Tregs In Peripheral Blood Research Articles

Quantification of Treg cells in peripheral blood and lymph nodes of dogs with multicentric lymphoma

ABSTRACT Lymphoma is a malignant tumor characterized by cell proliferation of lymphoid origin and corresponds to 90% of all hematopoietic neoplasms of dogs. Regulatory T cells (Tregs) have been the target of many investigations in oncology due to their potential of down-regulating immune responses, as well as ensuring the maintenance of active mechanisms of tumor suppression. The aims of the present study were to compare the percentage of Tregs in peripheral blood between dogs with multicentric lymphoma and healthy animals, together with the percentage of Tregs in peripheral blood and lymph nodes of dogs with multicentric lymphoma. Twenty-six animals were enrolled in the study: 10 healthy dogs comprised the control group (CG) and 16 dogs with multicentric lymphoma comprised the Lymphoma Group (LG). We observed that dogs in the LG showed a significantly higher Tregs expression in peripheral blood compared to the CG. No significant difference was observed between Tregs expression in lymph nodes and peripheral blood of the LG, however. With these results, it is possible to conclude that multicentric lymphoma is a neoplasm with high Tregs expression, which poses this as a condition of interest when investigating treatments that can suppress Regulatory T cells.

Enhanced TH17 Responses in Patients with IL10 Receptor Deficiency and Infantile-onset IBD.

IL10 receptor (IL10R) deficiency causes severe infantile-onset inflammatory bowel disease. Intact IL10R-dependent signals have been shown to be important for innate and adaptive immune cell functions in mice. We have previously reported a key role of IL10 in the generation and function of human anti-inflammatory macrophages. Independent of innate immune cell defects, the aim of the current study was to determine the role of IL10R signaling in regulating human CD4 T-cell function. Peripheral blood mononuclear cells and intestinal biopsies cells were collected from IL10/IL10R-deficient patients and controls. Frequencies of CD4 T-cell subsets, naive T-cell proliferation, regulatory T cell (Treg)-mediated suppression, and Treg and TH17 generation were determined by flow cytometry. Transcriptional profiling was performed by NanoString and quantitative real-time polymerase chain reaction. RNA in situ hybridization was used to determine the quantities of various transcripts in intestinal mucosa. Analysis of 16 IL10- and IL10R-deficient patients demonstrated similar frequencies of peripheral blood and intestinal Tregs, compared with control subjects. In addition, in vitro Treg suppression of CD4 T-cell proliferation and generation of Treg were not dependent on IL10R signaling. However, IL10R-deficient T naive cells exhibited higher proliferative capacity, a strong TH17 signature, and an increase in polarization toward TH17 cells, compared with controls. Moreover, the frequency of TH17 cells was increased in the colon and ileum of IL10R-deficient patients. Finally, we show that stimulation of IL10R-deficient Tregs in the presence of IL1β leads to enhanced production of IL17A. IL10R signaling regulates TH17 polarization and T-cell proliferation in humans but is not required for the generation and in vitro suppression of Tregs. Therapies targeting the TH17 axis might be beneficial for IL10- and IL10R-deficient patients as a bridge to allogeneic hematopoietic stem cell transplantation.

Relationship between HBeAg from HBsAg positive mothers and regulatory T cells in neonates and its influence on HBV intrauterine transmission

Objective: To explore the relationship between HBeAg in HBsAg positive mothers and CD(4)(+)CD(25)(+)Foxp3(+)regulatory T cells (Treg) in newborns, as well as how they would influence the increasing risk on HBV intrauterine transmission. Methods: We collected information on general demographic characteristics and delivery on 270 HBsAg positive mothers and their newborns from the Third People's Hospital of Taiyuan. Fluorescence quantitative polymerase chain reaction (FQ-PCR) and chemiluminescence immunoassay (CLIA) were used to detect HBV DNA and HBV serological markers in peripheral blood from both mothers and neonates. The expression of Treg and other immune cells in peripheral blood of neonates were detected with flow cytometry (FCM). Results: Maternal HBeAg positive rates were associated with an increased risk of intrauterine transmission (OR=4.08, 95%CI: 1.89-8.82). Rates of Treg in newborns born to HBsAg-positive mothers were higher than that of the negative group (Z=2.29, P=0.022). Each pair of the subjects was assigned to five different groups according to the HBeAg titers of mothers. Frequencies of both Treg and HBeAg in newborns and HBV DNA in mothers between the above said 5 groups showed similar trends of changing patterns and the differences between groups were statistically significant(χ(2)=18.73, P<0.001; χ(2)=181.60, P<0.001; χ(2)=183.09, P<0.001). Results from partial correlation analysis showed that after adjusting for neonatal HBeAg and maternal HBV DNA, mother's HBeAg titers were positively related to the percentage of Treg in their newborns (r(s)=0.19, P=0.039). In addition, the frequencies of Treg were negatively correlated with pDC and CD(4)(+) T cell in their newborns (r(s)=-0.21, P=0.017; r(s)=-0.23, P=0.009). Conclusion: HBeAg from HBsAg positive mothers might have inhibited the function of neonatal DC cells and T cells to reduce the immune response to HBV by up-regulating the proportion of Treg and finally increased the risk of HBV intrauterine transmission.

