Milk fat globule membrane supplementation protects againstβ-lactoglobul-ininduced food allergy in mice via upregulation of regulatory T cells and enhancement of intestinal barrier in a microbiota-derived short-chain fatty acids manner

Milk fat globule membrane(MFGM),which contains abundant glycoproteins and phospholipids,exerts beneficial effects on intestinal health and immunomodulation.The aim of this study was to evaluate the protective effects and possible underlying mechanisms of MFGM on cow’s milk allergy(CMA)in aβ-lactoglobulin(BLG)-induced allergic mice model.MFGM was supplemented to allergic mice induced by BLG at a dose of 400 mg/kg body weight.Results demonstrated that MFGM alleviated food allergy symptoms,decreased serum levels of lipopolysaccharide,pro-inflammatory cytokines,immunoglobulin(Ig)E,Ig G1,and Th2 cytokines including interleukin(IL)-4,while increased serum levels of Th1 cytokines including interferon-γand regulatory T cells(Tregs)cytokines including IL-10 and transforming growth factor-β.MFGM modulated gut microbiota and enhanced intestinal barrier of BLG-allergic mice,as evidenced by decreased relative abundance of Desulfobacterota,Rikenellaceae,Lachnospiraceae,and Desulfovibrionaceae,while increased relative abundance of Bacteroidetes,Lactobacillaceae and Muribaculaceae,and enhanced expressions of tight junction proteins including Occludin,Claudin-1 and zonula occludens-1.Furthermore,MFGM increased fecal short-chain fatty acids(SCFAs)levels,which elevated G protein-coupled receptor(GPR)43 and GPR109A expressions.The increased expressions of GPR43 and GPR109A induced CD103+dendritic cells accumulation and promoted Tregs differentiation in mesenteric lymph node to a certain extent.In summary,MFGM alleviated CMA in a BLG-induced allergic mice model through enhancing intestinal barrier and promoting Tregs differentiation,which may be correlated with SCFAs-mediated activation of GPRs.These findings suggest that MFGM may be useful as a promising functional ingredient against CMA.

Subpopulations of regulatory T cells are associated with subclinical atherosclerotic plaques,levels of LDL,and cardiorespiratory fitness in the elderly

Background:Atherosclerosis forms the pathological basis for the development of cardiovascular disease.Since pathological processes initially develop without clinically relevant symptoms,the identification of early markers in the subclinical stage plays an important role for initiating early interventions.There is evidence that regulatory T cells(Tregs)are involved in the development of atherosclerosis.Therefore,the present study aimed to identify and investigate associations with Tregs and their subsets in a cohort of healthy elderly individuals with and without subclinical atherosclerotic plaques(SAP).In addition,various lifestyle and risk factors,such as cardiorespiratory fitness,were investigated as associated signatures.Methods:A cross-sectional study was performed in 79 participants(male:n=50;age=63.6±3.7 years;body mass index=24.9±3.1 kg/m2;mean±SD)who had no previous diagnosis of chronic disease and were not taking medication.Ultrasound of the carotids to identify SAP,cardiovascular function measurement for vascular assessment and a cardiorespiratory fitness test to determine peak oxygen uptake were performed.Additionally,tests were conducted to assess blood lipids and determine glucose levels.Immunophenotyping of Tregs and their subtypes(resting(rTregs)and effector/memory(mTregs))was performed by 8-chanel flow cytometry.Participants were categorized according to atherosclerotic plaque status.Linear and logistic regression models were used to analyze associations between parameters.Results:SAP was detected in a total of 29 participants.The participants with plaque were older(64.8±3.6 years vs.62.9±3.5 years)and had higher peripheral systolic blood pressure(133.8±14.7 mmHg vs.125.8±10.9 mmHg).The participants with SAP were characterized by a lower percentage of rTregs(28.8%±10.7%vs.34.6%±10.7%)and a higher percentage of mTregs(40.3%±14.7%vs.30.0%±11.9%).Multiple logistic regression identified age(odds ratio(OR)=1.20(95%confidence interval(95%CI):1.011.42))and mTregs(OR=1.05(95%CI:1.021.10))as independent risk factors for SAP.Stepwise linear regression could reveal an association of peak oxygen uptake(β=0.441),low-density lipoprotein(LDL)(β=0.096),and SAP(β=6.733)with mTregs and LDL(β=0.104)with rTregs.Conclusion:While at an early stage of SAP,the total proportion of Tregs gives no indication of vascular changes,this is indicated by a shift in the Treg subgroups.Factors such as serum LDL or cardiopulmonary fitness may be associated with this shift and may also be additional diagnostic indicators.This could be used to initiate lifestyle-based preventive measures at an early stage,which may have a protective effect against disease progression.

