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Objective: To investigate the regulatory roles of cytokines expressed on peripheral blood T cells in chronic hepatitis B virus (HBV) infection., Methods: Sixty patients with chronic HBV infection were categorized into immune tolerance phase (IT), immune clearance phase (IC), and inactive carrier phase (IA) groups. Twenty healthy individuals were enrolled as healthy controls. Another 21 HBeAg-positive chronic hepatitis (CHB) patients were administrated with entecavir (0.5 mg/d) for 6 months. Serum alanine transaminase (ALT) levels were tested by automatic biochemistry analyzer, and HBV DNA loads were tested by PCR, and cytokines expressed on T cells were examined by flow cytometry., Results: There were negative correlations between IFN-γ levels expressed by peripheral blood CD4(+)T cells and CD8(+)T cells and serum HBV DNA loads in patients with chronic HBV infection, and there were negative correlations between the ratio of IFN-γ/IL-4 in peripheral blood CD4(+)T cells and CD8(+)T cells and serum HBV DNA loads. In addition, the expression levels of IFN-γ were gradually elevated and the expression levels of IL-4 were gradually lowered from IT to IA. In IT, the level of IFN-γ expressed by T cells in patients was lower than that in healthy controls and the level of IL-4 was higher than that in healthy controls. In IA, the levels of IFN-γ and IL-4 were recovered to the normal. Anti-virus therapy reduced serum HBV DNA load and ALT levels in patients, which was accompanied with the increase of IFN-γ level and IFN-γ/IL-4 ratio in CD8(+)T cells., Conclusion: During chronic HBV infection, IFN-γ and IL-4 expressed by peripheral blood T cells play dual immunoregulatory roles, which are correlated with the efficacy of entecavir.

This study was aimed to explore the effects of blocking B7/CD28 and CD40/CD154 co-stimulatory signals on immune function of sensitized mice', and provide the evidences of acquired immune tolerance for allogeneic bone marrow transplantation. The mice sensitized on 7 day before transplant were divided into 4 groups: (1)CTLA4Ig+ anti-CD154 isotype control IgG; (2)anti-CD154 +CTLA4Ig isotype control IgG; (3)CTLA4Ig and anti-CD154; (4)isotype control IgG of CTLA4Ig and anti-CD154. CTLA4Ig and anti-CD154 used in normal BALB/c mice as isotype control IgG. Each mouse in all groups received CTLA4Ig and anti-CD154 (or corresponding isotype control IgG) 500 µg respectively, and was injected via tail vein on 7 day before transplant. There were 5 mice in each group. The mice were sacrificed on day 0, then the number of CD19(+)CD69(+)B cells, CD44(high)/CD62L(high) and CD44(high)/CD62L(low)/- T cells were measured by flow cytometry. Changes of cytokines and sensitized antibody were tested by ELISA or flow cytometry. The results showed that the numbers of CD19(+)CD69(+)B cells were significantly increased in comparison with the normal group (P < 0.01) , whereas the numbers of cells were significantly decreased when blocking B7/CD28 or /and CD40/CD154 co-stimulatory signals (P < 0.01) . Blocking these 2 signals together displayed a synergistic effect (P < 0.01) . The central memory and effector T cells were defined as CD44(high)/CD62L(high) and CD44(high)/CD62L(low)/- respectively, those increased significantly after sensitized in comparison with those in normal group, whereas their numbers decreased when blocking B7/CD28 or/and CD40/CD154 co-stimulatory signals. Blocking these two signals together, displayed a synergistic effect (P < 0.01). Cytokines, IgG and IgM in all groups were not significantly different. Sensitizing antibody test showed that the fluorescence intensity of sensitized group significantly increased as compared with normal group, whereas fluorescence intensity of CTLA4Ig or/and anti-CD154 treated groups significantly decreased as compared with sensitized group (P < 0.01) . It is concluded that blocking the B7/CD28 or/and CD40/CD154 co-stimulatory signal can inhibit the cellular and humoral immune function, whereas blocking these two signals together displays a synergistic effect.

Aim: to observe whether the donor's marrow (BM) cells treated by GM-CSF and TGF-β in vitro were beneficial in inducing specific hypoergia to donor's lymphocyte in recipient., Methods: donor's marrow cell were cultured with GM-CSF and TGF-β in vitro and its maturities were tested by treating with LPS. BALB/c mice were divided into three groups and received treatment as following: (1) BM pretreatment group: donor's BM cell mentioned above were injected into caudal vein with 10(5); cell each mouse; (2) donor's splenocyte group: C57BL/6 mouse's splenocyte were injected through caudal vein with 10(5); cell each mouse; (3) Negative control group: PBS with the same volume were injected through caudal vein. The treatment were performed twice with one week internal in each group. One week after the second treatment, all of the mice were challenged by splenocyte of C57BL/6 mouse in abdominal cavity with 10(5); cell each mouse. Three days later, the allo-reactivities were tested: the proliferation of recipient mouse lymphocyte to donor's lymphocyte were test by single mix lymphocyte reaction, the level of serum IFN-γ and IL-10 were tested with ELISA methods. CD4(+);CD25(high); Treg cell and expression of killer cell lectin-like receptor G1 (KLRG1) on NK cell were tested with flow cytometric technique and the NK cytotoxicity were measured by LDH release method., Results: the donor's marrow cell treated by GM-CSF and TGF-β in vitro could resist the maturation promotion by LPS. Compared with donor's lymphocyte pretreatment, pre-treatment with BM cell induced decreased IL-10, increased CD4(+);CD25(high); Treg cell in spleen, decreased proliferation to donor's lymphocyte in vitro. Additionally, NK cell cytotoxicity was also decreased., Conclusion: the donor's marrow cell treated by GM-CSF and TGF-βcan induce the donor specific tolerance in some degree, and modulation of NK cell maybe participate in inducing immunological tolerance.