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

414 Influence of filaggrin genotype on regulatory T cells in atopic dermatitis

Atopic dermatitis (AD) is considered to result from skin barrier impairment coupled with environmental factors and abnormal immune reactivity. Although previous studies describe an important role of regulatory T cells (Tregs) in the pathogenesis of AD, the interaction between Tregs and epidermal barrier dysfunction in AD patients remains unclear. We here report a heterogeneous AD cohort, which has been characterized for filaggrin (FLG) mutation status by next generation sequencing and for phenotypical grading by assessment of EASI score, grade of erythema, transepidermal water loss (TEWL) and total serum IgE. AD patients revealed an increased EASI score, grade of erythema and TEWL, regardless of filaggrin genotype, when compared to healthy controls. Total serum IgE was increased in AD patients without filaggrin mutations when compared to healthy controls and AD patients carrying filaggrin null mutations. For characterization of circulating Tregs we identified demethylation of CpG dinucleotides in a conserved region of FoxP3 intron 1, representing stable Tregs with suppressive function, and percentages of CD4+ CD25+ CD127-/low Tregs in peripheral blood of AD patients and healthy controls. Although our cohort presented no significant differences in percentages of Tregs with demethylated FoxP3i1, we observed an increased proportion of CD4+ CD25+ CD127-/low Tregs within the CD4+ cell population only in FLG wt/wt AD patients. Within the Treg population, proportions of effector CCR4+CD45RAlow Tregs were enhanced, exhibiting increased proportions of Th2 Tregs, whereas proportions of Th1 Tregs were decreased in FLG wt/wt AD patients. Thus, in AD patients neither disease severity nor filaggrin genotype influences levels of suppressive Tregs, while Tregs with plasticity are increased in AD patients without filaggrin mutations.

Correlation analysis of the Epstein-Barr virus recruited regulatory T cells in nasopharyngeal carcinoma of immune escape

Objective: To investigate the relation of Epstein-Barr virus (EBV)-induced immune escape to regulatory T cells(Treg) in nasopharyngeal carcinoma(NPC). Methods: We recruited 83 patients who were pathologically diagnozed as nasopharyngeal carcinoma and treated in Nanfang Hospital from January to September 2016. CD4(+) CD25(+) FOXP3(+) Treg in peripheral blood was examined by flow cytometry and the level of EBV DNA in the blood was detected by quantitative real-time polymerase chain reaction(qRT-PCR)in 83 patients and 51 healthy adults as control. The expression of FOXP3(+) Treg and EBER in NPC in 32 of 83 patients and in nasopharyngitis in 16 control patients were tested by immunohistochemistry(IHC) and in-situ hybridization(ISH). Results: The ratio of Treg in blood in patients with NPC(5.29±1.45)% was higher than that in control group (4.78±1.19) %, with a statistically significant difference(P=0.035). The ratio of Treg in blood in patients with positive EBV was higher than that in patients with negative EBV(P=0.013), and there was a positive correlation between EBV DNA copies and Treg ratio(r=0.335, P=0.002). The density of FOXP3(+) Treg in NPC was higher than that in nasopharyngitis(P=0.001). Positive correlation was found between the density of FOXP3(+) Treg and the stain intensity of EBER(r=0.496, P=0.004). Conclusion: The ratios of Treg in blood and tissue in NPC patients with positive EBV are significantly increased, indicating a role of Treg in EBV-induced immune escape in NPC.

