreatic IFNγ⁺ T cells were significantly decreased while total Tregs in ST2^−/− recipient were increased (Fig. 1D, 1E). Second, using ST2 blockade with a neutralizing antibody from day -1 to day 9, 100 μg every over day for 6 doses), we showed significant improvement of survival in the treated group as compared to the isotype group (Fig. 2A), consistent with an increase in plasma IL-33 (Fig. 2B) as well as decreased plasma TNFα and IFNγ (Fig. 2C, 2D). Lastly, increased β-cell proliferation before immune cell invasion prevents progression of type 1 diabetes (<i>Ercument. et al, Nature Metabolism 2019</i>), and we found increase T cell infiltration. Thus, we specifically sought to analyze the ratio of β-cells/α-cells and both Teffs and regulatory cells in the pancreatic islets in anti-ST2 antibody treated vs. isotype groups. We found that the average ratio of β-cells/α-cells was decreased in the treated group in comparison to the isotype group (Fig. 3A). In addition, both frequencies of Tregs and ILC2s significantly increased while frequency of CD8⁺ Teffs decreased in the treated group as compared to the isotype group (Fig. 3B, 3C, 3D). We conclude that the effects of PTDM on glucose tolerance in the acute preclinical HCT model are not immediate while the effects on β-cells and immune cells are. Our data also suggest that PTDM display both type 1 and 2 diabetes phenotypes. Importantly, blockade of the IL-33/ST2 axis protected islet endocrine cells by regulating Teffs/ regulatory Tcells and ILC2s balance, and may provide an attractive approach for PTDM prevention.

Assessment of Anti-Oxidant Markers of Inflammation in Patients with Chronic Graft-Versus-Host Disease

Chronic graft-versus-host disease (cGVHD) is a late-occurring inflammatory condition post- allogeneic hematopoietic cell transplantation (HCT), which involves multiple distinct interactions among alloreactive dysregulated T and B cells. Prior studies have shown that autoantibodies to platelet-derived growth factor receptor (PDGFR) induce tyrosine phosphorylation and accumulation of Reactive Oxygen Species (ROS), causing expression of collagen type-1 gene through the Ha-Ras-Erk1/2–ROS signaling pathway. We hypothesize that antioxidant markers of inflammation (8-OHdG, total antioxidant levels, CXCL4 and pH2Ax) will be elevated in patients with scleroderma cGVHD compared to patients who do not develop cGVHD or have non-scleroderma cGVHD. Patients (n=36) were consented on an IRB-approved protocol for a cross-sectional single time point biospecimen collection. Patients with established cGVHD (n=29) or ≥2 years post-HCT without clinical manifestations of cGVHD (n=7) were included. Patients with cGVHD were divided into Scleroderma cGVHD (n=14) and Non-scleroderma cGVHD (n=15). Median age was 49 (range: 21-69) and 56 years (range: 18-72) for patients with scleroderma (group 1) and without scleroderma or no GVHD (group 2), respectively. Median prior lines of therapy was 3.2 (range: 2-5) for patients in group 1. All patients is group 1 and 77% of patients in group 2 received PBCSs as the graft source. Transplant was from a matched unrelated (group 1: 75%, group 2: 73%) or a matched sibling donor (group 1: 25%, group 2: 23%). Tacrolimus/sirolimus was the most commonly used GVHD prophylactic regimen in both groups (group 1: 83% and group 2: 64%). Peripheral blood samples were drawn at a single time point post-HCT, and serum samples were used for ELISA assays for levels of total antioxidants, 8-OHdG, and CXCL4. Flow cytometric analysis was performed to investigate percentage of Tregs and pH2AX+ cells. There were no differences in the total antioxidant levels, CXCL4 and Tregs between groups 1 and 2. Using a cutoff threshold of 10 ng/ml of 8-OHdG, 11 patients (78%) in group 1 and 9 patients (40.9%) in groups 2 had elevated levels of 8-OHdG (p=0.029, Fisher's exact). Percentage of pH2AX expressing cells was lower in group 2. At the cutoff threshold of 37%, only one patient (7%) in group 1 and 13 patients (59%) in group 2 had more than 37% pH2AX-expressing CD45+ cells in their peripheral blood (p=0.002, Fisher's exact). In conclusion, our pilot cross-sectional study showed that scleroderma cGVHD is associated with increased ROS levels (8-OHdG) without elevation of pH2AX cells in peripheral blood T cells. Our results indicate that ROS elevation in scleroderma patients may be a stress response to inflammatory milieu and is independent of DNA damage. Our data justify further prospective cohort studies to define the role of ROS markers in scleroderma cGVHD and to develop novel strategies to treat/prevent cGVHD.