Aim: To explore the effects of Delta1 gene on rat bone marrow-derived dendritic cells (DCs)., Methods: pcDNA3.1/delta1 plasmid was transfected into Rat DCs with lipofectamine gene transfection method. The expression of activation markers(Iad, CD80 and CD86) were examined by flow cytometry, the release of cytokines IL-12 were examined by ELISA and the stimulatory capacity of the resulting DCs in vitro were examined by mixed lymphocyte reaction(MLR)., Results: The biological character of DCs in experimental group, empty vector group and control group were examined by electron microscope, flow cytometry and cell growth rate counting. There were no differences of cell cycles, ultrastructure and cell growth rate among these three group cells (P>0.05). It stated that delta1 gene had no effect on the growth of DCs. Even at the stimulation of LPS, In the delta1 transfected DCs, we observed no significant effects on the expression of activation markers(Iad, CD80 or CD86). The release of IL-12 in DCs/delta1 was reduced compared with control DCs(P<0.05). The levels of IFN-gamma and IL-2 of the DCs/delta1 were significantly lower than those of the DCs(P<0.05), and the levels of IL-4 and IL-10 were significantly higher than that in control DCs(P<0.05)., Conclusion: Our data demonstrates that the delta1 gene transfection does not interfere with the differentiation or maturation of DCs, but reduce the ability to stimulating T cells, and the mechanism may be related to the decrease of Th1 /Th2 ratio by impairing the production of DCs-derived IL-12.

The interaction of tumor cells with host cells undergoes a progress of immunoediting, including surveillance, equilibrium and escape. The capability of escape from immune-surveillance is a hall marker of tumor cells, which greatly contributes to the tumor growth out of control and the therapy failure in tumor metastasis. It is indicated that tumor cells can recruit amounts of immune cells to tumor site and establish a suppressive immune microenvironment leading to tumor escape. The critical factor for tumor immunotolerance is the establishment of immunosuppressive microenvironment by interaction between pattern recognition receptors with pathogen-associated molecular patterns and damage-associated molecular patterns released from tumor tissue. Therefore, targeting tumor immunotolerance using small molecules or immunostimulatory biological agents such as monoclonal antibody, can inhibit tumor growth and invasion, and subsequently attenuate the tumor metastasis and decrease cancer-caused death.

Objective: To approach the effect of the donor antigenic specificity CD4+CD25+ regulatory T cell (Treg) on cellular immune tolerance function in rat composite tissue allotransplantation (CTA)., Methods: Use the method of immunomagnetic beads to separate CD4+CD25+ Treg, (1 x 10(6))CD4+CD25+ Treg was transfused to rat CTA model. Collected peripheral blood 30 days after operation, and used nylon wool column to separate B cell and T cell. With the stimulation of IgM, detected B cell proliferation and the level of IgG and IgA in serum. Observed the effect of CD4+CD25+ Treg on B cell and T cell function and the survival of allotransplants, and analyzed the data by statistics., Results: The purity of separated CD4+CD25+ Treg was 95.6%. The CPM of T cell of normal control group, topical intervention group, systemic intervention group and non-intervention group were (2436 +/- 358), (2273 +/- 136), (2338 +/- 228) and (3749 +/- 245). The CPM of B cells of normal control group, topical intervention group, systemic intervention group and non-intervention group were (2418 +/- 348), (2252 +/- 127), (2315 +/- 218) and (3720 +/- 224), there was a significant difference in these groups (P < 0.01). The serum level of IgG and IgA of topical intervention group and systemic intervention group were (12.56 +/- 1.30), (2.38 +/- 0.21), (13.48 +/- 1.23) and (2.86 +/- 0.24) g/L, and of normal control group was (12.35 +/- 1.28), (2.36 +/- 0.12) g/L, had no significant difference (P > 0.05). But Treg of non-intervention group was (16.58 +/- 1.12), (3.75 +/- 0.37) g/L, there was a significant difference in the non-intervention group and the three above groups (P < 0.01). The survival time of CTA in intervention of local and systemic groups were (97 +/- 13) and (63 +/- 10) d, which were significant longer than the non-intervention group [(22 +/- 8) d, P < 0.01]., Conclusions: Donor antigen specific CD4+CD25+ Treg has significantly inhibited B cell and T cell function. It can induce immune tolerance and extend the survival time of CTA; as well local application is better than systemic.