Changes in myeloid-derived suppressor cells (MDSC), Treg and Th17 cells in peripheral blood of patients with cervical human papillomavirus (HPV) infection

Objective To investigate the changes in myeloid-derived suppressor cells (MDSC), regulatory T cells (Treg) and T helper 17 (Th17) cells in peripheral blood of patients with HPV infection and cervical diseases including ectopic cervical columnar epithelium (CE), cervical intraepithelial neoplasia (CIN) and cervical squamous cell carcinoma (CC), and to analyze the roles of MDSC, Treg and Th17 cells in the development of cervical diseases. Methods This study enrolled 120 HPV-positive patients with cervical diseases (18 cases of CE, 50 cases of CIN, 52 cases of CC), 20 HPV-negative patients with cervical diseases (10 cases of CE and 10 cases of CIN) and 20 healthy subjects from March 2011 to December 2016. Changes in MDSC, Treg and Th17 cells in peripheral blood of all patients were detected by flow cytometry. Results Percentages of MDSC, Treg and Th17 cells in peripheral blood of patients who were positive for HPV were significantly higher than those in HPV-negative patients (P<0.05). Besides, percentages of these cells gradually increased with the exacerbation of cervical disease (CE-CINⅠ-CINⅡ-CINⅢ-CC) (P<0.05). Patients who were infected with high-risk HPV had significantly higher percentages of MDSC, Treg and Th17 cells in peripheral blood than those infected with low-risk HPV (P<0.05). Changes of MDSC, Treg and Th17 cells in peripheral blood of HPV-positive patients with cervical cancer were related to International Federation of Gynecology and Obstetrics (FIGO) stage, tumor differentiation, lymph node metastasis and vascular invasion (P<0.05). MDSC, Treg and Th17 cells in peripheral blood of all patients were significantly reduced after treatment (P<0.05). Patients whose status changed from HPV-positive to HPV-negative following treatment also showed significantly decreased MDSC, Treg and Th17 cells in peripheral blood as compared with those who remained HPV-positive (P<0.05). MDSC, Treg and Th17 cells in peripheral blood of HPV-positive patients with cervical diseases were positively correlated (r=0.599, 0.677, 0.648; P<0.05). Conclusion MDSC, Treg and Th17 cells in peripheral blood of patients with HPV infection are associated with the pathological process, severity and clinical pathological features of cervical diseases as well as therapeutic effects. Therefore, they can be used as biomarkers to detect the occurrence and development of HPV-positive cervical diseases. Key words: Human papillomavirus; Cervical cancer; Myeloid-derived suppressor cell; Regulatory T cell; T helper 17 cell

Effect of concurrent chemoradiotherapy and radiotherapy alone on peripheral myeloid-derived suppressor and T regulatory cells in patients with nasopharyngeal cancer

Objective: To investigate the percentage of myeloid-derived suppressor cells (MDSC) and T regulatory cells (Treg) in peripheral blood of nasopharyngeal cancer (NPC) patients undergoing concurrent chemoradiotherapy or radiotherapy alone. Methods: Sixty NPC patients who received radiotherapy or concurrent chemoradiotherapy from September 2012 to November 2015 and 20 healthy individuals were included in this study. For the patients, the blood samples were collected at four time points: pre-radiation (Pre-RT), reaching a dose of 40 Gy (RT-40 Gy), finishing radiation (RT-finish) and three months after finishing radiation (3m-post-RT). Flow cytometry was used to evaluate the percentage of Treg (CD4(+) CD25(+) CD127(low/-)) and MDSC (HLA-DR(-)CD11b(+) CD33(+) ) cells in peripheral blood. Results: Treg and MDSC cells were present in peripheral blood lymphocytes of healthy individuals as a percentage of (7.50±1.62)% and (1.08±0.48)%, respectively. The proportions of peripheral Treg cells in patients at Pre-RT, RT-40 Gy, RT-finish and 3m-post-RT time points were (8.42± 1.52)%, (9.10±1.57)%, (8.87±1.56)% and (7.31±1.43)%, respectively, showing a statistically significant difference between Pre-RT and the other groups (P<0.05). At Pre-RT point, the percentage of Treg cells in Stage Ⅲ-Ⅳ patients [(8.63±1.39)%] was higher than that in Stage Ⅰ-Ⅱ [(7.65±1.94)%, P=0.042]. Moreover, the proportions of peripheral MDSC cells in patients at Pre-RT, RT-40 Gy, RT-finish and 3m-post-RT time points were (2.14±1.21)%, (4.08±1.90)%, (3.76±1.31)% and (1.52±0.88)%, respectively. The percentages of MDSC cells at RT-40 Gy and RT-finish points were significantly higher than those at Pre-RT, while the percentage of MDSC cells at 3m-post-RT was significantly lower than those at Pre-RT (P<0.05). At Pre-RT point, the percentage of MDSC cells in Stage Ⅲ-Ⅳ patients [(2.25±1.26)%] was higher than that in Stage Ⅰ-Ⅱ [(1.35±0.66)%, P=0.007]. At RT-finish point, the proportions of MDSC and Treg cells in patients with Ⅲ-Ⅳ grade of radiation induced oral mucositis [(4.41±1.27)% and (9.91±1.23)%] were significantly higher than those in Ⅰ-Ⅱ grade patients [(3.15±1.04)% and (8.41±1.52)%, both of P<0.05]. Conclusions: The proportions of MDSC and Treg cells in initial treated NPC patients are higher than healthy individuals, and they are also associated with the tumor stages. During the concurrent chemoradiotherapy and radiation, the percentage of MDSC and Treg cells is elevated, suggesting a decreased immune activity. The increase of MDSC and Treg cells is related to radiation induced oral mucositis.

