Treg Counts Research Articles

Reconstitution of Th17, Tc17 and Treg cells after paediatric haematopoietic stem cell transplantation: Impact of interleukin-7

Successful reconstitution of T lymphocytes after allogeneic haematopoietic stem cell transplantation (HSCT) is needed to establish the graft-versus-leukaemia effect and an effective anti-microbial defense, but the ratio between functionally different T-cell subsets needs to be balanced to avoid graft-versus-host disease (GVHD).IL-7 is essential for T-cell generation in the thymus and peripheral T-cell homeostasis. High IL-7 levels have been associated with impaired T-cell reconstitution, increased risk of acute GVHD and treatment-related mortality, but the underlying cellular mechanisms behind these associations have not been investigated previously. We hypothesized that increased levels of IL-7 post-transplant alters the balance between immune-regulatory T cell subsets during the post-transplant lymphocyte recovery towards a more pro-inflammatory profile.We quantified Th17 cells, Tc17 cells and Tregs in 29 children following HSCT. Th17 cell and Treg counts rose significantly from day +90 to +180 post-HSCT, and prior acute GVHD was associated with significant changes in the concentration of Tregs (9.4×106/L vs. 1.3×106/L, P=0.0052) and the Th17/Treg ratio (1.5 vs. 4.2, P=0.025). The plasma level of IL-7 at day +90 correlated inversely with Th17 cell counts (rs=−0.65, P=0.0002) and the proportion of Tc17 cells (rs=0.64, P=0.0005) at day +90, but not with Tregs. Furthermore, high IL-7 levels at day +7 were predictive of a less naïve T-cell phenotype at day +90.These findings add further evidence that IL-7 is a key regulatory factor that may tune the balance between functionally different T-cell subsets following HSCT.

Increased Expression of Programmed Cell Death-1 in Regulatory T Cells of Patients with Severe Sepsis and Septic Shock: An Observational Clinical Study.

While regulatory T cells (Tregs) constitutively express programmed cell death-1 (PD-1), the role of PD-1 expression in Tregs of patients with sepsis remains unclear. Thus, we determined PD-1 expression in Tregs from the peripheral blood of patients with severe sepsis and septic shock. Seventy-eight patients with severe sepsis and 40 with septic shock, as well as 21 healthy subjects, were enrolled in this study. The percentage of peripheral blood PD-1+ Tregs, as well as absolute Treg counts, were compared between these three groups. PD-1 expression in Tregs and absolute Treg counts were also compared between survivors and non-survivors in patients with sepsis. PD-1 expression in Tregs increased in patients with sepsis compared to healthy controls. Conversely, absolute Treg counts were significantly decreased in patients with sepsis compared to healthy controls; patients with septic shock had the lowest absolute Treg counts. Among patients with sepsis, survivors had lower levels of PD-1 expression in Tregs, as well as higher absolute Treg counts, than non-survivors. Additionally, the percentage of PD-1+ Tregs correlated positively with lactate levels as well as the Acute Physiology and Chronic Health Evaluation II and Sequential Organ Failure Assessment scores in patients with sepsis. PD-1 was upregulated in Tregs of patients with sepsis and may indicate a state of immune dysfunction. Overexpression of PD-1 in Tregs was associated with more severe sepsis as well as poor outcomes.

Effects of tripterygium wilfordii polyglycoside tablets on expression of CD4+ /CD8+ and CD4+ CD25+ Treg in elderly patients with relapsing refractory immune thrombocytopenic purpura