Objective: To investigate the in situ expression of regulatory T cells (Tregs) in thymus and its significance for myasthenia gravis (MG)., Methods: Thirty nine MG patients who had not accepted immunosuppressive therapy before thymectomy were recruited as study group and 19 patients undergoing cardiosurgery as control. There was no significant statistical difference in age and gender between the two groups (P > 0.05). The location and proportion of Tregs in thymus were analyzed using indirect immunofluorescence double labeling immunohistochemistry and image analysis software., Results: (1) Tregs were all located in medulla zone of thymus in control and MG groups. (2) Cells of germinal center had a high expression of CD25 and a low expression of CD4. It indicates that a majority of germinal center cells were B cells. (3) There were fewer Tregs around germinal center. (4) There was no statistical significant difference in absolute number of Tregs between the two groups (P > 0.05). But the proportion of Tregs was higher in control group (P < 0.05)., Conclusion: The proportion of thymus Tregs decreases in MG so as to weaken the function of immuno-regulation and suppression. This is probably a precipitating factor of MG.

Objective: To investigate the mechanism of autologous mesenchymal stem cells (MSCs) in prolonging the survival of dogs receiving living donor liver transplantation., Methods: Canine models of allogenic living donor liver transplantation was established in 14 beagle dogs by non-venous by-pass method, and in 7 of the recipients, autologous MSCs labeled by BrdU was infused into the portal vein, with the other 7 dogs as the control. The survival time of the two groups of the dogs was observed after the operation. The liver function (AST and ALT levels), liver pathologies and the differentiation of the transplanted cells were also evaluated postoperatively., Results: Compared with the control group, the dogs receiving MSC transplantation showed significantly increased median survival time (P<0.001) with lowered levels of AST and ALT (P<0.01). The two groups exhibited similar graft rejection after the operation. In dogs with MSC transplantation, the BrdU-labeled MSCs differentiated into liver-like cells in the liver and secreted albumin., Conclusion: Autologous MSCs infusion through the portal vein during allogenic living donor liver transplantation can prolong the survival of the recipient dogs. The stem cells transplanted can differentiate into mature liver-like cells and secrete albumin in the hepatic tissue.

Objective: To investigate the role of indoleamine 2,3-dioxygenase (IDO) in immune tolerance in patients with chronic hepatitis B (CHB)., Methods: Peripheral venous blood samples were collected from 50 CHB patients, and HBV viral load, T lymphocyte subsets and of IDO mRNA, protein and activity were detected with the blood samples from 50 healthy donors as the control. The correlations between HBV viral load, T lymphocyte subsets and IDO were statistically analyzed., Results: In CHB patients, the mRNA and protein levels and activity of IDO were all significantly higher than those in control group [mRNA: (2.110-/+0.615)x10(3) vs (0.143-/+0.026)x10(3); protein: 0.22-/+0.06 vs 0.02-/+0.0017; activity: 26.07-/+8.12 vs 4.98-/+1.65; P<0.05]. IDO mRNA level was positively correlated to HBV viral load (r=0.502, P<0.001) and ALT (r=0.65, P<0.01), and IDO mRNA, protein and activity were inversely correlated to CD4+ T cells (r=-0.622, -0.682, and -0.549, respectively, P<0.05), CD8+ T cells (r=-0.487, -367, and -294, respectively, P<0.05) and the ratio of CD4/CD8 (r=-0.426, -0.533, and -0.397, respectively, P<0.05)., Conclusion: IDO is closely correlated to HBV viral load and responsible for immunotolerance against HBV. Suppression of IDO can be a new therapeutic approach to reverse immunotolerance in CHB.

Aim: To study the NKT cell subsets and their differentiation., Methods: Splenic lymphocytes from C57BL/J mice that had received SEB treatment were collected as effector cells on the 10(th) day. The cells were cultured in medium containing ConA, LPS and IL-2 for 3 days and measured their response to mitogens and cytokine. The inhibitory action of the effector cells was examined. The effector cells were cultured with normal lymphocytes and above mitogens or cytokine for 3 day. The cells proliferation was assessed with MTT method.The NKT cell subsets among these effector cells with the tolerance function were analyzed and their differentiation sources and correlation of functions were detected by flow cytometry., Results: The response of SEB-activated effector cells to ConA, LPS and IL-2 was significantly decreased compared with that of normal lymphocytes. The A values of cell proliferation were decreased from 0.80+/-0.04, 0.60+/-0.03 and 0.55+/-0.07 in control groups to 0.60+/-0.05, 0.30+/-0.05 and 0.27+/-0.04 in effector groups, respectively (P<0.01, n=3).The inhibitory ability of effectors cells against the response of normal lymphocytes to ConA, LPS and IL-2 were clearly observed. They inhibited the response of normal lymphocytes to several mitogens and cytokine. And the A values of cell proliferation were decreased to 0.26+/-0.02, 0.48+/-0.04 and 0.34+/-0.02, respectively (P<0.01, n=3). The CD4(+)NK1.1(+), CD8(+)NK1.1(+), TcRV8(+)NK1.1(+) NKT cell subsets among SEB-activated effector cells with tolerance function were significantly increased and shown that they come from T cell population. And the CD4(-)CD8(-)/NK1.1(+)CD3(+)NKT cells by ConA or SEB-activated were shown coming from NK cell population., Conclusion: The effector cells with tolerance function activated by superantigen SEB relate to CD4(+)NK1.1(+), CD8(+)NK1.1(+), TcRVbeta8(+)NK1.1(+) NKT cell subsets. The NKT cell subsets come from T cells. The CD4(-)CD8(-)/NK1.1(+)CD3(+)NKT cells differentiating from NK cells are not involved in the regulation of tolerance.