Enrichment of Human CCR6+ Regulatory T Cells with Superior Suppressive Activity in Oral Cancer.

Human oral squamous cell carcinoma (OSCC) constitutes an inflammatory microenvironment enriched with chemokines such as CCL20, which promote cancer cell invasion and tumor progression. We found that in OSCC there is a correlation between the expression of CCL20 and FOXP3 mRNA. Therefore, we hypothesized that OSCC may favor the recruitment and retention of regulatory T (Treg) cells that express the CCL20 receptor, CCR6. Interestingly, most (∼60%) peripheral blood Treg cells express CCR6, and CCR6+ Treg cells exhibit an activated effector/memory phenotype. In contrast, a significant portion (>30%) of CCR6- Treg cells were found to be CD45RA+ naive Treg cells. Compared to CCR6- naive or memory Treg cells, CCR6+ Treg cells exhibit stronger suppressive activity and display higher FOXP3 expression along with lower methylation at the Treg-specific demethylated region of the FOXP3 gene. This predominance of CCR6+ Treg cells was also found in the draining lymph nodes and tumor-infiltrating lymphocytes of OSCC patients with early or late clinical staging. Moreover, CCR6+ Treg cells isolated from tumor-infiltrating lymphocytes or draining lymph nodes maintained similar phenotypic and suppressive characteristics ex vivo as did their counterparts isolated from peripheral blood. These results suggest that CCR6 marks activated effector or memory Treg phenotypes with superior suppressive activity in humans.

Abstract 5613: PD-1 and TIGIT are major immune checkpoint receptors expressed in breast cancer-infiltrating T cells

Abstract Immune checkpoint blockers, which target co-inhibitory receptors of T cells, have provided promising responses against various tumors. However, the expression of immune checkpoint receptors (ICRs) in breast cancer remains poorly understood. We aimed to investigate the expression pattern of multiple ICRs in tumor-infiltrating and peripheral blood (PB) lymphocytes in human breast cancer. We isolated lymphocytes from fresh breast tumor and paired PB from 21 patients who underwent surgery between July 2016 and November 2016. Multi-color flow cytometry was performed primarily focusing on expression of multiple ICRs in CD8+ T cells and regulatory T cells (Tregs). In CD8+ T cell subsets, PD-1+ or TIGIT+ cells were more frequently observed in tumor-infiltrating CD8+ T cells than in PB CD8+ T cells (p&amp;lt;0.0001 and p=0.02, respectively). Around half of the tumor infiltrating CD8+ T cells expressed PD-1 (median 58.20%) and TIGIT (45.95 %). In contrast, LAG3 and TIM3 were hardly detected in both tumor-infiltrating and PB CD8+ T cells. Intriguingly, about 40% of tumor-infiltrating CD8+ T cells were PD-1+TIGIT+, and &amp;gt;70% of PD-1+ tumor-infiltrating CD8+ T cells co-expressed TIGIT, indicating that functional exhaustion of breast tumor-infiltrating CD8+ T cells might be mediated not by PD-1 alone but by multiple receptors including TIGIT. However, the expression of PD-1 and TIGIT showed different patterns in detail. PD-1 was frequently expressed by CCR7-CD45RA- effector memory T cells (TEM) (p=0.001) whereas TIGIT was frequently expressed by CCR7-CD45RA+ effector memory RA T cells (TEMRA) (p=0.03), suggesting that TIGIT is expressed during terminal differentiation of CD8+ T cells. Next, we examined breast tumor-infiltrating Tregs. The frequency of CD25+FoxP3+ Tregs among CD4+ T cells were higher in tumor than in PB (16.60% vs. 7.86%; p=0.002). In particular, CD45RA-FoxP3hi activated suppressive Tregs account for 77.6% of tumor-infiltrating Tregs (vs. PB 32.2%; p=0.005). Tumor-infiltrating Tregs showed higher expression of CD39 (p=0.005), a marker of the suppressive activity of Tregs. We also examined the expression of ICRs on Tregs as upregulation of these receptors is associated with enhanced suppressive activity of Tregs. CD4+CD25+FoxP3+ Tregs in tumors showed higher expression levels of PD-1, TIGIT and CTLA-4 compared to PB Tregs (p&amp;lt;0.05). There were no noteworthy correlations between ICR expression and clinical features, such as age, stage and subtype. We show that PD-1 and TIGIT are major ICRs expressed in breast tumor-infiltrating CD8+ T cells. Moreover, we found that CD4+CD25+FoxP3+ Tregs are abundant in breast tumors and overexpress PD-1, TIGIT and CTLA-4. Our data provide an understanding of comprehensive phenotypes of immune checkpoint expression on T cells in breast cancer. Functional changes of CD8+ T cells and Tregs by blocking of single or multiple ICRs are being investigated. Citation Format: Jee Ye Kim, Minsuk Kwon, Sung Mook Lim, Joo Heung Kim, Hyung Seok Park, Seho Park, Seung Il Kim, Young Up Cho, Soonmyung Paik, Eui-Cheol Shin. PD-1 and TIGIT are major immune checkpoint receptors expressed in breast cancer-infiltrating T cells [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 5613. doi:10.1158/1538-7445.AM2017-5613