CD4^(+)CD25^(+) regulatory T cells decreased future liver remnant after associating liver partition and portal vein ligation for staged hepatectomy

BACKGROUND Associating liver partition and portal vein ligation for staged hepatectomy(ALPPS)is an innovative surgical approach for the treatment of massive hepatocellular carcinoma(HCC),the key to successful planned stage 2 ALPPS is future liver remnant(FLR)volume growth,but the exact mechanism has not been elucidated.The correlation between regulatory T cells(Tregs)and postoperative FLR regeneration has not been reported.AIM To investigate the effect of CD4^(+)CD25^(+)Tregs on FLR regeneration after ALPPS.METHODS Clinical data and specimens were collected from 37 patients who developed massive HCC treated with ALPPS.Flow cytometry was performed to detect changes in the proportion of CD4^(+)CD25^(+)Tregs to CD4^(+)T cells in peripheral blood before and after ALPPS.To analyze the relationship between peripheral blood CD4^(+)CD25^(+)Treg proportion and clinicopathological information and liver volume.RESULTS The postoperative CD4^(+)CD25^(+)Treg proportion in stage 1 ALPPS was negatively correlated with the amount of proliferation volume,proliferation rate,and kinetic growth rate(KGR)of the FLR after stage 1 ALPPS.Patients with low Treg proportion had significantly higher KGR than those with high Treg proportion(P=0.006);patients with high Treg proportion had more severe postoperative pathological liver fibrosis than those with low Treg proportion(P=0.043).The area under the receiver operating characteristic curve between the percentage of Tregs and proliferation volume,proliferation rate,and KGR were all greater than 0.70.CONCLUSION CD4^(+)CD25^(+)Tregs in the peripheral blood of patients with massive HCC at stage 1 ALPPS were negatively correlated with indicators of FLR regeneration after stage 1 ALPPS and may influence the degree of fibrosis in patients’livers.Treg percentage was highly accurate in predicting the FLR regeneration after stage 1 ALPPS.

Pien Tze Huang alleviates Concanavalin A-induced autoimmune hepatitis by regulating intestinal microbiota and memory regulatory T cells

BACKGROUND Traditional Chinese medicine has used the drug Pien Tze Huang(PTH),a classic prescription,to treat autoimmune hepatitis(AIH).However,the precise mode of action is still unknown.AIM To investigate the mechanism of PTH in an AIH mouse model by determining the changes in gut microbiota structure and memory regulatory T(mTreg)cells functional levels.METHODS Following induction of the AIH mouse model induced by Concanavalin A(Con A),prophylactic administration of PTH was given for 10 d.The levels of mTreg cells were measured by flow cytometry,and intestinal microbiota was analyzed by 16S rRNA analysis,while western blotting was used to identify activation of the toll-like receptor(TLR)2,TLR4/nuclear factor-κB(NF-κB),and CXCL16/CXCR6 signaling pathways.RESULTS In the liver of mice with AIH,PTH relieved the pathological damage and reduced the numbers of T helper type 17 cells and interferon-γ,tumor necrosis factor-alpha,interleukin(IL)-1β,IL-2,IL-6,and IL-21 expression.Simultaneously,PTH stimulated the abundance of helpful bacteria,promoted activation of the TLR2 signal,which may enhance Treg/mTreg cells quantity to produce IL-10,and suppressed activation of the TLR4/NF-κB and CXCL16/CXCR6 signaling pathways.CONCLUSION PTH regulates intestinal microbiota balance and restores mTreg cells to alleviate experimental AIH,which is closely related to the TLR/CXCL16/CXCR6/NF-κB signaling pathway.