Objective: To investigate the expression of tolerance-associated interleukin (IL)-2 mRNA and IL-10 mRNA in rats after allogeneic liver transplantation., Methods: The experimental rats were randomly divided into 3 groups: acute rejection model group (group I), CD4(+)CD25(+)T cell treatment group (group II), donors of the two groups were Wistar while recipients were Sprague-Dawley (SD) rats, and group III as control group, both donors and recipients of this group were SD rats, with 12 rats in each group. Splenic lymphocyte of donor in group II were injected through vena dorsalis penis 7 days before liver transplantation; equal volume normal saline (NS) were injected in rats of group I and III. The contents of IL-2 mRNA and IL-10 mRNA in the liver tissue were determined by reverse transcription-polymerase chain reaction (RT-PCR), and the number of lymphocytes in the donor liver was quantified with flow cytometry 7 days after transplantation. Meanwhile the pathologic change in the donor tissue were observed, and the recipients' life span was recorded., Results: IL-2 mRNA was expressed strongly in the liver of group I, but it was expressed weakly in group II, and no expression was detected in the liver of group III. IL-10 mRNA was expressed only in group II, and the level was high. The rats in group II and III survived over 30 days, but rats in group I had a shorter life (8-11 days, both P<0.01). There was heavy lymphocytic infiltration in the liver of group I,and was much higher than that of the other groups [group I:(14.31+/-3.41)x10(6)/g, group II: (5.04+/-1.13) x10(6)/g, group III: (1.55+/-0.40) x10(6)/g, all P<0.01], and pathology showed moderate rejection. Rats in group II had milder lymphocytic infiltration, and pathology showed no signs of rejection or uncertain rejection, and the ratio of CD4(+) T cell and CD4(+)CD25(+) were higher than those of group I [(43.31+/-8.07)% vs. (33.65+/-7.25)% and (11.39+/-1.92)% vs. (3.05+/-0.62)%] and group III [(43.31+/-8.07)% vs. (31.18+/-6.25)% and (11.39+/-1.92)% vs. (3.37+/-0.72)%, P<0.05 or P<0.01]. In group III no lymphocytic infiltration was found, and pathology showed no sign of rejection., Conclusion: IL-2 may participate in the immune rejection in allogeneic liver transplantation, but IL-10 plays an important role in CD4(+)CD25(+) T cell inducing immune tolerance of allogeneic liver transplantation in the rats.

The study was aimed to compare the effects of T-cell suppression mediated by mesenchymal stem cells (MSC) from normal individuals and myelodysplastic syndromes (MDS) patients. MSC were cultured from the bone marrow of 12 healthy volunteers and 12 MDS patients, the morphology, surface markers and expression of several cytokines of MSC from normal individuals and MDS patients were compared, and the effects of T-cell suppression were tested in the following assays: phytohemaglutinin (PHA)-primed cultures, mixed lymphocyte reaction (MLR), cell cycle of T-cell after PHA-primed cultures and apoptosis of T-cell as well. The results showed that the MSC from normal individuals and MDS patients were similar in morphology, proliferation and surface markers. The suppressions of T-cell proliferation induced by PHA and alloantigens mediated by MDS-MSC were significantly lower than that of normal MSC. More T-cells were arrested in G0/G1 phase by normal MSC, while the effects were deficient by MDS-MSC. The suppression of T-cell activation mediated by MDS-MSC was also lower than that of normal MSC, but suppression effect on T-cell apoptosis increased. The cytokines TGF-beta1, 3, FasL expressed by MDS-MSC were reduced as compared with normal MSC, but TGF-beta2 expression increased in MDS-MSC. It is concluded that although the morphology, proliferation and cell surface markers of MDS-MSC are normal, the T-cell suppression mediated by MDS-MSC is deficient as compared with normal controls. Whether these abnormalities are relevant to the pathogenesis of aplastic anemia remains to be determined.

The study was aimed to explore the roles of exosomes derived from regulatory dendritic cells of mice in the induction of immune tolerance. Immature DC (iDC) from mouse bone marrow cells and regulatory DCs (rDC) were induced by treating iDC with TGF-beta1 and IL-10. The phenotype of regulatory DCs and normal DCs were assayed by flow cytometry. Exosomes from immature DCs (iDex) and regulatory DCs (rDex) were isolated by ultracentrifugation and ultrafiltration. A skin transplantation model was established with the recipients BALB/c mice and the donor C57BL/6 mice. Recipients were divided into PBS control group, iDex group (injection 10 microg iDex of donor C57BL/6 mice via tail vein at days 7 and 3 before skin transplantation), rDex group (injection 10 microg rDex of donor C57BL/6 mice via tail vein at days 7 and 3 before skin transplantation). The capacity of the donor mice and the unrelated allogeneic donor mice to stimulate allogeneic T lymphocyte proliferation was examined by mixed lymphocyte culture (MLR). The results showed that TGF-beta1 and IL-10 could down-regulate the expressions of costimulatory molecules, including CD80, CD86 and CD40. The graft mean survival time (MST) in control group, iDex group and rDex group was 7.8, 10.7 and 18.8 days, respectively. There was significant difference in MST between iDex group and control group (p<0.05), and between rDex group and iDex group (p<0.01). The results of MLR assays indicated donor-specific hyporeactivity especially in rDex group, while the tolerant B/C mice were still immunocompetent to unrelated allogeneic DBA mouse. It is concluded that injection iDex or rDex of donor mice via tail vein before skin transplantation induces immunotolerance, and the effect of rDex is more significant.