Differential profile of activated regulatory T cell subsets and microRNAs in tolerant liver transplant recipients.

Regulatory T cells (Tregs) play a potential role in operational tolerance in liver transplantation (LT) patients, and microRNAs (miRNAs) are known to be involved in immunological responses and tolerance. Thus, we analyzed the implication of different peripheral blood Treg subsets and miRNAs on LT tolerance in 24 tolerant (Tol) and 23 non-tolerant (non-Tol) LT recipients by cellular, genetic, and epigenetic approximation. Non-Tol patients had a lower demethylation rate of the forkhead box P3 (FOXP3) regulatory T cell-specific demethylated region (TSDR) than Tol patients that correlated with the frequency of circulating Tregs. Tol patients presented a different signature of Treg subset markers compared with non-Tol patients with increased expression of HELIOS and FOXP3 and a higher proportion of latency-associated peptide (LAP)+ Tregs and CD45RA- human leukocyte antigen D related (HLA-DR)+ activated effector-memory Tregs. The expression of miR95, miR24, miR31, miR146a, and miR155 was higher in Tol than in non-Tol patients and was positively correlated with activated Treg markers. In conclusion, these data suggest that activated effector-memory Tregs and a TSDR-demethylation state of Tregs may play a role in the complex system of regulation of LT tolerance. In addition, we describe a set of miRNAs differentially expressed in human LT Tol patients providing suggestive evidence that miRNAs are implied in the preservation of self-tolerance as mediated by Tregs. Liver Transplantation 23 933-945 2017 AASLD.

Crosstalk Between Enzyme Matrix Metalloproteinases 2 and 9 and Regulatory T Cell Immunity in the Global Burden of Atherosclerosis.

Changes in immune and inflammatory responses may play a crucial role in the development and progression of atherosclerosis, as an autoimmune, chronic and progressive inflammatory disease. Immunological activity and vascular inflammation during atherosclerosis can be modulated by autoimmune responses against self-antigens, according to changeable risk factors (cholesterol, oxidized low-density lipoprotein (ox-LDL) in the vascular wall, fatty acids, etc.), and accompanied by accumulation of leucocytes and proinflammatory cytokines, which stimulate the transcription of matrix metalloproteinases (MMPs), whose concentration are increased in foam cell-rich regions. Regulatory T cells (Tregs) represent a unique subpopulation of T cells specialized in the regulation of immune response and in the suppression of proatherogenic T cells. The aim of our study was to examine the interactions between the concentration of enzyme matrix metalloproteinases 2 and 9 (MMP-2 and 9) in urine and the percentage of Tregs in peripheral blood of two groups of patients: with carotid artery stenosis (CAS), undergoing surgery and with mild atherosclerosis (A) from general practice. The method of enzyme immunoassay (ELISA) was used to determine enzyme MMP expression, and Tregs was examined by flow cytometric analysis. Our data have showed a large increase in the enzyme MMP-2 and 9 in the urine of CAS and A patients in comparison with healthy controls and indicated this method as an easy marker for the monitoring of the development of atherosclerosis. Simultaneously, the diminished number of Tregs in the same patients pointed the importance of these regulatory mechanisms in the etiopathogenesis of atherosclerosis and possible Tregs-mediated therapy.

Prospective evaluation of toceranib phosphate in metastatic canine osteosarcoma.