Mechanism of T cell hyporesponsiveness to HBcAg is associated with regulatory T cells in chronic hepatitis B

AIM： To study the mechanisms of hyporesponsiveness of HBV-specific CD4^＋ T cells by testing TH1 and TH2 commitment and regulatory T cells. METHODS： Nine patients with chronic hepatitis B were enrolled. Peripheral blood mononuclear cells were stimulated with HBcAg or HBsAg to evaluate their potential to commit to TH1 and TH2 differentiation. HBcAg-specific activity of regulatory T cells was evaluated by staining with antibodies to CD4, CD25, CTLA-4 and interleukin-10. The role of regulatory T cells was further assessed by treatment with anti-interleukin-10 antibody and depletion of CD4^＋CD25^＋ cells. RESULTS： Level of mRNAs for T-bet, IL-12R β2 and IL-4 was significantly lower in the patients than in healthy subjects with HBcAg stimulation. Although populations of CD4^＋CD25^highCTLA-4^＋ T cells were not different between the patients and healthy subjects, IL-10 secreting cells were found in CD4^＋ cells and CD4^＋CD25^＋ cells in the patients in response to HBcAg, and they were not found in cells which were stimulated with HBsAg. Addition of anti-IL-10 antibody recovered the amount of HBcAgspecific TH1 antibody compared with control antibody （P 〈 0.01, 0.34% ± 0.12% vs 0.15% ± 0.04%）. Deletion of CD4^＋CD25^＋ T cells increased the amount of HBcAgspecific TH1 antibody when compared with lymphoo/tes reconstituted using regulatory T cells （P 〈 0.01, 0.03% ± 0.02% vs 0.18% ± 0.05%）.CONCLUSION： The results indicate that the mechanism of T cell hyporesponsiveness to HBcAg includes activation of HBcAg-induced regulatory T cells in contrast to an increase in TH2-committed cells in response to HBsAg.

High mobility group box-1 protein inhibits regulatory T cell immune activity in liver failure in patients with chronic hepatitis B

BACKGROUND: Liver failure in chronic hepatitis B (CHB) patients is a severe, life-threatening condition. Intestinal endotoxemia plays a significant role in the progress to liver failure. High mobility group box-1 (HMGB1) protein is involved in the process of endotoxemia. Regulatory T (Treg) cells maintain immune tolerance and contribute to the immunological hyporesponsiveness against HBV infection. However, the roles of HMGB1 and Treg cells in the pathogenesis of liver failure in CHB patients, and whether HMGB1 affects the immune activity of Treg cells are poorly known at present, and so were explored in this study. METHODS: The levels of HMGB1 expression were detected by ELISA, real-time RT-PCR, and Western blotting, and the percentage of CD4(+)CD25(+)CD127(low) Treg cells among CD4(+) cells was detected by flow cytometry in liver failure patients with chronic HBV infection, CHB patients, and healthy controls. Then, CD4(+)CD25(+)CD127(low) Treg cells isolated from the peripheral blood mononuclear cells from CHB patients were stimulated with HMGB1 at different concentrations or at various intervals. The effect of HMGB1 on the immune activity of Treg cells was assessed by a suppression assay of the allogeneic mixed lymphocyte response. The levels of forkhead box P3 (Foxp3) expression in Treg cells treated with HMGB1 were detected by RT-PCR and Western blotting. RESULTS: A higher level of HMGB1 expression and a lower percentage of Treg cells within the population of CIA(+) cells were found in liver failure patients than in CHB patients (82.6+/-20.1 mu g/L vs. 34.2+/-13.7 mu g/L; 4.55+/-1.34% vs. 9.52+/-3.89%, respectively). The immune activity of Treg cells was significantly weakened and the levels of Foxp3 expression were reduced in a dose- or time-dependent manner when Treg cells were stimulated with HMGB1 in vitro. CONCLUSIONS: The high level of HMGB1 and the low percentage of Treg cells play an important role in the pathogenesis of liver failure in patients with chronic HBV infection. Moreover, HMGB1 can weaken the immune activity of Treg cells. It is suggested that effectively inhibiting HMGB1 expression could be a feasible way to treat liver failure by suppressing endotoxemia and enhancing Treg cell activity.

Gastric cancer cells induce human CD4^+ Foxp3^+ regulatory T cells through the production of TGF-β1