Objective: To explore the expression of CD4+CD25+ regulatory T cells in corneal graft, in the iris and peripheral blood in the high risk rat corneal transplant tolerance induced by superantigen staphylococcal enterotoxin B (SEB)., Methods: A high risk rat penetrating corneal transplantation model was established, whereas SD rat donor corneas were implanted into Wistar rat recipients. The recipient rats were divided into two groups. Group control and group SEB had intraperitoneal injection of normal saline solution or SEB (75 microg/kg), respectively. Orthotopic corneal transplantation was performed 1 day after the injection. The corneal graft survival time was evaluated. The expression of CD4+CD25+ T cells in corneal grafts and the iris was evaluated by immunohistochemical staining. The percentage of CD4+CD25+ regulatory T cells in peripheral blood was determined by flow cytometry., Results: Corneal graft mean survival time was (11.63+/-2.83) d in group control and (14.13+/-0.99) d in group SEB. The difference of survival time between these two groups was statistically significant (P<0.05). Histopathology showed remarkable inflammatory cells infiltration in the rejected grafts. The intensity of the infiltration was compatible with the severity of rejection reaction. Immunohistochemical staining of grafts showed that the CD4+CD25+ regulatory T cells were expressed in rejected grafts in both groups 20 days after the surgery, but not before the surgery. The iris whole mount staining showed that before the surgery, no expression of CD4+CD25+ regulatory T cells was observed in group control but relatively remarkable expression was observed in group SEB. Twenty days after the transplantation, CD4+CD25+ regulatory T cells were observed in both groups while group SEB had more pronounced expression. Peripheral blood flow cytometry analysis revealed that the mean percentages of CD4+CD25+ regulatory T cells were stable before and after surgery in group control. However, in group SEB, an increase shift appeared and followed by a decrease to normal level 20 d after the surgery., Conclusion: CD4+CD25+ regulatory T cells take part in immune tolerance in rat immune tolerance induced by superantigen SEB in high risk rat corneal transplantation.

Objective: To evaluate the therapeutic effect of mucosal tolerance on inflammatory bowel disease (IBD)., Methods: Forty-two SD rats underwent enema of 2, 4, 6-trinitro-benzene-sulfonic acid (TNBS) so as to establish models of experimental colitis and then divided into 7 equal groups: colitis group (without treatment), oral ovalbumin (OVA) group (undergoing gastric perfusion of OVA as inducing antigen), nasal OVA group (undergoing OVA nose dropping), oral OVA plus adjuvant group [undergoing gastric perfusion OVA with lipopolysaccharide (LPS) as adjuvant], nasal OVA plus adjuvant group, adjuvant control group, and blank control group (administrated with PBS orally). Another 6 rats were used as blank control group. All of the rats were executed 21 days after enema with their spleens taken out. Splenic lymphocytes were isolated and co-cultured with OVA for 96 h. MTT method was used to calculate the proliferation index so as to detect the mucosal tolerance. Macroscopical and histolopathological scores of colon were estimated so as to evaluate the therapeutic effects., Results: Typical manifestations of colitis occurred 3-4 days after enema and persisted to the day 21 after enema. The macroscopical and histolopathological scores of colon of the colitis group (mean rank were 9.5 and 9.5) were both significantly higher than those of the normal control group (mean rank were 3.5 and 3.5 both P < 0.01). The splenic lymphocyte proliferation indexes of the oral OVA plus LPS, nasal OVA, and nasal OVA plus LPS groups were 0.11 +/- 0.05, 0.12 +/- 0.07, and 0.06 +/- 0.04 respectively, all significantly lower than that of the normal control group (0.30 +/- 0.13, all P < 0.05), however, without significant differences among these 3 groups (all P > 0.05). The splenic lymphocyte proliferation indexes of the oral OVA group was 0.25 +/- 0.10, not significantly different from that of the normal control group (P > 0.05). The macroscopical scores of the oral OVA plus LPS group, nasal OVA plus LPS group, and oral LPS alone group were 3.0 +/- 1.3, 2.0 +/- 0.6, and 4.3 +/- 1.0 respectively, all significantly lower than that of the colitis group (6.3 +/- 0.8, all P < 0.05); and the histological score of the oral OVA plus LPS group, nasal OVA plus LPS group, and oral LPS alone group were 3.0 +/- 1.1, 2.7 +/- 1.0, and 4.6 +/- 1.6 respectively, all significantly lower than that of the colitis group (7.5 +/- 1.0, all P < 0.05). The colonic score of the nasal OVA plus LPS group was most significantly decreased., Conclusion: Colitis interferes with the induction of oral tolerance, but not the induction of nasal tolerance. Both oral tolerance combined with adjuvant and nasal tolerance combined with adjuvant have therapeutic effects on experimental colitis. Oral adjuvant alone also has therapeutic effect on colitis, and the therapeutic effect of nasal tolerance combined with adjuvant is the best.

In order to study how to induce tolerogenic dendritic cells in vitro and its mechanism, the K562 cells transduced with HLA-G construct were used to co-culture with DC. Then their related immunological changes, such as membrane molecules CD80, CD86, ILT3 and ILT4 expression levels were detected by flow cytometry. Allogeneic proliferation of peripheral blood mononuclear cells (PBMNC) was detected by mixed lymphocyte reaction. The results showed that CD80 and CD86 expressions on DC were downregulated, while ILT3 and ILT4 expressions were upregulated after co-culturing with K562-HLA-G cells. The DCs were less able to stimulate the allogenic PBMNC. It is concluded that the membrane-bound HLA-G can upregulate expression of inhibitory receptors ILT3 and ILT4, inducing tolerogenic DC in vitro, which may provide a novel strategy for transplant tolerance induction.