Efficacious therapies for measurable metastatic canine osteosarcoma (OSA) are generally lacking. Preliminary retrospective studies suggested that approximately 50% of dogs with measurable metastatic OSA experienced clinical benefit (objective response or clinically meaningful disease stabilisation) following toceranib (TOC) treatment. The purpose of this clinical trial was to prospectively evaluate the clinical outcome following TOC treatment in dogs with measurable pulmonary metastatic OSA. A secondary goal was to identify potential biomarkers of clinical benefit by measuring changes in plasma vascular endothelial growth factor (VEGF) and circulating regulatory T-cell (Treg) percentage. Twenty-two dogs with pulmonary metastasis from appendicular OSA having undergone previous amputation were treated prospectively with TOC. Adverse events (AEs) were common but predominantly low grade. Nine patients were withdrawn from the study prior to the week 8 assessment of response either due to progressive disease (PD), decreased quality of life or owner perceived unacceptable AEs. Of the patients evaluable for disease progression at week 8 (or earlier), 3/17 (17.6 %) had stable disease with the remainder having PD. The median progression-free survival time for all patients was 57 days (range 7-176 days) with a median overall survival time of 89 days (range 7-574 days). Plasma VEGF concentrations were significantly elevated in patients after 4 weeks of TOC treatment, but no changes were observed in percentage of Treg in peripheral blood. Overall, the results of this clinical trial do not support the use of TOC as single agent therapy for canine metastatic OSA.

T Helper Cells in the Immunopathogenesis of Systemic Sclerosis – Current Trends

AbstractSystemic sclerosis (SSc) is a chronic progressive autoimmune disease characterized by skin and multiorgan involvement with alterations in both the innate and adaptive immunities. The hallmark of the disease is widespread fibrosis engaging the skin and multiple internal organs, as well as the musculoskeletal system. There is mounting evidence that T cells are key players in the pathogenesis of scleroderma. The current review discusses the role of the different T helper (Th) lymphocyte subsets in the processes of inflammation and fibrosis, characteristics for the pathogenesis of the disease. Cytokines produced by Th cell populations have a major effect on endothelial cells and fibroblasts in the context of favoring/inhibiting the vasculopathy and the fibrosis spread. The Th2 pro-fibrotic cytokines IL-4 and IL-13 have been shown to induce collagen synthesis by fibroblasts, whereas IFN-γ demonstrates an inhibitory effect. Increased Th17 cells are present in the scleroderma skin infiltrates. The combination of IL-17, IFN-γ and TGF-β levels in CD45RO and CD45RA cells from patients with SSc is useful to distinguish between the limited and the diffuse phenotype of the disease. There are accumulating data for functional and numerical alterations in the Tregs in SSc. High levels of TNF-α which might reduce the suppressive ability of Tregs have been described. According to some studies, the number of Tregs in scleroderma skin biopsies has been decreased against the normal absolute number of Tregs in peripheral blood of the same patients, which suggests suppressed immunomodulatory response. Other studies reported increased frequency of Tregs in peripheral blood of patients with systemic sclerosis and established a correlation with disease activity. The main immunological challenge remains the identification of the trigger of the autoimmune response in SSc, the causes for preferential Th2-type cell responses and the immunological differences between the diffuse and the limited cutaneous form of the disease.

Role of PIM2 in allergic asthma.

T cell-associated inflammation, particularly type 2 inflammation, has an important role in asthma pathogenesis, which is suppressed by regulatory T cells (Tregs). Proviral integration site for Moloney murine leukemia virus 2 (PIM2), a member off the serine/threonine kinase family, promotes the growth and survival of T cells and influences the function of Treg cells. However, whether PIM2 affects asthma pathogenesis remains unclear. Peripheral blood mononuclear cells and Treg cells from asthmatic and healthy subjects were obtained, and the expression level of PIM2 was measured by reverse transcription-quantitative polymerase chain reaction and immunocytochemistry. In addition, BALB/c female mice sensitized and challenged by ovalbumin were used as an asthma model, and PIM2 inhibitor was injected during the challenge period to observe the effect of PIM2 on asthma. The asthma symptoms were recorded, and airway hyper-responsiveness (AHR), expression levels of cytokines in the serum or bronchoalveolar lavage fluid (BALF), and the number of BALF leukocytes were evaluated. In addition, hematoxylin and eosin staining and immunohistochemistry of lung tissues was performed. The results demonstrated that PIM2 was overexpressed in patients with asthma in natural Treg cells. Inhibition of PIM2 attenuated asthma symptoms, and improved AHR and airway inflammation compared with asthmatic mice without inhibition of PIM2. In addition, expression levels of interleukin (IL)-10 and forkhead box protein 3 (FOXP3) in BALF were increased following PIM2 inhibition (IL-10, 470.3±21.78 vs. 533.7±25.55 pg/ml, P<0.05; FOXP3, 259±4.68 vs. 279.3±3.68 pg/ml; asthma and PIM2 inhibition groups, respectively; P<0.05). In conclusion, PIM2 may exhibit an important role in asthma pathogenesis and exacerbate AHR, airway inflammation and asthma symptoms. These effects of PIM2 may be dependent on Treg cells and the secretion of IL-10 by Tregs. The results of the present study suggest that PIM2 may be a potential target molecule for asthma treatment.