AIM: To elucidate the molecular and cellular features responsible for the increase of regulatory T cells (Tregs) in gastric cancer. METHODS: The frequencies of CD4 + Foxp3 + Tregs and the level of transforming growth factor-β1 (TGF-β1) were analyzed from 56 patients with gastric cancer byflow cytometry and enzyme-linked immunosorbent assay respectively. Foxp3 gene expression was analyzed by real-time polymerase chain reaction. The gastric cancer microenvironment was modeled by establishing the coculture of gastric cancer cell line, MGC-803, with sorting CD4 + T cells. The normal gastric mucosa cell line, GES-1, was used as the control. The production of TGF-β1 was detected in supernatant of MGC and GES-1. The carboxyfluorescein diacetatesuccinimidyl ester (CFSE) dilution assay was performed to evaluate the proliferation characteristics of induced Tregs. Neutralizing anti-TGF-β1 antibody was added to the co-culture system for neutralization experiments. RESULTS: The level of serum TGF-β1 in gastric cancer patients (15.1 ± 5.5 ng/mL) was significantly higher than that of the genderand age-matched healthy controls (10.3 ± 3.4 ng/mL) (P < 0.05). Furthermore, the higher TGF-β1 level correlated with the increased population of CD4 + Foxp3 + Tregs in advanced gastric cancer (r = 0.576, P < 0.05). A significant higher frequency of CD4 + Foxp3 + Tregs was observed in PBMCs cultured with the supernatant of MGC than GES-1 (10.6% ± 0.6% vs 8.7% ± 0.7%, P < 0.05). Moreover, using the purified CD4 + CD25 T cells, we confirmed that the increased Tregs were mainly induced from the conversation of CD4 + CD25 naive T cells, and induced Tregs were functional and able to suppress the proliferation of effector T cells. Finally, we demonstrated that gastric cancer cells induced the increased CD4 + Foxp3 + Tregs via producing TGF-β1. Gastric cancer cells upregulated the production of TGF-β1 and blockade of TGF-β1 partly abrogated Tregs phenotype. CONCLUSION: Gastric cancer cell can induce Tregs development via producing TGF-β1, by which the existence of cross-talk between the tumor and immune cells might regulate anti-tumor immune responses.

Analysis of circulating regulatory T cells （CD4 ＋CD25 ＋CD 127-）after cryosurgery in prostate cancer

This study was performed to assess the response of regulatory T cells （Tregs） following cryosurgery in prostate cancer （PCa） patients by measuring their frequency and immune function. Blood was collected prior to and at 4 and 8 weeks after treatment in 30 patients with high-risk PCa who underwent cryosurgery and from 15 healthy volunteers. Circulating CD4＋CD25＋CD127- Tregs were isolated. Their frequency was detected by flow cytometry, and immune suppressive function was evaluated by measuring the proliferation of CD4＋CD25- T cells cocultured with Tregs. The results showed that the percentage of circulating CD4＋CD25＋CD127- Tregs was increased in PCa patients compared to healthy volunteers （7.6%±0.73% vs. 5.8%±0.54%, P〈0.001）. The frequency of circulating CD4＋CD25＋CD127- Tregs was reduced 4 weeks after cryosurgery compared to before surgery （6.3%__.0.58% vs. 7.6%±0.73%, P〈0.001）, and the decrease persisted for 8 weeks. However, the suppressive function of Tregs was increased in eight of 12 patients, which might contribute to cancer recurrence. Then the response of circulating Tregs is complicated after cryosurgery for PCa, and further studies are warranted.

Immunosuppressive effects of rat mesenchymal stem cells:involvement of CD4^+ CD25^+ regulatory T cells

BACKGROUND: Recent studies show that mesenchymal stem cells (MSCs) have immunomodulatory properties. They suppress the immune response to alloantigen and modify the proliferation of T cells. CD4(+)CD25(+) regulatory T cells have strong immunomodulatory potential. However, little is known about the effects of rat MSCs (rMSCs) on the development of regulatory T cells. METHODS: MSCs were obtained from bone marrow of male Sprague-Dawley rats, and co-cultured with CD3(+) T cells from allogeneic spleen cells. The proportion of CD4(+)CD25(+) regulatory T cells was analyzed by flow cytometry. To further confirm the immunosuppressive activity of rMSCs, we used MTT assay and flow cytometry of CD3(+) T cells to investigate the proliferative responses of CD3(+) T cells to mitogenic stimuli. Enzyme-linked immunosorbent assay was performed to detect alterations of the cytokines TNF-alpha, TGF-beta and IL-10. RESULTS: The proliferation of CD3(+) T cells decreased when co-cultured with rMSCs, and the degree of inhibition was concentration-dependent. The percentage of CD4(+)CD25(+) regulatory T cells increased when CD3(+) T cells were co-cultured with different concentrations of rMSCs. The levels of pro-inflammatory cytokine (TNF-alpha) decreased while anti-inflammatory JGF-beta, IL-10) cytokines increased in mixed lymphocyte reaction. CONCLUSIONS: rMSCs inhibit allogeneic T cell proliferation in mixed cell cultures. This immunosuppressive effect seems to be mediated by inducing the generation of CD4(+)CD25(+) regulatory T cells and soluble factors.