Objective To explore the mechanism of tripterygium wilfordii polyglycoside tablets for elderly patients with relapsing refractory immune thrombocytopenic purpura(ITP), and to seek the theoretical basis for Chinese medicine treatment of this disease. Methods The clinical data of 79 patients with relapsing refractory ITP were retrospectively analyzed.According to whether the combined use of tripterygium wilfordii polyglycoside, they were divided into the control group(35 cases) and the observation group(44 cases). The control group was treated with platelet and tranexamic acid, sulfasalazine, sulforaphane sodium, hemagglutinin and other symptomatic hemostasis treatment.The observation group in symptomatic hemostasis support on the basis of treatment with tripterygium wilfordii polyglycoside tablets.The CD4+ /CD8+ ratio and CD4+ CD25+ Treg expression were compared between the two groups. Results The CD4+ /CD8+ ratio, CD4+ CD25+ Treg and platelet count in the control group before treatmentwere (0.96±0.36), (1.21±0.67)%, (13.14±6.92)×109/L, respectively, which of the observation group were (0.92±0.37), (1.19±0.59)%, (11.51±6.21)×109/L, respectively, there were no statistically significant differences between the two groups(all P>0.05). The CD4+ /CD8+ ratios in peripheral blood of the observation group at 2 weeks, 3 weeks and 4 weeks after treatmentwere (1.04±0.56), (1.55±0.34), (1.59±0.41), respectively, there were statistically significant differences between the two groups(t=9.994, 9.797, all P 0.05). There were statistically significant differences in the CD4+ /CD8+ ratio and CD4+ CD25+ Treg expression between the observation group and control group at 4 weeks after treatment(t=8.589, P 0.05), but the symptoms of bleeding of the observation group was lighter at 3 weeks after treatment. Conclusion Tripterygium wilfordii polyglycoside improves the expression of CD4+ /CD8+ and CD4+ CD25+ Treg in peripheral blood of elderly patients with relapsed or refractory ITP.It is an ideal drug for the treatment of relapsed and refractory ITP in the elderly, it is worth further study. Key words: Tripterygium; Purpura, thrombocytopenic, idiopathic; T-Lymphocytes, regulatory; Aged

High density lipoproteins selectively promote the survival of human regulatory T cells

HDLs appear to affect regulatory T cell (Treg) homeostasis, as suggested by the increased Treg counts in HDL-treated mice and by the positive correlation between Treg frequency and HDL-cholesterol levels in statin-treated healthy adults. However, the underlying mechanisms remain unclear. Herein, we show that HDLs, not LDLs, significantly decreased the apoptosis of human Tregs in vitro, whereas they did not alter naïve or memory CD4+ T cell survival. Similarly, oleic acid bound to serum albumin increased Treg survival. Tregs bound and internalized high amounts of HDL compared with other subsets, which might arise from the higher expression of the scavenger receptor class B type I by Tregs; accordingly, blocking this receptor hindered HDL-mediated Treg survival. Mechanistically, we showed that HDL increased Treg ATP concentration and mitochondrial activity, enhancing basal respiration, maximal respiration, and spare respiratory capacity. Blockade of FA oxidation by etoxomir abolished the HDL-mediated enhanced survival and mitochondrial activity. Our findings thus suggest that Tregs can specifically internalize HDLs from their microenvironment and use them as an energy source. Furthermore, a novel implication of our data is that enhanced Treg survival may contribute to HDLs' anti-inflammatory properties.

Low utility of serum 25–hydroxyvitamin D3 and 1, 25-dihydroxyvitamin D3 in predicting peripheral Treg and Th17 cell counts in ESRD and renal transplant patients

BackgroundVitamin D has shown an immune-modulatory effect in different studies. Vitamin D stimulates Tregs and inhibits Th17 cells. The immune-modulatory role of vitamin D in chronic kidney disease (CKD) and renal transplant patients is unclear. We measured whether different serum levels of vitamin D were associated with an increased or decreased presence of lymphocyte subsets including Treg and Th17 cells in end-stage renal disease (ESRD) and renal transplant recipients. MethodsEighty-seven renal transplant recipients and 53 end-stage renal disease (ESRD) patients were enrolled in this study. The absolute counts of CD4+ and CD8+ T, CD16+ CD56+ NK, CD19+ B, CD4+ CD25+ CD127- Foxp3+ (Tregs), Helios+ Tregs, CD38+ Tregs, and CD4+ CD17+ (Th17) cells were analyzed in peripheral blood in both patient groups. In addition, serum 25 (OH) D3, 1, 25 (OH)2 D3, IL-6, IL-17, IL-23, and TGF-β1 were measured. The association between lymphocyte subset counts and 1, 25 (OH)2 D3 or 25 (OH) D3 was studied, as was the association between serum IL-6, IL-17, IL-23, or TGF-β1 and 1,25 (OH)2 D3 or 25 (OH) D3. ResultsSerum 25 (OH) D3 and 1,25 (OH)2 D3 levels were not independently associated with peripheral CD4+ T, CD19+ B, CD16+ CD56+ NK, Treg, or Th17 cell counts. In contrast to serum 25 (OH) D3, serum1, 25 (OH)2 D3 was positively associated with CD8+ T cells counts in renal transplant recipients. ConclusionOur findings indicate low utility of serum 25 (OH) D3 and 1, 25 (OH)2 D3 levels in predicting a change in lymphocyte subset counts in ESRD and renal transplant patients.