Objective: To explore the role of combined heart-thymus transplantation for heart allograft in rats., Methods: Vascularized heart-thymus combined transplantation was performed with microsurgical technique. Graft survival, histopathology, infiltration of CD4+, CD8+ T cells, level and mRNA expressions of IL-2 and IL-4 in the serum and cardiac grafts were investigated., Results: Heart allograft in the controls had a survival time of (6.0+/-0.76) d. heart-thymus combined transplantation in non-thymectomized rats had a survival time of (6.88+/-0.64)d (P<0.05). Heart-thymus combined transplantation in thymectomized rats led to an evident survival time of (14.13+/-5.82)d (P<0.01) for cardiac graft, which further obtained long term survival after short course of treatment with cyclosporine. Pathologic lesion and infiltration of CD4+ and CD8+ T cells in cardiac grafts showed mitigated in the long term survival group. IL-2 level in the serum and cardiac grafts maintained low level in the long term survival group, whereas IL-4 maintained high level., Conclusion: Whether thymectomized or not in recipient rats, heart-thymus combined transplantation has a positive effect to protect cardiac graft. Furthermore, in thymectomized rats heart-thymus combined transplantation may lead to evident survival prolongation of the heart grafts, which induces immune tolerance in short course of treatment with cyclosporine.

Objective: To explore the role of anti-alphabeta T cell receptor (TCR) and anti-CD80 monoclonal antibodies (mAbs) combined with donor bone marrow cells (BMCs) infusion in the induction of murine skin allografts tolerance., Methods: On day 0, 2 x 10(8) BMCs of BALB/c mice were injected into recipient C57BL/6 mice via the tail vein, meanwhile, an intraperitoneal injection of TCRalphabeta mAb (500 microg) was given. On day 2, CD80 mAb was administered intraperitoneally. Skin grafting was performed on day 6. Delayed type hypersensitivity (DTH), mixed lymphocyte reaction (MLR), IL-2 reverse assay of MLR, adoptive transfer assay and chimerism detection were performed at different time points and tolerance mechanisms were investigated., Results: The mean survival time (MST) of BALB/c skin allografts in C57BL/6 recipients that were treated by anti-TCRalphabeta and anti-CD80 mAbs combined with donor BMCs infusion was 70 days. DTH and MLR assay indicated that the tolerant mice displayed significant hyporesponsiveness. The result of IL-2 reverse test showed that clone anergy was probably involved in the formation of tolerance in the tolerant C57BL/6 mice. In vivo and in vitro adoptive transfer assay, suppressive activity in the spleens of tolerant C57BL/6 mice was observed. Chimerism existed in both the thymus and spleen of the tolerant C57BL/6 mice. The chimerism level gradually declined with time., Conclusion: Treatment of anti-TCRalphabeta and anti-CD80 mAbs combined with donor BMCs infusion can successfully induce a long-term tolerance in BALB/c mice to C57BL/6 skin graft. Multiple mechanisms, including clone anergy, suppressor cells and chimerism are involved in the tolerance.

Objective: To explore the roles of CD4+ CD25+ and CD8+ CD28- T regulatory cells in spontaneous tolerance of inbred rats orthotopic liver Transplantation., Methods: The orthotopic liver transplantation models of spontaneous tolerance were established between BN (donor) and LEW (recipient) inbred rats whose survival time was more than 120 days, The expression of CD8+ CD28- T and CD4+ CD25+ T cells in PBMC and spleen were determined by flow cytometry. CD8+ CD28- T and CD4+ CD25+ T cells were sorted by immunomagnetic beads. The expressions of CD25, CD28, CD152 and CD45RO on CD8+ CD28- T and CD4+ CD25+ T regulatory cells were also determined by flow cytometry., Results: Compared with control groups, the proportion of CD8+ CD28- T regulatory cells in spontaneous models was significantly increased in peripheral blood mononuclear cells (PBMC) and spleen [PBMC (30.2+/-5.8)% vs (16.4+/-6.1)%; Spleen (23.7+/-7.2)% vs(13.4+/-5.5)% P<0.01]. CD4+ CD25+ T regulatory cells had no change; CD11b was expressed on CD8+ CD28- T regulatory cells in both control groups and tolerance groups, and CD25, CD152 (CTL-4) were not expressed on CD8+ CD28- T regulatory cells. The expression of CD45RO on CD8+ CD28- T regulatory cells was 76.1% in control groups and more than 90% in tolerance groups; CD11b, CD152 (CTL-4) and CD45RO were expressed on CD4+ CD25+ T regulatory cells in both control groups and tolerance groups., Conclusion: The roles of CD8+ CD28- T cells were more important than CD4+ CD25+ T cells in spontaneous tolerance of rat liver transplantation. the different expressions of CD152 showed the functions of CD8+ CD28- and CD4+ CD25+ T cells were different. The different expressions of CD45RO suggested CD8+ CD28- T regulatory cells was changed before and after liver transplantation.