Analysis of CD4^+CD25^+ Regulatory T Cells and Foxp3 mRNA in the Peripheral Blood of Patients with Asthma

The changes of CD4^＋CD25^＋ regulatory T cells （CD4^＋CD25^＋ Treg） and Foxp3 mRNA in peripheral blood mononuclear cells （PBMCs） from patients with asthma were investigated in order to elucidate the possible roles of CD4^＋CD25^＋ Treg in the development of asthma. The peripheral blood samples were collected from 29 healthy controls （normal control group） and 78 patients with asthma which included 30 patients in exacerbation group, 25 patients in persistent group, and 23 patients in remission group. By using flow cytometry and RT-PCR, the CD4^＋CD25^＋ Treg ratio and Foxp3 mRNA in PBMCs were detected. The CD4^＋CD25^＋ Treg ratio and Foxp3 mRNA in PBMCs of exacerbation and persistent groups were lower than that of remission and normal control groups （P〈0.05）. Although the CD4^＋CD25^＋ Treg ratio and Foxp3 mRNA of remission group were also lower than that of normal control group, there was no significant difference between them （P〉0.05）. As compared with persistent group, exacerbation group had lower CD4^＋CD25^＋ Treg ratio and Foxp3 mRNA （P〈0.05）. It was indicated that the decrease of CD4^＋CD25^＋ Treg ratio and its function in PBMCs may be responsible for pathogenesis of asthma.

Regulatory T cells in patients with inflammatory bowel diseases treated with adacolumn granulocytapheresis

AIM: To investigate if the clinical efficacy of granulocytes and monocytes by adsorption (GMA) is associated with an increased frequency of peripheral regulatory T cells (Tregs), as these cells have proven to be successful in suppressing inflammatory bowel disease (IBD) in animal models. METHODS: We report four cases of corticosteroid- dependent ulcerative colitis (UC) and two Crohn’s disease (CD) cases with severe cutaneous lesions who received GMA therapy. The frequency of CD4+ CD25high (Tregs) in peripheral blood was analyzed by flow cytometry and the expression of FoxP3 and TGF beta in purified CD4+ T cells was determined by real time PCR prior to and one month after the last apheresis session, and at the time of endoscopic and clinical assessing. RESULTS: Increased expression of Fox P3 mRNA was found in all five patients who responded to cytapheresis with remission of clinical symptoms, mucosal inflammation and cutaneous lesions, and an increased frequency of circulating Tregs was found in four patients. These changes were not observed in the patient with UC who did no respond to GMA. Variations in TGF-β (mRNA) did not parallel that of FoxP3 mRNA. CONCLUSION: The clinical efficacy of GMA on IBD and related extra intestinal manifestations was associated with an expansion of circulating CD4+ CD25+ Tregs and higher expression of FoxP3 in CD4+ T cells. Accordingly, an elevated CD4+ CD25+ FoxP3 may be a valuable index of remission in patients with IBD and other chronic relapsing-remitting inflammatory conditions during treatment with GMA.

Regulatory T cells in viral hepatitis

The pathogenesis and outcome of viral infections are significantly influenced by the host immune response. The immune system is able to eliminate many viruses in the acute phase of infection. However, some viruses, like hepatitis C virus (HCV) and hepatitis B virus (HBV), can evade the host immune responses and establish a persistent infection. HCV and HBV persistence is caused by various mechanisms, like subversion of innate immune responses by viral factors, the emergence of T cell escape mutations, or T cell dysfunction and suppression. Recently, it has become evident that regulatory T cells may contribute to the pathogenesis and outcome of viral infections by suppressing antiviral immune responses. Indeed, the control of HCV and HBV specific immune responses mediated by regulatory T cells may be one mechanism that favors viral persistence, but it may also prevent the host from overwhelming T cell activity and liver damage. This review will focus on the role of regulatory T cells in viral hepatitis.

Regulatory T cells in inflammatory bowel diseases and colorectal cancer

Regulatory T cells(T regs) are key elements in immunological self-tolerance.The number of T regs may alter in both peripheral blood and in colonic mucosa during pathological circumstances.The local cellular,microbiological and cytokine milieu affect immunophenotype and function of T regs.Forkhead box P3+ T regs function shows altered properties in inflammatory bowel diseases(IBDs).This alteration of T regs function can furthermore be observed between Crohn's disease and ulcerative colitis,which may have both clinical and therapeutical consequences.Chronic mucosal inflammation may also influence T regs function,which together with the intestinal bacterial flora seem to have a supporting role in colitis-associated colorectal carcinogenesis.T regs have a crucial role in the immunoevasion of cancer cells in sporadic colorectal cancer.Furthermore,their number and phenotype correlate closely with the clinical outcome of the disease,even if their contribution to carcinogenesis has previously been controversial.Despite knowledge of the clinical relationship between IBD and colitis-associated colon cancer,and the growing number of immunological aspects encompassing sporadic colorectal carcinogenesis,the molecular and cellular links amongst T regs,regulation of the inflammation,and cancer development are still not well understood.In this paper,we aimed to review the current data surrounding the role of T regs in the pathogenesis of IBD,colitis-associated colon cancer and sporadic colorectal cancer.