Analysis of long term CD4+CD25highCD127- T-reg cells kinetics in peripheral blood of lung transplant recipients

BackgroundThe role of CD4+CD25highCD127− T-reg cells in solid-organ Transplant (Tx) acceptance has been extensively studied. In previous studies on kidney and liver recipients, peripheral T-reg cell counts were associated to graft survival, while in lung Tx, there is limited evidence for similar findings. This study aims to analyze long term peripheral kinetics of T-reg-cells in a cohort of lung recipients and tests its association to several clinical variables.MethodsFrom jan 2009 to dec 2014, 137 lung Tx recipients were submitted to an immunological follow up (median: 105.9 months (6.7–310.5)). Immunological follow up consisted of a complete blood peripheral immuno-phenotype, inclusive of CD4+CD25highCD127− T and FOXP3+ cells. We tested the association between T-reg and relevant variables by linear OR regression models for repeated measures, adjusting for time from Tx. Also, by ordered logistic models for panel data, the association between Chronic Lung Allograft Dysfuncton (CLAD) onset/progression and T-reg counts in the previous 3 months was tested.ResultsAmong all variables analyzed at multivariate analysis: Bronchiolitis Obliterans Syndrome (OR −6.51, p < 0.001), Restrictive Allograft Syndrome (OR −5.19, p = 0.04) and Extracorporeal photopheresis (OR −5.65, p < 0.001) were significantly associated to T-reg cell. T-reg cell counts progressively decreased according to the severity of CLAD. Furthermore, patients with higher mean T-reg counts in a trimester had a significantly lower risk (OR 0.97, p = 0.012) of presenting CLAD or progressing in the graft dysfunction in the following trimester.ConclusionsOur present data confirm animal observations on the possible role of T-reg in the evolution of CLAD.

Role of regulatory T cells in acute myeloid leukemia patients undergoing relapse-preventive immunotherapy

Regulatory T cells (Tregs) have been proposed to dampen functions of anti-neoplastic immune cells and thus promote cancer progression. In a phase IV trial (Re:Mission Trial, NCT01347996, http://www.clinicaltrials.gov) 84 patients (age 18–79) with acute myeloid leukemia (AML) in first complete remission (CR) received ten consecutive 3-week cycles of immunotherapy with histamine dihydrochloride (HDC) and low-dose interleukin-2 (IL-2) to prevent relapse of leukemia in the post-consolidation phase. This study aimed at defining the features, function and dynamics of Foxp3+CD25highCD4+ Tregs during immunotherapy and to determine the potential impact of Tregs on relapse risk and survival. We observed a pronounced increase in Treg counts in peripheral blood during initial cycles of HDC/IL-2. The accumulating Tregs resembled thymic-derived natural Tregs (nTregs), showed augmented expression of CTLA-4 and suppressed the cell cycle proliferation of conventional T cells ex vivo. Relapse of AML was not prognosticated by Treg counts at onset of treatment or after the first cycle of immunotherapy. However, the magnitude of Treg induction was diminished in subsequent treatment cycles. Exploratory analyses implied that a reduced expansion of Tregs in later treatment cycles and a short Treg telomere length were significantly associated with a favorable clinical outcome. Our results suggest that immunotherapy with HDC/IL-2 in AML entails induction of immunosuppressive Tregs that may be targeted for improved anti-leukemic efficiency.

The variation of circulating regulatory T cells count of lung carcinoma and its prognostic value for evaluating treatment response

