Association between development of CD4+CD25+ regulatory T cells and thymus CD4-CD25+ cells

AIM: To explore the correlation between development of CD4⁺CD25⁺ regulatory T cells (CD4⁺CD25⁺ Tr) and thymus CD4^-CD25⁺ cells. METHODS: The ratios of CD4⁺CD25⁺ regulatory T cells to CD4+ T cells in thymus, spleen, lymph node and peripheral blood of mice from birth to mature and also the ratios of CD4^-CD25⁺ cells to CD4^- T cells in thymus were measured by flow cytometry. Purified CD4⁺CD25⁺ T cells and CD4⁺CD25^- T cells were labeled with CFDA-SE, and then stimulated with various kinds of stimulators. RESULTS: The percentages of CD4⁺CD25⁺ Tr in mouse spleen, lymph nodes and peripheral blood increased gradually, but not in thymus, from day one to week 10 of the age with rapid rising from day one to week 1. The percentages of CD4^-CD25⁺ cells in mouse thymus were quite high on day one after birth, and decreased rapidly from day one to week 1. Both CD4⁺CD25⁺ Tr and CD4⁺CD25^- T cells showed no proliferation in response to ConA, while CD4⁺CD25⁺ Tr showed a transient enlargement of cell size. Both CD4⁺CD25⁺ Tr and CD4⁺CD25^- T cells underwent proliferation in response to PDB plus ionomycin. CD4⁺CD25^- T cells, but not CD4⁺CD25⁺ Tr, showed a proliferative response to the stimulation of coated anti-CD3 plus soluble anti-CD28 antibody, however, CD4⁺CD25⁺ Tr showed significant proliferation and CD4⁺CD25^- T cells showed a stronger response in addition of high dose of IL-2. CONCLUSION: The thymus CD4^-CD25⁺ cells are probably the precursor of CD4⁺CD25⁺ Tr during cell development.     

Proliferation of CD4+CD25+ T cells from PBMCs of the gastric cancer patients and inhibitory effect on CD4+CD25- T cells in vitro

AIM:To investigate the proliferation of CD4+CD25+ T cells from PBMCs of the gastric cancer patients and the inhibitory effect on CD4+CD25- T cells in vitro. METHODS:Magnetic activated cell sorting (MACS) method was used to separate CD4+CD25+T and CD4+CD25-T cells from peripheral blood monocytic lymphocytes in the gastric cancer patients, and then the purity and activity of CD4+CD25+T cells were analyzed with flow cytometer. After stimulated with anti-CD3 Ab, anti-CD28 Ab and rh IL-2, CD4+CD25- and CD4+CD25+ T cells were cocultured. The inhibitory effect of CD4+CD25+T on CD4+CD25-T cells was assayed by ［3H］ thymidine proliferation experiment. RESULTS:(1)After sorting, CD4+CD25+ T cells purity in healthy control and gastric cancer patients were 83.80%±1.84% and 84.13%±2.77%, respectively. No significant difference between the two groups (P>0.05) was observed. (2)The activity of CD4+CD25+ and CD4+CD25- T cells in healthy control and the gastric cancer patients after sorting were 98.52%±0.72% and 97.80%±0.95%. There was no significantly difference between the two groups (P>0.05). (3) CD4+CD25+ T cells obviously inhibited the CD4+CD25-T cell proliferation in vitro. The inhibition achieved to maximum in coculture of CD4+CD25+ T cells together with CD4+CD25- T cells (ratio of 1∶〖KG-*2〗1). CONCLUSION:The MACS system can effectively isolate CD4+CD25+ and CD4+CD25- T cells. After sorting, CD4+CD25+T cells obviously inhibit the proliferation of CD4+CD25- T cells in vitro and the inhibitory effect display an effect-target ratio relationship.     

Analysis of activation of T cells from draining lymph node and peripheral blood in allotransplantation mice

AIM:To study the activation of T cells from local lymph node and peripheral blood early after allotransplantation.METHODS:Transplant of myocardio-tissue into mouse forearm subcutaneously was used as a model to analyze the expression of CD69 by T subpopulations from draining lymph node and peripheral blood by flow cytometry.RESULTS:The expression rates of CD69 by both CD4⁺T cells and CD8⁺T cells from the draining lymph node were raised (P＜0.01) 72 h after allotransplantation, and it was higher on CD8⁺T cells than on CD4⁺T cells (P＜0.01). No significant difference in CD69 expression was found on CD4⁺T and CD8⁺T cells from peripheral blood among the groups, topical complete Freund's adjuvant (CFA) and systemic cyclosporin(CsA) enhanced and inhibited expression of CD69 by both CD4⁺T cells and CD8⁺T cells after allotransplantation, respectively (P＜0.05 or P＜0.01).CONCLUSION:To detect the expression of CD69 by T cells from draining lymph node can keep insight to the allorecognition early after transplantation.     