Rapamycin ameliorates experimental autoimmune uveoretinitis by inhibiting Th1/Th2/Th17 cells and upregulating CD4+CD25+ Foxp3 regulatory T cells

· AIM: To determine the effects of rapamycin on experimental autoimmune uveoretinitis(EAU) and investigate of role of rapamycin on T cell subsets in the disease.·METHODS: EAU was induced in rats using peptides1169 to 1191 of the interphotoreceptor binding protein(IRBP). Rapamycin(0.2 mg/kg/d) was administrated by intraperitoneal injection for a consecutive 7d after immunization. Th1/Th2/Th17 cytokines, TGF-β1, and IL-6produced by lymphocyteswere measured by ELISA, while Th17 cells and CD4 +CD25 + regulatory T cells(Tregs)from rat spleen were detected by flow cytometry.·RESULTS: Intraperitoneal treatment immediately after immunization dramatically ameliorated the clinical course of EAU. Clinical responses were associated with reduced retinal inflammatory cell infiltration and tissue destruction. Rapamycin induced suppression of Th1/Th2/Th17 cytokines, including IFN-γ, IL-2, IL-17, IL-4, and IL-10 release from T lymphocytes of EAU rats, in vitro.Rapamycin also significantly increased TGF-β1production but had no effect on IL-6 productionof T lymphocytes from EAU rats in vitro. Furthermore,rapamycin decreased the ratio of Th17 cells/CD4 +T cells and upregulated Tregs in EAU, as detected by flow cytometry.·CONCLUSION: Rapamycin effectively interferes with T cell mediated autoimmune uveitis by inhibiting antigen-specific T cell functions and enhancing Tregs in EAU.Rapamycin is a promising new alternative as an adjunct corticosteroid-sparing agent for treating uveitis.

Establishment of Nasal Tolerance to Heat Shock Protein-60 Alleviates Atherosclerosis by Inducing TGF-β-dependent Regulatory T Cells

Mounting evidence supports that a newly identified regulatory T cell (Treg),CD4+LAP+ Treg,is associated with oral tolerance induction and following inhibition of atherosclerosis,but little is described about whether nasal tolerance to antigen likewise induces the novel Tregs production and the relevant antiatherosclerotic benefit.We investigated the effect of nasal administration of heat shock protein-60 (HSP60) on atherogenesis.HSP60 or phosphate buffer solution (PBS) was nasally adminis-tered to six-week-old male ApoE-/-mice.At the 10th week after the nasal administration,there was a significant decrease in atherosclerotic plaque areas of aortic roots in the HSP60-treated mice as com-pared with those in the PBS-treated mice.Atherosclerosis suppression was accompanied with a signifi-cant increase in CD4+LAP+ and CD4+CD25+Foxp3+ Tregs and a concurrently increased production of TGF-β in the HSP60-treated mice.The protective effect of HSP60 was offset by injection of anti-TGF-βantibody.It is concluded that nasal administration of HSP60 can inhibit atherosclerotic formation through immune tolerance which is established by Tregs depending on the induction of anti-inflammatory cytokine TGF-β.Immune tolerance induced by nasal administration of HSP60 may provide an alternative therapeutic method for atherosclerosis.