This study was aimed to analyze the proportion of T cell subsets, CD4(+) CD25(high) regulating T cells (Tr) in peripheral blood of B-NHL patients and their change regularity, and to investigate the immunosuppression mechanism and influence of chemotherapy on immunosuppression function of B-NHL patients. The peripheral blood was collected from 42 patients with B-NHL, 36 patients with B-NHL who achieved partial remission (PR) or complete remission (CR) after 4 - 6 cycles of chemotherapy and 15 healthy controls. By using monoclonal antibodies, the blood samples were evaluated with the flow cytometry for lymphocyte subsets and Tr cells. The results showed that the proportion of CD3(+) and CD4(+) T cells, and the ratio of CD4/CD8 in patients with B-NHL group was significantly less than those in the healthy controls, i.e. (68.33 +/- 15.27)% versus (72.06 +/- 9.26)%; (34.47 +/- 12.84)% versus (42.45 +/- 9.2)%; 1.36 +/- 0.26 versus 1.92 +/- 0.20, but the prevalence of the CD4(+) CD25(high) Tr cells was significantly higher than those in the healthy group [(4.10 +/- 1.21)% versus (2.04 +/- 1.03)%, P < 0.001]. The ratio of CD4/CD8 in chemotherapy group was lower than that in control, but the proportion of CD4(+) CD25(high) Treg cells in chemotherapy group was higher than those before chemo-/radio-therapy and the control. It is concluded that the relative increase of CD4(+) CD25(high) Tr cells in peripheral blood of B-NHL patients may be related to immunosuppression and tumor progression.

Objective: To establish a parabiosis model between allogenic conspecific adult mice to study two-way paradigm., Methods: Fifty-four female Balb/c mice and 54 male C57BL/6 mice were paired and equally divided into 3 groups, namely group 1 with normal saline (NS) injection, group 2 with injections of spleen cells and cyclophosphamide (CP), and group 3 injected with spleen cells, CP, and cyclosporin A (CsA). The treatments were performed by injecting the spleen cells from one of the mice in a pair into the other via tail vein and vise versa, and two days after the operation, CP (150 mg/kg) was injected intraperitoneally. Intraperitoneal CsA (30 mg/kg daily) injection was given starting from 2 days before till 7 days after the operation. Twelve of the 18 pairs of parabiosis mice in each group were separated after 1 week, and part of the skin were transplanted to each other. The maintenance of parabiosis was observed in the other 6 pairs of parabiosis mice were observed. Mixed lymphocyte reaction (MLR) and delayed-type hypersensitivity (DTH) were observed and studied in the separated mice., Result: The duration of parabiosis maintenance and skin survival of the group 3 was significantly longer than those in the other two groups, and group 3 showed suppressed MLR and DTH., Conclusion: With the application of immunosuppressants, we have successfully established the two-way paradigm model in mice.

Aim: To explore the role of T cell immunoglobulin- and mucin- domain containing molecule-3 (Tim-3) in the induction of tumor immune tolerance by investigating the expression of two types of Tim-3 at the early stage of tumorigenesis and the temporal-spatial relationship between the expression of Tim-3 and other immuno-inhibitory genes in tumor., Methods: By using semi-quantitative RT-PCR method, we investigated the expression of Tim-3, forkhead box P3 (Foxp3), cytotoxic T lymphocyte antigen 4 (CTLA-4), glucocorticoid-induced tumor necrosis factor receptor family-related receptor (GITR), TGF-beta, IL-10 and IFN-gamma at different time in tumor of tumor-bearing mice. Meanwhile, the growth of tumor was determined., Results: At the early stage of tumorigenesis, sTim-3 was expressed before flTim-3 in tumor. When flTim-3 was expressed, the expression of sTim-3 was down-regulated. The expression of Foxp3, and GITR was simultaneous with that of flTim-3 and was up-regulated gradually with tumor growth. The expression of CTLA-4 was earlier than that of flTim-3 and also up-regulated gradually. The expression of flTim-3 and Foxp3 was also consistent with each other in tissue distribution. The growth of tumor was postively correlated with the expression of flTim-3, Foxp3, and CTLA-4 and negatively with the expression of sTim-3., Conclusion: Along with the growth of tumor, immune tolerance was gradually established in tumor. flTim-3 but not sTim-3 may induce tumor immunological tolerance.

To study the effect of adoptive transfer of paternal antigen-tolerant T cells on recipient reactive T cells, CBA/JxDBA/2 mating was recruited as an abortion-prone model, and CBA/JxBALB/c mating as a successful pregnancy model. The abortion-prone CBA/J females mated with DBA/2 males were injected intraperitoneally with rat anti-mouse CD80 and CD86 mAb or rat isotype IgG at day 4 after gestation (time of implantation). The purified T cells were obtained from spleen of the pregnant CBA/J mice using magnetic beads at day 9 after gestation and labeled with CFSE in vitro. The CFSE-labeled T cells were intravenously injected into other CBA/J females mated with DBA/2 males at day 4 after gestation. The proliferation of recipient splenocytes in response to DBA/2 stimulator cells was evaluated at day 9 after gestation in vitro, and the expressions of intracellular cytokines and costimulatory molecules in CFSE +/- T cells were analyzed by flow cytometry. The results showed that adoptive transfer of either paternal antigen-tolerant T cells or T cells from BALB/c-mated CBA/J mice significantly suppressed the proliferation of recipient splenocytes in response to DBA/2 stimulator cells and resulted in lower frequency of cells positive for IL-2, IFN-gamma, CD28 and higher frequency of IL-10,CTLA-4-producing cells in both CFSE+ CD3+ population and CFSE- CD3+ population compared with adoptive transfer of T cells from isotype IgG-treated CBA/J mice, whereas the frequency of IL-4-producing cells did not appear significant change. Our findings suggest that paternal antigen-tolerant T cells transferred in recipient not only function as antigen-specific suppresser cells but also disable the recipient reactive T cells, which co-suppresses maternal rejection to the allogeneic fetus, thus resulting in the decrease of the embryo resorption rate of the abortion-prone mice to that of the normal pregnancy mice.