Immunosuppressive effect of dihydroartemisinin on murine T lymphocytes

AIM: To investigate the effect of dihydroartemisinin (DHA) on the proliferation of murine T lymphocytes stimulated by Con A in vitro and its related immunosuppressive mechanism. METHODS: Murine T lymphocytes were stimulated by Con A and treated with different concentrations of DHA. Cell proliferation was measured by carboxyl fluoresce in diacetate succinmidyl ester (CFDA-SE) staining. The expression of CD69, CD25 and CD71,which was the marker of early, middle, later activation of CD3+ T lymphocytes, was measured by flow cytometry (FCM) combined with two-color immunofluorescent staining of cell surface antigen. Fluorescence calcium indicator fluo-4/AM was used to measure the change of the intracellular calcium concentration (［Ca2+］i) of murine T lymphocytes. The distribution of the cell cycle was analyzed by PI staining. The expression of CD69, the early activation antigen on CD4+CD25high Treg was also measured by FCM combined with three-color immunofluorescent staining. RESULTS: The result of CFDA-SE staining showed that DHA efficiently inhibited the Con A-induced proliferation of T-lymphocytes in a time-and dose-dependent manners. DHA showed modestly increased proportions of CD69 and CD25 on Con A-stimulated CD3+T cells, but inhibited the expression of CD25 in a dose dependent manner. DHA with Con A, but not DHA alone, caused an increase in intracellular calcium concentration of T cells. The results of FCM analysis with PI staining showed that DHA imposed a total cell cycle arrest in G0/G1 and prevented cells entering S phase and G2/M phase. Furthermore, DHA reduced the expression of CD69 on CD4+CD25high Treg. CONCLUSION: DHA, which exhibits immunosuppressive effect on the proliferation of murine T-lymphocytes, is promising to be developed as an immunosuppressive reagent.     

Regulatory T cells regulate function of effector T lymphocytes in tumor-draining lymph node of mouse hepatocelluar carcinoma

AIM: To investigate the roles of regulatory T cells (Tregs) on the function of effector T lymphocytes in tumor-draining lymph nodes (TDLNs). METHODS: The number expansion of Foxp3+ Tregs and CD4+ or CD8+ T cells in the TDLNs from mouse hepatocellular carcinoma model was detected by immunohistochemical staining and flow cytometry. Foxp3 mRNA expression was determined by real-time quantitative PCR. The ability of IFN-γ secretion in CD8+ T cells in the TDLNs was measured by enzyme-linked immunosorbent spot technique(ELISPOT). RESULTS: The expansions of Tregs and effector T cells were significantly increased in the TDLNs during tumor development. Tregs diffusely distributed in the CD8+ T cells occupancy area. The level of Foxp3 mRNA expression was significantly higher in the TDLNs than that in the inguinal lymph node (P<0.01) and spleen (P<0.01) from the same mouse inoculated Hepa1-6 cells. Tregs trended to accumulate within the TDLNs exclusively, but not in other peripheral lymph nodes(LNs) of the same host. Foxp3 mRNA expression was significantly higher in the spleen from the tumor mice than that from mice injected with LPS (P<0.01). Tregs suppressed the CD8+ T cells primed in the TDLNs that retained the ability to secrete IFN-γ via anti-CD3 stimulation. CONCLUSION: Tregs play an important role in regulating the function of CD8+ T cells. Deletion of Tregs could be crucial for establishment of tumor-specific immunotherapy.     

Effect of oxymatrine on mouse allergic contact dermatitis induced by DNFB and lymphocyte proliferation stimulated by Con A

AIM: To explore the inhibitory effect of oxymatrine （OMT） on the allergic contact dermatitis （ACD） stimulated by dinitrofluorobenzene （DNFB） and its effects on the proliferation of the lymphocytes. METHODS: ① An ACD mouse model was established by stimulation with DNFB, and then the mice were injected intraperitoneally with different dosages of OMT， PBS and hydrocortisone （HCT） respectively, the swelling degree of their auricles was examined. ② Carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) dye and flow cytometer were used to examine the fluorescence intensity changes of lymphocytes stimulated by polyclonal stimulator ConA and OMT. RESULTS: ① compared with PBS group, OMT possessed the strong inhibitory effect on the ACD caused by DNFB in a dose-dependent manner, and its inhibitory effect was equivalent to the HCT of the same dosage with fewer side effects. ② In vitro experiments proved that OMT (500, 125 and 31 mg/L) had the ability to restrain the proliferation of lymphocytes of mouse. CONCLUSION: OMT possesses an inhibitory effect on the ACD induced by DNFB, and OMT is a kind of immunosuppressor.     