Clinicopathologic and prognostic significance of regulatory T cells in patients with hepatocellular carcinoma: a meta-analysis.

The clinicopathologic and prognostic significance of regulatory T cells (Tregs) in patients with hepatocellular carcinoma (HCC) remains controversial. We performed a meta-analysis to resolve this issue. PubMed, Embase, Cochrane library, and the Web of Science were searched to identify eligible studies performed up to November 2016. A total of 3,854 HCC patients from 27 cohort studies were included. The meta-analysis revealed that high levels of Tregs were associated with poor overall survival (OS; HR = 1.95, P < 0.00001) and disease-free survival (DFS; HR = 1.82, P < 0.00001). However, the prognostic effect varied greatly according to the site of the Tregs. Higher intratumoral and peripheral blood levels of Tregs were associated with shorter OS and DFS, whereas a high peritumoral Tregs level was not associated with decreased OS and DFS. Trial design, therapy and method of detection had no effect on prognosis of Tregs. Moreover, the patients with high Tregs infiltration had multiple tumors, high AFP level, poor differentiation, later TNM stage, and vascular invasion. The present study demonstrates that high levels of intratumoral and peripheral blood Tregs predict multiple tumors, high AFP level, poor differentiation, later TNM stage, and vascular invasion and might be a promising prognostic factor in patients with HCC.

Sequential monitoring of lymphocyte subsets and of T-and-B cell neogenesis indexes to identify time-varying immunologic profiles in relation to graft-versus-host disease and relapse after allogeneic stem cell transplantation.

T and B lymphocyte subsets have been not univocally associated to Graft-versus-host disease (GVHD) and relapse of hematological malignancies after stem cell transplantation (SCT). Their sequential assessment together with B and T cell neogenesis indexes has been not thoroughly analysed in relation to these changing and interrelated immunologic/clinic events yet.Lymphocyte subsets in peripheral blood (PB) and B and T cell neogenesis indexes were analysed together at different time points in a prospective study of 50 patients. Principal component analysis (PCA) was used as first step of multivariate analysis to address issues related to a high number of variables versus a relatively low number of patients. Multivariate analysis was completed by Fine-Gray proportional hazard regression model. PCA identified 3 clusters of variables (PC1-3), which correlated with acute GVHD: PC1 (pre-SCT: KRECs≥6608/ml, unswitched memory B <2.4%, CD4+TCM cells <45%; HR 0.5, p = 0.001); PC2 (at aGVHD onset: CD4+>44%, CD8+TCM cells>4%; HR 1.9, p = 0.01), and PC3 (at aGVHD onset: CD4+TEMRA<1, total Treg<4, TregEM <2 cells/μl; HR 0.5, p = 0.002). Chronic GVHD was associated with one PC (TregEM <2 cells/μl at day+28, CD8+TEMRA<43% at day+90, immature B cells<6 cells/μl and KRECs<11710/ml at day+180; HR 0.4, P = 0.001). Two PC correlated with relapse: PC1 (pre-SCT: CD4+ <269, CD4+TCM <120, total Treg <18, TregCM <8 cells/μl; HR 4.0, p = 0.02); PC2 (pre-SCT mature CD19+ >69%, switched memory CD19+ = 0 cells and KRECs<6614/ml at +90; HR 0.1, p = 0.008). All these immunologic parameters were independent indicators of chronic GVHD and relapse, also considering the possible effect of previous steroid-therapy for acute GVHD. Specific time-varying immunologic profiles were associated to GVHD and relapse. Pre-SCT host immune-microenvironment and changes of B cell homeostasis could influence GVH- and Graft-versus-Tumor reactions. The paradoxical increase of EM Treg in PB of patients with GVHD could be explained by their compartmentalization outside lymphoid tissues, which are of critical relevance for regulation of GVH reactions.