Depletion of CD4^+CD25^+ regulatory T cells can promote local immunity to suppress tumor growth in benzo[a]pyrene-induced forestomach carcinoma

AIM： To elucidate the distribution of CD4^＋CD25^＋ regulatory T cells （Tregs） in different lymphoid tissues and its local enhancement on tumor growth before and after depletion of CD4^＋CD25^＋ Tregs. METHODS： Female ICR mice were garaged with benzo[a]pyrene （BaP） to induce forestomach carcinoma. CD4^＋CD25^＋ Tregs were intraperitoneally depleted with monoclonal antibody PC61. These mice were divided into BaP-only, BaP ＋ IgG, BaP ＋ PC61, and control groups. The forestomach of mice was dissected for histological analysis, and tunnel test was performed for apoptosis of tumor cells. CD4^＋CD25^＋ Tregs were sorted from different lymphoid tissues and expression of Foxp3, IL-10, and chemokine receptors was analyzed by flow cytometry, semi-quantitative and veal-time polymerase chain reaction. RESULTS： The mice gavaged with only BaP showed increased forestomach papilloma and carcinoma at wk 16 and 32. The proportion of CD4^＋CD25^＋ Tregs was significantly higher in peri-stomach regional lymph nodes than in other lymphoid tissues. These CD4^＋CD25^＋ Tregs in regional lymph nodes expressed higher levels of Foxp3 and IL-10, enriched in the CD62L-subset, and CCR1 and CCR5 chemokine receptors. In mice gavaged with BaP ＋ PC61, the number of tumor nodules and tumor volume decreased significantly with massive infiltrating cells and apoptosis of tumor cells. In the draining regional lymph nodes, the number of CD4^＋CD25^＋ Tregs also decreased significantly. CONCLUSION： Inducible and activated CD4^＋CD25^＋ Tregs in the draining regional lymph nodes suppress host local immunity during tumor growth. Depletion of CD4^＋CD25^＋ Tregs can promote host local immunity to suppress tumor growth.

Changes of CD4^+ CD25^+ Regulatory T Cells in Peripheral Blood in Patients with Hepatocellular Carcinoma Before and after TACE

This study investigated the changes of CD4＋ CD25＋ regulatory T cells （Tregs） in periph-eral blood of patients with hepatocellular carcinoma before and after transcatheter arterial chemoem-bolization （TACE）. The proportion of CD4＋ CD25＋ Tregs among CD4＋ T lymphocytes in peripheral blood of 33 patients with hepatocellular carcinoma was determined by flow cytometry before, 1 week and 1 month after TACE. And 25 healthy volunteers served as control. One month after TACE, the patients were divided into two groups： 22 in group A, who were in stable condition or getting better; and 10 in group B, who were deteriorating. One patient died and was excluded. The results showed that the percentage of CD4＋CD25＋ Tregs among CD4＋ T lymphocytes did not significantly change in the 33 patients 1 week after TACE as compared with that before TACE, however, the difference was significant （P〈0.01） between the patients with hepatocellular carcinoma and the healthy subjects. The percentage of CD4＋ CD25＋ Tregs among CD4＋ T lymphocytes in group A 1 month after TACE was decreased significantly in comparison with that before and 1 week after TACE （P〈0.01）, whereas, that in group B was increased significantly 1 month after TACE （P〈0.01）. It was concluded that patients with hepatocellular carcinoma had a higher proportion of CD4＋CD25＋ Tregs in peripheral blood. TACE did not significantly affect the level of CD4＋ CD25＋ Tregs within short time （such as 1 week）. The proportion of CD4＋CD25＋ Tregs in peripheral blood 1 month after TACE was related to the prognosis of hepatocellular carcinoma.

Regulatory T Cells and Their Molecular Markers in Peripheral Blood of the Patients with Systemic Lupus Erythematosus

CD4＋CD25＋ regulatory T cells （Tregs） and the expression of their molecular markers （GITR, Foxp3） in peripheral blood of the patients with systemic lupus erythematosus （SLE） were investigated in order to reveal the pathogenesis of SLE on the cellular and molecular levels. The level of Tregs in peripheral blood was detected by flow cytometry. The expression levels of GITR and Foxp3 mRNA in peripheral blood mononuclear cells （PBMCs） were assayed by reverse transcriptase-polymerase chain reaction （RT-PCR）. The level of IL-6 in the plasma was measured by ELISA. Comparisons were made among 3 groups： the active SLE group, the inactive SLE group, and normal control group. The level of Tregs in the active SLE group and the inactive SLE group was significantly lower than in the normal control group （P〈0.01）. The level of Tregs in the active group was lower than in the inactive group with the difference being not significant （P〉0.05）. The level of Tregs in SLE patients was significantly negatively correlated with the disease active index in SLE （SLEDAI） （r=-0.81, P〈0.01）. The expression levels of GITR mRNA in PBMCs of the active SLE group and the inactive SLE group were significantly higher than in the normal control group （P〈0.05）, and those of Foxp3 mRNA in SLE patients of both active and inactive SLE groups were significantly lower than in the normal control group （P〈0.05）. There was no significant difference in the expression of GITR and Foxp3 mRNA between the active SLE group and inactive SLE group （P〉0.05）. The plasma levels of IL-6 in both the inactive SLE group and active SLE group were significantly higher than in the normal control group （P〈0.01）. The plasma level of IL-6 in the active SLE group was sig- nificantly increased as compared with that in the inactive SLE group （P〈0.05）, and the plasma level of IL-6 in SLE was significantly positively correlated with SLEDAI scores （r=0.58, P〈0.01） and significantly negatively correlated with the ratio of CD4＋CD25＋ cells/CD4＋ cells （r=-0.389, P〈0.05）. It was concluded that the levels of Tregs and Foxp3 mRNA in peripheral blood of SLE patients were decreased and the levels of GITR mRNA and plasma IL-6 were increased. The Tregs and their molecular markers GITR, Foxp3 as well as the plasma IL-6 might play an important role in the pathogenesis of SLE.