Objective To investigate the prognostic value of CD4+ CD25+ CD127low/- regulatory T cells (Treg) in peripheral blood samples, tumor markers for lung cancer (TM) and associated clinicopathologic features for evaluating pre-treatment predictors of non-small cell lung cancer (NSCLC). Methods The percentages of Treg were measured by flow cytometry, the level of serum TM was analyzed by enzyme linked immunosorbent assay (ELISA); and then Treg cells, serum TM level and associated clinicopathologic features were used to evaluate the predictive value of NSCLC treatment. Results The percentages of Treg cells in patients with NSCLC were significantly higher than those in healthy control [(7.02±2.31)% vs. (4.53±0.86)%, t=6.949, P=0.000]. In the areas of lung cancer, a significant positive correlation was found among the percentages of Treg cells and degree of differentiation (F=15.229, P=0.000), tumor size (t=-2.972, P=0.003), TNM stage (t=-4.856, P=0.001), vascular tumor thrombus (t=-0.653, P=0.000). The baseline circulating Treg count and the serum TM level before treatment in NSCLC were lower in the patient group with positive treatment response than in the group with negative response [Treg: (5.77±1.71)% vs. (7.02±2.02)%, t=-3.983, P=0.001; cytokeratin 19 fragment (CYFRA21-1): 2.88 (2.13-4.44) vs. 4.76 (2.82-8.95), t=4.032, P=0.000; carcinoembryonic antigen (CEA): 2.72 (1.54-4.01) vs. 3.53 (1.99-8.42), t=2.385, P=0.017; carbohydrate antigen (CA)125: 9.90 (6.50-14.70) vs. 19.20 (11.50-57.25), t=5.148, P=0.000]. Through logistic regression analysis, it revealed that pre-treatment Treg count and TNM stage were independent prognostic factors for clinical response to NSCLC treatment [odds ratio (OR)=3.993, P=0.001; OR=1.742, P=0.000]. Conclusion The percentage of Treg cells was closely related to the severity of NSCLC development, and pre-treatment Treg count and TNM stage were a feasible method to predict clinical response to NSCLC treatment. Key words: Lung carcinoma; Regulatory T cells; Tumor marker; Treatment response

Abstract 2624: An FKBP5-based immunophenotype for assessment of the immunosuppression status and possible prediction of immunotherapy response in melanoma patients

Abstract Background and aim: FKBP51 is an immunophilin encoded by FKBP5 gene on chromosome 6. It is a protein resident in lymphocytes and involved in immune response. Recently, our group demonstrated that the inhibitory checkpoint PD-L1/PD1 promoted the alternative splicing of FKBP5 gene, resulting in increased expression of its variant 4, in PBMC of melanoma patients. The aim of this study was to address whether such a molecular signature could help in identifying an immune profile associated with increased probability of immunotherapy response. Experimental Design: The splicing FKBP51 isoform or FKBP51s was measured in peripheral blood T lymphocytes subsets (CD3/CD4, CD3/CD8, CD25 and PD-L1) and CD14 monocytes from a cohort of 118 patients and 77 age- and sex-matched healthy controls, by flow cytometry. Blood samples were collected before patients underwent ipilimumab treatment. Furthermore, in 64 out of 118 patients, expression of FKBP51s was also assessed in regulatory T cells. Results: Physiologically, each PBMC subset analyzed contained a fraction of an FKBP51spos component, which resulted expanded in melanoma patients. We also measured an increase in CD3/CD8 and PD-L1 lymphocytes in patients. CD4 T lymphocytes showed the FKBP51sneg fraction significantly impaired, which might reflect the condition of impaired T cell help. Treg count was increased, in accordance with previous studies. The count of FKBP51sposTregs defined a subgroup of nonresponder patients to ipilimumab, by 92.6%. A 2D hierarchical partitioning of data from FKBP51s-immunophenotype (heatmap) revealed 3 main clusters: C1 (33 pts, 51,5%), C2 (14 pts, 22%) and C3 (17 pts, 26,5%). FKBP51spos Treg subset appeared globally increased in all clusters. In C1, values of effector T cells and monocytes were not different from normal donors. C2 showed a significant increase of FKBP51spos PD-L1 monocytes and a significant probability of not responding (Chi-square=5.46; p=0.019). This finding is consistent with the hypothesis that response to ipilimumab is prevented by accessory cells exerting a negative immune regulatory control. C3 showed a significant increase of FKBP51s in overall lymphocyte subsets analyzed. Patients in this cluster showed reduced overall survival. This finding suggests that a prevalence of activation (CD25) and co-inhibitory (PD-L1) markers together with the expansion of FKBP51spos effector T cells might reflect a condition of chronic lymphocyte stimulation in some advanced melanoma patients, contributing to T cell-exhaustion. Conclusions: FKBP51s-based immunophenotype of melanoma patients revealed several profiles virtually related to a negative immune regulatory control and highlights an impairment of a Treg subset endowed with increased suppressive potential. Such FKBP51sTreg subset is likely to be associated with immunotherapy response (Chi-square=9.916, p=0.002) . Citation Format: Simona Romano, Ester Simeone, Anna D'Angelillo, Paolo D'Arrigo, Mario Capasso, Vito Alessandro Lasorsa, Martina Tufano, Anna Rea, Michele Russo, Nicola Zambrano, Paolo Antonio Ascierto, Maria Fiammetta Romano. An FKBP5-based immunophenotype for assessment of the immunosuppression status and possible prediction of immunotherapy response in melanoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2624. doi:10.1158/1538-7445.AM2017-2624

Correlation between unexplained recurrent spontaneous abortion with CD4+CD25+ regulatory T-cell and killer cell immunoglobulin-like receptor levels.