CTLA-4 haploinsufficiency in a patient with an autoimmune lymphoproliferative disorder

CTLA-4 haploinsufficiency in a patient with an autoimmune lymphoproliferative disorder

Relationship between peripheral Treg/Th17 cells and pregnancy outcomes in the first trimester of patients with unexplained recurrent spontaneous abortion

Objective To investigate Treg and Th17 cell numbers in the peripheral blood of patients with unexplained recurrent spontaneous abortion (URSA), with an aim to elevate their potential for predicting pregnancy outcomes. Methods Peripheral blood was collected from 60 URSA pregnant women and 56 healthy pregnant women during their first trimester in the Department of Reproductive Endocrinology of the Second Affiliated Hospital of Soochow University from January 2014 to June 2015. The counts of regulatory T cell (Treg), T helper cell 17 (Th17), CD4 positive T cell (CD4+ T cell) and total lymphocytes in the peripheral blood were determined by flow cytometry. Calculated the ratios of Treg/CD4+ T cells, Treg/total lymphocytes, Th17/CD4+ T cells, Th17/total lymphocytes and Treg/Th17, then the pregnancy outcomes were followed up. According to the different pregnancy outcomes, the patients were divided into three groups: URSA successful delivery group (23 cases), URSA re-miscarriage group (37 cases) and normal successful delivery group (44 cases). Non-parametric test were used to compare the differences between groups. Results There were no difference between the URSA re-miscarriage group and URSA successful delivery group in the CD4+ T cell counts[(1 570.16±519.56) vs (1 571.83±450.37), t=0.01, P=0.99]and total lymphocytes[(4 497.11±975.48) vs (4 558.52±1 032.54), t=0.23, P=0.82]. However, the ratios of Treg/CD4+ T cells[(1.09±0.62) vs (3.32±2.30), t=4.55, P=0.000 1], Treg/total lymphocytes[(0.42±0.24) vs (1.31±1.14), t=3.67, P=0.001 3]and Treg/Th17[(0.56±0.41) vs (2.51±2.38), t=3.89, P=0.000 8]in the URSA miscarriage again group were significantly lower whereas the ratios of Th17/CD4+ T cells[(1.85±0.92) vs (0.93±0.36), t=5.40, P<0.001]and Th17/total lymphocytes[(0.64±0.31) vs (0.31±0.14), t=5.52, P<0.001]were significantly higher. Similarly, There were no difference between the URSA re-miscarriage group and normal successful delivery group in the CD4+ T cell counts[(1 570.16±519.56) vs (1 566.86±651.36), t=0.03, P=0.98]and total lymphocytes[(4 497.11±975.48) vs (4 720.91±1 572.92), t=0.78, P=0.44]. However, the ratios of Treg/CD4+ T cells[(1.09±0.62) vs (3.17±1.81), t=7.16, P<0.001], Treg/total lymphocytes[(0.42±0.24) vs (1.03±0.51), t=7.08, P<0.001]and Treg/Th17[(0.56±0.41) vs (1.87±1.68), t=4.98, P<0.001]in the URSA re-miscarriage group were significantly lower whereas the ratios of Th17/CD4+ T cells[(1.85±0.92) vs (1.12±0.50), t=4.29, P<0.001]and Th17/total lymphocytes[(0.64±0.31) vs (0.36±0.15), t=5.01, P<0.001]were significantly higher. Conclusions Treg and Th17 cell counts in the peripheral blood of patients with URSA might be useful in predicting their pregnancy outcomes. The decrease of Treg cell counts and the increase of Th17 cell counts might imply another abortion.(Chin J Lab Med, 2017, 40: 174-179) Key words: Abortion, habitual; Pregnancy trimester, first; T-lymphocytes, regulatory; Th17 cells

Analysis of miR-146a and miR-142-3p as Potential Markers of Freshly Isolated or In Vitro-Expanded Human Treg cells.

Regulatory CD4+ T cells (Tregs) are pivotal for prevention of autoimmunity. The use of Tregs is therefore of increasing interest in invitro drug screening assays as well as for a cytotherapy per se against autoimmune disorders. For both purposes, invitro expansion of peripheral blood Tregs is necessary and there is an increasing need to identify novel markers that can discriminate natural thymic-derived Tregs (tTregs) from other T cell subsets, and ideally, such markers should be stably expressed during invitro expansion procedures. We screened for novel miRNAs differentially expressed in tTregs and identified miR-146a and 142-3p as possible candidates. We analysed freshly isolated naïve and activated tTregs and non-Treg subsets after or prior to invitro expansion. We observed a tTreg-specific profile of these miRNAs together with FOXP3 and Helios in freshly isolated tTregs, but observed a decline in the same markers in activated tTregs as opposed to naïve tTregs. In vitro-expanded Tregs could be identified based on FOXP3 expression, but with loss of a discriminate profile for miRNA candidates and a decline in FOXP3 when activated tTregs were expanded. Our data demonstrate miR-146a and 142-3p as potential miRNA markers for discrimination between non-Treg cells and tTregs, but these miRNAs are not stable markers for invitro-expanded Treg cells. In addition, the loss of FOXP3 in expansion of activated tTregs has implication for invitro use of this cell subset in immunopharmacological assays and cytotherapy as FOXP3 is pivotal for suppressive function.