Regulating regulatory T cells to achieve transplant tolerance

BACKGROUND:Regulatory T cells(Tregs) play crucial roles in both induction and maintenance of tolerance. This active immune regulation may contribute not only to the control of immune responses to self-antigens and thereby prevent autoimmune diseases,but also the control of responses to non-self molecules in adaptive immunity. Numerous experimental and clinical studies indicate that manipulating the balance between regulatory and responder T cells is an effective strategy to control immune responsiveness after transplantation. DATA SOURCES:Literature search was conducted using PubMed on the related subjects. Part of the material was based on the most recent work in the authors’ laboratory. RESULTS:We propose some new strategies to achieve transplant tolerance in rodent animals via manipulating Treg function,including using histone deacetylase(HDAC) inhibitor to regulate Foxp3 transcription and enhance Treg suppression,induction of Treg-sparing apoptosis via Nur77,and identification of the co-inhibitory molecule herpes virus entry mediator(HVEM) as an effector molecule for Treg function. CONCLUSION:Regulation of Treg function will definitely provide us very promising tools to achieve clinical tolerance in the future.

Effect of Tiotropium Bromide on Expression of CD_8^+CD_(25)^+FoxP_3^+ Regulatory T Cells in Patients with Stable Chronic Obstructive Pulmonary Disease

The expression of CD8＋CD25＋FoxP3＋ regulatory T cells（CD8＋Tregs） in the peripheral blood of patients with stable chronic obstructive pulmonary disease（COPD）,and the effect of muscarinic cholinergic receptor antagonist tiotropium bromide on the expression of CD8＋Tregs were investigated.Twenty-three patients with moderate to severe stable COPD were enrolled in this study.All patients inhaled tiotropium bromide（18 μg daily） for 3 months.Before and after inhalation of tiotropium bromide,peripheral blood samples were collected from the patients,and T cells were labeled by three-color labeled monoclonal antibodies.Flow cytometry was used to detect the quantity and percentage of CD8＋T cells,CD8＋CD25＋T cells,CD8＋Tregs,CD4＋T cells,CD4＋CD25＋T cells and CD4＋CD25＋FoxP3＋ regulatory T cells（CD4＋Tregs） respectively.The percentage of CD4＋T cells was increased from（27.82±2.18）% to（35.53±1.3）%（t=3.20,P=0.004） in the peripheral blood of patients with stable COPD after inhalation of tiotropium bromide for 3 months,that of CD4＋CD25＋T cells was decreased from（10.03 ±1.42）% to（4.21 ±0.65）%（t=3.78,P=0.001）,and that of CD8＋Tregs was increased from（8.41 ±1.68）% to（21.34 ±4.20）%（t=2.72,P=0.013）.At baseline,CD8＋T cells,CD8＋CD25＋T cells and CD4＋Tregs were detectable in the peripheral blood,but no significant changes were observed after treatment.Linear correlation analysis revealed that the difference before and after treatment in CD4＋T cells and CD4＋CD25＋T cells was negatively correlated with the ratio of change in CD8＋Tregs before and after treatment（r=-0.61,P=0.013;r=-0.72,P=0.001 respectively）.In the peripheral blood of patients with stable COPD,there was the expression of CD8＋Tregs and CD4＋Tregs.Muscarinic receptor antagonist,tiotropium bromide,can promote the amplification of CD4＋T cells,inhibit the expression of CD25＋T cells,and enhance the expression of CD8＋Tregs.CD8＋Tregs and CD4＋Tregs can be used as new indicators to understand the immune status of patients.They are helpful in judging the treatment efficacy and disease immunophenotype.