The present study investigated the correlation between unexplained recurrent spontaneous abortion (URSA) with CD4+CD25+ regulatory T-cell (Treg) and killer cell immunoglobulin-like receptor (KIR)-2DL1 levels. A total of 76 URSA patients were enrolled (35 without pregnancy, Group A, and 41 with early abortion, Group B). Additionally, 30 patients who received a regular abortion as planned (Group C) and 30 healthy volunteers (Group D) were selected. Peripheral venous blood and fresh decidual tissue samples were obtained from all the patients, and flow cytometry was performed to detect CD4+CD25+Treg and Foxp3 transcription factor levels. mRNA and protein KIR-2DL1 expression levels were assayed using quantitative PCR and western blot analysis, respectively. No statistically significant differences in peripheral venous blood CD4+CD25+Treg/CD4+ and Foxp3+/CD4+CD25+Treg cell proportions were found among the groups (P>0.05). However, the decidual tissues of Group C presented significantly higher levels of both cell types versus other groups (P<0.05). No statistically significant differences were found in comparisons among Groups A, B, and D (P>0.05). In peripheral venous blood, mRNA and protein KIR-2DL1 expression levels in Group C were significantly higher than those in the other three groups (P<0.05), but again, there were no statistically significant differences among Groups A, B, and D (P>0.05). In decidual tissues, KIR-2DL1 levels were significantly higher in Group C relative to Groups A, B, and D (P<0.05). Decreased CD4+CD25+Treg counts and KIR-2DL1 expression levels were closely associated with the onset of URSA. CD4+CD25+Tregs mainly exert their effects on decidual tissues, while KIR-2DL1 can act on peripheral venous blood and decidual tissues. These may present new targets for early intervention in URSA.

Urinary levels of prostaglandin E2 are positively correlated with intratumoral infiltration of Foxp3+ regulatory T cells in non-small cell lung cancer.

The immune microenvironment of primary tumors has been reported to be one of the factors influencing the prognosis of patients with cancer. The tumor-infiltrating regulatory T cell (Treg) count has previously been revealed to be positively correlated with intratumoral cyclooxygenase-2 (Cox-2) expression, and was also associated with poor survival among patients with non-small cell lung cancer (NSCLC). In addition, the urinary levels of a prostaglandin E2 (PGE2) metabolite (PGE-M) were used as a biomarker in clinical trials of the Cox-2 inhibitor celecoxib. In the current prospective study, the association of urinary PGE2 and PGE-M levels with intratumoral Cox-2 expression and Treg count was examined in patients with NSCLC. A total of 21 patients with NSCLC who underwent complete resection of the tumor at Kawasaki Medical School Hospital (Kurashiki, Japan) were enrolled. Urine specimens were obtained prior to surgery in order to examine urinary PGE2 and PGE-M levels. A significant positive association was observed between urinary PGE2 levels and the intratumoral Treg count (P=0.023), but not the intratumoral Cox-2 expression levels. No significant associations were identified between urinary PGE2 levels and any of the other clinicopathological characteristics examined, including age, sex, smoking history, histology, tumor size, nodal status and disease stage. However, no significant association was observed between urinary PGE-M levels and the intratumoral Treg count (P=0.069) or Cox-2 expression. In conclusion, urinary PGE2 levels were positively correlated with intratumoral Treg counts in patients with NSCLC in the current study. This indicates that urinary PGE2 may be an improved biomarker, compared with PGE-M, for the prediction of intratumoral Treg numbers.

FKBP51s signature in peripheral blood mononuclear cells of melanoma patients as a possible predictive factor for immunotherapy.