There has been an increasing interest in recent years on mesenchymal stem cell (MSC). It is well known that MSCs are capable of self-renewal and differentiating into many cell lineages. MSC can be expended to a large quantity that is required for clinical transplantation. Recent studies show that MSC have potential application in immune diseases due to their unique immunologic characteristics, such as low immunogenicity and immunoregulatory function. But their immunoregulatory mechanism is not yet clear. This review discusses the advances in researches on the mechanism of MSCs' immunoregulatory function and potential clinical application in immune disease and organ transplantation.

Objective: To investigate the influence of local application of cytotoxic lymphocyte antigen 4-Ig (CTLA4-Ig) adenovirus on the burn wound with alloskin grafting upon the murine immune function., Methods: Sixty BALB/c mice were randomly divided into A (operation control), B (CTLA4-Ig transfection) and C (normal control) groups, with 20 mice in each group. Skin wounds (full-thickness loss) sized 1.5 cm x 1.5 cm were created on the backs of mice in A and B groups. Then the skin grafts of the same size obtained from C57BL mice were grafted into the skin wounds. 0.1 g of cross-linking polyacrylic resin (carbomer cream) without adenovirus was daubed onto the wounds in A group, and the same amount of carbomer cream with adenovirus in titers of 5 x 10(9)/L was daubed onto the wounds in B group, while no treatment was given in C group. 1 ml of 10% SRBC (sheep red blood cell) was injected intraperitoneally to all the mice of the three groups on the 1st post injury day (PID). Splenocytes from BALB/c, C57BL and Kunming mice were harvested for mixed lymphocyte culture on 7, 14, 21 and 28 PIDs. Agglutination assay was used in the same time to detect the SRBC antibody titers., Results: The reaction of murine splenocytes in B group to the donor (C57BL) splenocytes was suppressed in a specific way (P < 0.05) within 14 PIDs. There was no difference in the titers of anti-SRBC antibody among the 3 groups (P > 0.05)., Conclusion: Local application of CTLA4-Ig recombinant adenovirus exhibited no influence on the murine humoral immunity, but might induce systemic and specific T cell tolerance in immunity system.

Objective: To study effect of blockade of costimulatory signal CD(80), CD(86) at the early stage of gestation on the maternal peripheral immuno-tolerance status to paternal antigen and pregnant outcome of murine abortion-prone model., Methods: The experiments were performed in two groups, using CBA/J x BALB/c matings as the normal pregnancy model and CBA/J x DBA/2 matings as the abortion-prone model. The pregnant CBA/J mice which had mated with DBA/2 or BALB/c male mice respectively were injected intraperitoneally with purified rat isotype IgG or purified rat anti-mouse CD(80), CD(86) mAb at day 4 of gestation (time of implantation). The proliferation response of maternal splenic immuno-competent cells to paternal splenic cells as stimulator was analyzed by one way mixed lymphocyte reaction, and IL-2 production was assayed by ELISA to evaluate the immuno-tolerance of maternal peripheral immuno-competent cells to paternal antigen at day 9 of gestation. The embryo resorption rate was counted at day 14 of gestation., Results: In the CBA/J x DBA/2 matings, anti-CD(80), CD(86) mAb administered at day 4 of gestation reduced significantly the embryo resorption rate, and induced a significantly lower proliferation response and IL-2 production by maternal splenic immuno-competent cells to paternal antigen compared with rat isotype IgG., Conclusions: In vivo blockade of costimulatory signal at day 4 of gestation can induce the immuno-tolerance of maternal peripheral immuno-competent cells to paternal antigen, thus leading the embryo resorption rate of the abortion-prone model to that of normal pregnancy model.

Objective: To investigate and evaluate a clinically perspective way of inducing transplantation tolerance., Methods: Multi-cord blood transfusion was performed before kidney transplantation in 20 cases. Donor-specific mixed lymphocyte reaction(MLR) and allograft rejection were observed and studied., Results: The donor-specific MLR was suppressed in every recipient. No allograft rejection took place in the cord blood transfusion group three months after kidney transplantation., Conclusion: Multi-cord blood transfusion is a practical way of inducing transplantation tolerance.

HLA-G is a non-classical major histocompatibility complex class I molecule that differs from the classical HLA I class molecules by (1) a limited polymorphism, (2) a tissue-restricted distribution and (3) a transcription of spliced messenger RNAs encoding for at least four membrane-bound and two soluble HLA-G isoforms. Extensive studies over the past few years have identified HLA-G as a molecule involved in immune tolerance. In this review, attempts were made to summarize the current state of knowledge of the polymorphisms, expression, function, the effects of HLA-G on immuno-associated disease, evolution of HLA-G and its utility in disease therapy.