Reduced activated regulatory T cells and imbalance of Th17/activated Treg cells marks renal involvement in ANCA-associated vasculitis

The role of naturally occurring regulatory T cells (Treg) in the control of the immune tolerance of ANCA-associated vasculitis (AAV) has not been well defined. Therefore, we separate the phenotypically heterogeneous Treg cells into different subsets based on the expression of FOXP3 and CD45RA during AAV pathogenesis. Fifty-four AAV patients (38 patients with renal involvement) and 19 healthy controls (HCs) were enrolled in this study. Levels of CD4+T cell subsets and cytokines were detected by flow cytometry. Treg immunesuppression capacity was measured in co-culture experiments. The diagnostic value for Treg subsets was evaluated by the areas under the receiver operating characteristic curves (AUC). Patients with AAV had lower percentages and numbers of activated Treg cells (aTreg, P = 0.044, P = 0.002), while higher levels of total Treg cells (P = 0.001, P = 0.026) with diminished immunosuppression capacity. The proportions of effector memory T-cell subpopulation (P < 0.001) were increased in AAV patients. Interestingly, the AUC of the aTreg improved significantly the diagnostic potential of AAV. Furthermore, the ratio of Th17/aTreg was significantly increased in active and renal vasculitis patient and positive correlation between Th17/Treg subset ratio and creatinine or BUN. In addition, we found that cytokine IL-2 and IL-4 exhibited a downward while IL-6, IL-10, TNF-α, IFN-γ and IL-17A trend upward in AAV patients. Increase in total Treg levels, along with functional deficiency, and decrease in aTreg cells constitute potential novel biomarkers for AAV. And the ratio of Th17/aTreg might serve as an important tool to recognize and monitor AAV patients with renal involvement and disease remission.

Identification of the immunological profile in rejection-free heart transplantation

BackgroundTolerance induction following organ transplantation can be achieved by adoptive cell transfer of regulatory T-cells (Tregs) or dendritic cells (DCs). However, the target immunological profile is unknown. The present study aimed to identify an immunological profile connected to tolerance induction following heart transplantation (HTx). MethodsBlood samples of long-term rejection-free HTx patients (LT-HTx, n = 20) and patients on the HTx waiting list (pre-HTx, n = 20) were compared. Flow cytometric and multiplex analyses of DCs, Tregs, subsets of both cell types and serum cytokines were performed. Furthermore, principle component and cluster analysis was used to identify a target immunological profile using a multiparametric dataset. ResultsPlasmacytoid DCs expressing blood DC antigen (BDCA) 2 and BDCA4 were significantly increased in LT-HTx patients (BDCA2+: 29.4 ± 10.1%, p = .022; BDCA4+: 26.4 ± 9.3%, p = .008) compared to pre-HTx patients (BDCA2+: 22.8 ± 7.2%; BDCA4+: 18.9 ± 7.4%). The percentage of total Tregs and of their CD62L+ subset was reduced in LT-HTx patients (%Tregs: 9.1 ± 3.7%, p = .026; %CD62L+: 85.1 ± 11.9%, p = .009) compared to pre-HTx patients (%Tregs: 11.8 ± 3.6%; %CD62L+: 93.3 ± 4.5%). LT-HTx patients showed different cytokine levels than pre-HTx patients. Principle component and cluster analysis revealed that the total DCs, BDCA2+ and BDCA4+ DCs and CD147+ Tregs had the strongest influence to distinguish among long-term rejection-free and pre-HTx patients. ConclusionIn conclusion, we defined the immune status of pre-HTx patients and the target immunological profile of LT-HTx patients. These data may help to establish a monitoring tool that is based on a multiparametric dataset.