The inhibitory immune checkpoint PD-L1/PD1 promotes the alternative splicing of the FKBP5 gene, resulting in increased expression of its variant 4 in the peripheral blood mononuclear cells of melanoma patients. The variant 4 transcript is translated into the truncated FKBP51s protein. Given the importance of co-inhibitory signalling in tumour immune escape, here we tested the potential for using FKBP51s expression to predict immunotherapy outcomes. To do this, we immunophenotyped PBMCs from 118 melanoma patients and 77 age- and sex-matched healthy controls. Blood samples were collected before patients underwent ipilimumab treatment. In 64 of the 118 patients, FKBP51s expression was also assessed in regulatory T cells (Tregs). We found that each PBMC subset analysed contained an FKBP51spos fraction, and that this fraction was greater in the melanoma patients than healthy controls. In CD4 T lymphocytes, the FKBP51sneg fraction was significantly impaired. Tregs count was increased in melanoma patients, which is in line with previous studies. Also, by analyses of FKBP51s in Tregs, we identified a subgroup of ipilimumab nonresponder patients (p=0.002). In conclusion, FKBP51s-based immunophenotyping of melanoma patients revealed several profiles related to a negative immune regulatory control and identified an unknown Treg subset. These findings are likely to be useful in the selection of the patients that are candidate for immunotherapy.

Significance of Thl7/Treg Ratio in aGVHD Occurence after Allo-HSCT

To explore the relationship between Th17 and Treg levels and aGVHD occurence in patients after allo-HSCT. Thirty-nine patients received allo-HSCT were divided into 2 groups: aGVHD group(17 cases) and non-aGVHD group (22 cases). For aGVHD group, the peripheral blood was collected before aGVHD occurence, in occurence and after aGVHD improvement; for non-aGVHD group, the peripheral blood was collected after 1, 2 and 3 months of transplantation. In addition, 16 healthy donors were used as controls, their peripheral blood was collected before mobilization. The Th17 and Treg counts as well as Th17/Treg ratio were detected by flow cytometry. Among patients with aGVHD after transplantation, the Th17 count increased, and the Treg count decreased, the Th17/Treg ratio increased before aGVHD occurence, as compared with the patients without aGVHD(P<0.05); but after aGVHD occurence, the Th17 count decreased, Treg count increated, and the Th17/Treg ratio decreased as compared with that before aGVHD occurence (P<0.05). After aGVHD was improved, the Th17/Treg ratio decreased as compared with level before aGVHD occurence (P<0.05). After aGVHD was improved, the Th17/Treg ratio was no statistical different from healthy donors (P>0.05). Among patients without aGVHD after transplantation, the Th17/Treg ratio at 2 and 3 months after transplantation was no statistical different from that of healthy donos(P>0.05). The Th17 and Treg levels recoverel showly after transplantation, but the Th17/Treg ratio recoveres after 2 months in the patients after transplantation. The Th17 cells may initiate aGVHD; when aGVHD happened, the Treg level increases, which may regulate the aGVHD ontcome through inhibiting the Th17 cells. The detection of Th17/Treg ratio after transplantation can predict the occurence and outcome of aGVHD.

Integrated stress response activation by sleep fragmentation during late gestation in mice leads to emergence of adverse metabolic phenotype in offspring

BackgroundLate gestational sleep fragmentation (SF) is highly prevalent particularly in obese women, and induces metabolic dysfunction in adult offspring mice. SF induces activation of the integrated stress response (ISR), which might be involved in metabolic disorders. We hypothesized that adult offspring of double mutant mice (DM) involving the critical ISR genes CHOP and GADD34 would be protected from developing obesity and insulin resistance following SF. MethodsTime-pregnant CHOP/GADD34 DM and wild type (WT) mice were randomly assigned to sleep control (SC) or SF conditions during the last 5days of gestation. At 24-weeks of age, body weight, fat mass, and HOMA-IR were assessed in the offspring. Tregs lymphocytes, Lyc6chigh, M1 and M2 macrophages were examined in visceral white adipose tissues (vWAT) using flow cytometry. The effects of plasma exosomes on adipocyte cell line proliferation, differentiation and insulin sensitivity were also evaluated. ResultsSF-WT male showed significant increases in body weight, vWAT mass and HOMA-IR compared to SC-WT mice, while SF had no effect in SF-DM mice. Inflammatory macrophages (Ly-6chigh) and the ratio of M1/M2 macrophages were increased while FoxP3+ Tregs counts were decreased in SF-WT but not in SF-DM mice. Exosomes from SF-WT, but not from the SF-DM offspring increased pre-adipocyte proliferation and differentiation, and decreased in vitro adipocyte insulin sensitivity. ConclusionActivation of the ISR during late gestation, as induced by late gestational SF, appears to underlie some of the transgenerational modifications in metabolic genes ultimately contributing to a metabolic syndrome phenotype in adult offspring.

Evaluation of T Regulatory Lymphocytes Transcription Factors in HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP) Patients.

HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is an aggressive neurological disease. The CD4+CD25+ T cell population plays pivotal roles in the maintenance of immunological tolerance and prevention of such autoimmune diseases. In the current study, proviral load (PVL), factor forkhead box p3 (Foxp3), and glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) gene expression and regulatory T cells (Tregs) counts of 21 HAM/TSP patients and 16 HTLV-1 healthy carriers (ACs) were measured using real-time PCR, TaqMan method, and flow cytometry. The demographic, history of disease, and severity of myelopathy were assessed by a checklist and the Osame motor disability score (OMDS). The mean OMDS for HAM/TSP was 4.82±2.37 which had no significant correlation with Treg count or the expression of Foxp3, GITR, and PVL. The CD4+CD25+ cell counts had no significant differences between HAM/TSP and ACs. Findings revealed a higher PVL in HAM/TSPs (313.36 copies/104) compared to ACs (144.93 copies/104, p=0.035). The Foxp3 and GITR mRNA levels were lower in HAM/TSP patients (11.78 and 13.80, respectively) than those in healthy carriers (18.44 and 21.00, p=0.041 and 0.03, respectively). There was a significant correlation between Treg frequency and Foxp3 gene expression (R=0.67, p=0.006) and GITR and Foxp3 (R=0.84, p=0.042) in HAM/TSP patients. Furthermore, the transcription factors have strong correlations with CD4+CD25+ T cell frequencies. These findings suggest that HTLV-1 infection can modify the expression of main functional transcription factors, FOXP3 and GITR, which may lead to immune response deterioration of Tregs and consequently HAM/TSP manifestation.

The establishment of cow's milk protein allergy in infants is related with a deficit of regulatory T cells (Treg) and vitamin D.

Cow's milk protein allergy (CMPA) is the most common food allergy in infants. However, little is known about which specific immune mechanisms are related with the CMPA onset. The objective was to investigate which immune alterations constitute differential factors between allergy and tolerance, and hence could be implicated in the CMPA establishment in infants. An extensive analysis of immune subsets, including Treg and cytokine-secreting cells was performed in blood samples from 28 infants younger than 9 mo obtained 1-4 d after the first adverse reaction to milk. Less than 4 d after first allergic reaction, infants who developed CMPA had decreased Treg counts and increased frequency of IL4-secreting CD4 T cells compared to controls. The deficit of Tregs was correlated with decreased serum levels of vitamin D. Values of Tregs, IL4-secreting cells and vitamin D were good predictors of CMPA diagnosis. Basal vitamin D levels in CMPA infants also predicted those CMPA patients developing spontaneous tolerance in the first year. Establishment of CMPA in infants was related with lower Treg and vitamin D levels. These immune alterations would be crucial factors behind the CMPA establishment and they could constitute a therapeutic target for treatment of CMPA.

Mesenchymal Stem Cells Induced In Vitro Generated Regulatory-T Cells: Potential Soldiers of Transplantation Biology

Introduction: CD4+ regulatory T-cells that constitutively express CD25 have a potent mechanism of action of immunomodulation and hence hold a great therapeutic potential for the treatment of autoimmune diseases and tolerance induction in the context of solid organ transplantation. Methods: For in vitro generation of Tregs volunteer donor’s adipose tissue was resected and mesenchymal stem cells (MSC) were generated. MSC were co-cultured with post-renal transplantation recipient’s peripheral blood derived peripheral blood mononuclear cells (PBMC) and with irradiated PBMC from same recipient with the addition of exogenous IL-2. Controls were carried out without use of MSC. In vitro generated Tregs were subjected for quantitative and qualitative analysis of Tregs specific markers. Results: Quantitative analysis revealed increased cell counts of Tregs in comparison to PBMC. Tregs specific markers evaluation using flocytometer showed rise in all Treg specific cell population: CD4+25+127Neg with mean 7.6 ± 6.1%, CD4+FoxP3+ mean of 21.7 ± 15.3% and CD4+CTLA-4+ mean of 16.9 ± 13.7%. Conclusion: We conclude that MSC induce in vitro generation of Tregs. In vitro generated Tregs hold a great promise as “magic bullet” for various disease prevention and/or treatment. Keywords: Regulatory-T cells, In vitro generation, mesenchymal stem cells, co-culture, IL-2.

Reversible immune abnormality and regulatory T cells in offspring of Der p 1-exposed female mice.