Abstract 4043: Application of high complexity flow cytometry for Monitoring activated T-cells, T and B regulatory subsets in combination immunotherapy trials, melanoma case study

Abstract Background Ever increasing knowledge of the interplay between immune composition and a novel drugs’ treatment efficacy or the lack thereof leads to continual advancements in immunotherapies. Given the exponential increase in combination therapies, tools to determine the most promising regimens are critical. Immunotherapeutic agents either reverse T-cell inhibitory pathways (e.g., PD-1) or activate the immune system to induce cytotoxic T-cell activation and proliferation through agonistic stimulation (e.g., Cytokines). These treatment strategies have proven to be efficacious by enhancing the balance between immune-effector and suppressive/regulatory cells that can directly affect the outcome of immunotherapy. To concurrently monitor multiple immune cell subsets responsible for either immuno-suppression or activation, we developed a high-complexity (13-color) flow cytometry panel to reliably enumerate activated T-cells, Tregs (A &amp; B subsets) and Bregs for clinical trial application. Methods The panel includes CD3, CD4, CD8, CD19, FoxP3, CD25, CD127, CD45RA, HLA-DR, CD38, CD24, and CD27 to monitor total Tregs, Treg-subsets, Bregs and T-cell activation. After robust analytical method validation following industry guidelines for exploratory biomarker assessments, we confirmed the clinical utility of this panel in stage III or IV melanoma patients (N= 14) enrolled in a randomized phase II clinical trial receiving high-dose of IL-2 with or without Afliberecept (VEGF inhibitor). Results We observed up-to 20-fold increase in activated CD8+ T-cells following treatment. Interestingly, while no discernible change was observed in the naive Treg-A cells (CD45RAhiFoxp3lo), a remarkable 5-fold expansion of effector Treg-B cells (CD45RAloFoxp3hi) was observed following treatment. Additionally, frequencies of Bregs (CD24hiCD38hi) remained unchanged after treatment. Conclusions In summary, we demonstrated the utility of a powerful high complexity clinical flow cytometry tool for precisely monitoring immune cell subsets involved in tumor escape such as Tregs and Bregs or those involved in tumor killing like CD8+ cytotoxic T-cells. More notably, this tool may be utilized to deprioritize agents that cause immunosuppression and focus on combination agents that lead to additive immune activation. Citation Format: Jennifer Tsau, Michael Abadier, Brittney Atzmiller, Christine Vaupel, Naveen Dakappagari, Ghanashyam Sarikonda, Ahmad Tarhini. Application of high complexity flow cytometry for Monitoring activated T-cells, T and B regulatory subsets in combination immunotherapy trials, melanoma case study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4043.

Helicobacter pylori pediatric infection changes FcεRI expression in dendritic cells and Treg profile in vivo and in vitro

H. pylori infection shows an inverse relationship with allergies. Dendritic cells regulate mucosal immune responses including the induction of T regulatory cells which are fundamental in Helicobacter pylori-induced dampening of allergies. In this respect expression of high-affinity IgE receptor (FcεRI) has been associated with a regulatory dendritic cell profile. Therefore we aimed to evaluate possible mechanisms by which H. pylori infection might modify atopy in pediatric patients. Here we show that H. pylori-infected children exhibited both increased expression of FcεRI on peripheral myeloid and plasmacytoid dendritic cells and higher levels of Foxp3 and Latency Associated Peptide on T regulatory cells. Moreover, exposure to H. pylori drove increased FcεRI expression and IL-10 secretion by both pediatric H. pylori-exposed monocyte derived dendritic cells and T cells. Finally, we show a positive correlation between expression of FcεRI in circulating myeloid DCs and total Treg cells, suggesting that in children, H. pylori infection may have a modulating role in atopy, mediated by both altered surface expression of FcεRI on children's DC and an increased T regulatory cell profile.