Maternal allergic diseases have an important influence on the origin of allergic rhinitis (AR) in offspring, but the mechanism and the duration of the maternal effect are unknown. Previous researches prompted the important roles of Tregs and Foxp3 DNA methylation in the development of allergic diseases. To investigate the immune state and Tregs in the offspring of Der p 1-exposed female mice. BALB/c female mice were exposed to Der p1 to construct the mouse AR model, then mated with normal male mice. Offspring were kept in an allergen-free environment after birth. At postnatal weeks 3, 5 and 8, mice were culled for testing. Compared with the offspring of PBS-exposed female mice (N-N), the offspring of Der p 1-exposed female mice (A-N) showed increased IL-4 and decreased IL-10 levels in serum at postnatal weeks 3 and 5. Correspondingly, the percentage of Tregs in spleen CD4+ cells declined significantly at postnatal week 5 in A-N. Further analysis of the methylation status of spleen lymphocytes revealed hypermethylation of the Foxp3 promoter in A-N mice at postnatal weeks 3 and 5. However, by 8 weeks of age, all abnormalities in cytokines, Treg counts and Foxp3 DNA methylation in A-N mice had returned to normal levels. Under the influence of maternal AR, offspring have an abnormal immune state at birth. However, without exposure to allergens, the immune state in AR offspring recovered by maturity. Changes in Tregs and Foxp3 DNA methylation may be the mechanism for this reversible immune abnormality in AR offspring.

Prognostic significance of regulatory T lymphocytes in patients with hepatocellular carcinoma.

We investigated the prognostic role of regulatory T cells (Tregs) in patients with hepatocellular carcinoma (HCC). Relevant evidence regarding prognostic significance of Tregs was systematically searched in MEDLINE and Embase databases. A meta-analysis was performed to compare survival in patients with high or low Tregs level (either in peripheral blood or tumor). Eighteen studies were identified that fulfilled for the eligibility criteria and were included for data synthesis. Our pooled hazard ratios (HRs) demonstrated that increased Tregs intratumoral accumulation was significantly associated with worse overall survival (HR=2.04, 95% confidence interval (CI): 1.72-2.42) and disease-free survival (HR=1.82, 95% CI: 1.58-2.09). Three studies evaluated the role of Tregs in peripheral blood, and all of them showed that increased peripheral Tregs correlated with shortened disease-free and overall survival. Collectively, our results showed that the increased Tregs count is tightly associated with the shortened survivals. Its measurement in either primary tumor or even circulation might be a candidate marker of prognostic significance in HCC patients.

IFNy + and IFNy − Treg subsets with stable and unstable Foxp3 expression in kidney transplant recipients with good long-term graft function

BackgroundTreg are a heterogenous cell population. In the present study we attempted to identify Treg subsets that might contribute to stable and good long-term graft function. MethodLymphocyte and Treg subsets were studied in 136 kidney transplant recipients with good long-term graft function and in 52 healthy control individuals using eight-color-fluorescence flow cytometry. Foxp3 TSDR methylation status was investigated in enriched IFNy+ and IFNy− Treg preparations using high resolution melt analysis. ResultsCompared with healthy controls, patients showed strong associations of IFNy secreting Helios+ and Helios− Treg with Treg that co-expressed perforin and/or CTLA4 (CD152; p<0.01). Moreover they showed associations of IFNy−Helios+ Treg with Treg that produced TGFβ and/or perforin and of IFNy−Helios− Treg with TGFβ production (all p<0.01). Only in patients, but not in healthy controls, were IFNy− Helios+ and Helios− Treg associated with higher CD45+, CD3+, (CD4+), CD19+ lymphocyte counts (p<0.001). In addition IFNy−Helios+ Treg were associated with CD16+56+ lymphocytes (p<0.001). Enriched IFNy− Treg from female but not male patients showed an association of Foxp3 methylation with higher total Treg and higher Helios+IFNy−, CXCR3+Lselectin+ (CD183+CD62L+), CXCR3−Lselectin+ and CD28+HLADR+ Treg subsets (p<0.01). Enriched IFNy+ Treg from male patients showed an association of demethylated Foxp3 with total Treg and IL10−TFGβ+ Treg counts, and in enriched IFNy− Treg an association of methylated Foxp3 with APO1/FasR+FasL+ (CD95+CD178+) Treg (p<0.01). ConclusionsKidney recipients with good long-term graft function possess IFNy+ and IFNy− Treg with stable and unstable Foxp3 expression in the blood. They co-express CD28, HLADR, CTLA4, CXCR3, Lselectin, TGFβ, perforin and FasL and might contribute to the establishment and maintenance of good long-term graft function.