Atrophied thymus creates tTreg repertoire holes altering an antigen-specific population in the periphery

Abstract We have shown that there is an increased ratio of total thymic T regulatory (tTreg) cells to thymic T conventional (tTcon) cells generated by the atrophied thymus related to aging. This observation, coupled with the accumulation of peripheral Treg (pTreg) cells with age, poses the difficult question of why Tregs in the periphery of the elderly are unable to suppress amplified self-reactivity-induced inflammaging. We utilized a chimeric mouse model with immune system reconstitution in which lethally irradiated rat insulin promotor-driven (RIP) mOVA host mice received mixed OT-II TCR transgenic and wild-type bone marrow, each expressing distinct congenic identifiers (CD45.1 vs CD45.2). In this system, OVA serves as a mock self-antigen, which is expressed mainly in the pancreas of the host mice. Further, our mOVA host mice carried a FoxN1-floxed gene, for induction of conditional FoxN1 knock-out, resulting in thymic atrophy analogous to age-related thymic atrophy. We observed that chimeric mice with induced thymic atrophy exhibited significantly decreased OVA-specific (OT-II) Tregs, but not total (pan) Tregs, in the spleen and pancreas. The specific Treg cells of mice with thymic atrophy were also more instable, showing relatively decreased FoxP3 expression, and had greater plasticity, showing an increased Th1-like (IFNγ+FoxP3+) Treg phenotype. Overall, our preliminary results suggest that thymic atrophy creates changes in the antigen-specific repertoire during tTreg generation resulting in potential “holes” that may contribute to inflammaging in the elderly and negatively impact Treg cell-mediated regulation in the aged immune system. (Supported by NIH/NIAID 1R01AI121147)

Distinct CD28 signaling requirements determine the alternative fates of thymic agonist selection

Abstract During thymic T cell development, T cells strongly reactive to self-Ag are eliminated from the conventional T cell repertoire by clonal deletion. Alternatively, however, a number of T cell subsets are positively selected by strong recognition of self-Ag/MHC by TCR in thymus, termed agonist selection. Those T cell subsets include 1) Foxp3+ Treg cells, 2) invariant NKT (iNKT) cells, and 3) αβTCR+ PD-1+ CD4− CD8− precursors of CD8αα IELs (pIEL). In addition to a strong TCR signal, B7-CD28 co-stimulation has a role in these agonist selections. This role is context dependent. For instance, B7-CD28 has a positive role for thymic Treg and iNKT cell generation, while it has a negative role for thymic pIEL generation. These differences may indicate involvement of distinct CD28 signaling in these events. To test this hypothesis, we analyzed CD28 cytoplasmic tail mutant mice in which tyrosine 170 residue was substituted to phenylalanine (Y170F), proline residues in PYAP motif were substituted to alanine (AYAA), or both mutation were present (Y170F/AYAA). Generation of self-Ag-specific Treg, defined by pMHCII tetramer binding, was attenuated in AYAA mice but not in Y170F mice, consistent with the reported role of the PYAP motif for total Treg generation. Meanwhile, interestingly, the negative role of CD28 for self-Ag-specific pIEL generation was not affected by Y170F and/or AYAA substitution, indicating that this CD28 effect was mediated by motif(s) other than Y170 and PYAP. Analysis of thymic iNKT cell (NKT1, 2 and 17) development in CD28 mutant mice is currently in progress. These results suggest that the differing roles of B7-CD28 costimulation for agonist selection fates are mediated via qualitatively distinct CD28 signaling.