Influence of allogeneic cornea on human peripheral blood T lymphocyte subtype in vitro

AIM: To observe the immunoregulation of allogeneic cornea on the human peripheral blood T lymphocytes in vitro. METHODS: After Co-culture of human peripheral blood lymphocytes and allogeneic cornea in vitro, T lymphocytes were labeled by monoclonal antibody, and analyzed by fluorescent activated cell sorter (FACS). RESULTS: CD25 expression on T lymphocytes in control was 25.2%, after stimulated by the allogeneic cornea or PDB, CD25 expression on T lymphocytes was 56.8% and 80.9%, respectively. After stimulated by the allogeneic cornea, CD25 expression on CD4+ or CD8+ T lymphocytes were 67.3% and 52.3%, respectively. CONCLUSION: Allageneic cornea stimulates CD25 expression on human peripheral blood T lymphocytes, and the CD25 expression on CD4+ T lymphocytes is more prominent than CD8+ T lymphocytes.     

Effects of deep-frozen treatment on the immune response of bone-patellar tendon-bone xenograft rejection

AIM: To investigate the effects of deep-frozen treatment on the immune response of bone-patellar tendon-bone (BPB) xenograft rejection. METHODS: A muscle pocket model was used to study the immune response of rat to deep-frozen treated BPB xenograft from guinea pig, autograft and fresh xenograft served as controls. The expression of T-cell surface activation antigen CD25 in the peripheral blood and the morphological changes of the implants were used to measure the immune response. RESULTS: The expression of CD25 in CD4+, CD8+ cells greatly increased 3 d after fresh BPB xenograft, the obvious difference to that of autograft (P<0.05) was observed. It reached a peak level 14 d after fresh BPB xenograft and didnt decrease 35 d after transplantation. The expression of CD25 in CD4+, CD8+ cells was greatly inhibited in deep-frozen treated BPB xenograft. Its expression was delayed and just had a small increase 14 d after transplantation. The P values were all less than 0.05 compared to those of fresh xenograft at every time-point after transplantation. Histologic examination showed that there were a few lymphocytes surrounding the bone and tendon tissue in deep-frozen treated BPB xenograft, but the lymphocytes hadnt invaded into the tissues. No tissue necrosis, but a clear tendon structure and regular fibrogen arrangement were observed. There were large amount of fragmentary lymphocytes infiltrating into or surrounding the bone and fibrogen tissues after fresh xenograft, the bone was broken into pieces, the tendon structure was unclear and the fibrogen arrangement was irregular, quite a few of inflammatory cells such as acidophilic granulocytes and macropolycytes were found in some slices. CONCLUSION: Deep-frozen treatment markedly reduces BPB xenograft antigenicity and inhibits the immune rejection.     

Effect of camptothecin on the activation,proliferation and cell cycle distribution of the mouse T lymphocytes in vitro

AIM: To study the effects of camptothecin (CPT) on the activation, proliferation and cell-cycle distribution of the mouse T lymphocytes stimulated by concanvalin A (ConA) in vitro. METHODS: A model of T cell activation and proliferation was established by stimulated the cells with Con A. T cells were treated with different concentrations of CPT. The expression of CD69, the early marker of CD3+ T cell activation, was measured by FACS. The proliferation index was determined by carboxyl fluorescin diacetate succinmidyl ester by flow cytometry. The cell-cycle distribution was analyzed by propidium iodide staining. RESULTS: After stimulation with Con A for 6 h, the activation rate of CD69+ T cell in Con A group was (58.88±0.55)%. The percentages of CD69 positive cells were (55.48±0.98)%, (54.67±1.05)%, (50.40±0.82)%, (42.47±1.32)%, correspond to the treatments with different concentrations of CPT (10 nmol/L, 20 nmol/L, 50 nmol/L, 100 nmol/L), respectively. After 48 h treatment with Con A, the proliferation index in different concentrations of CPT treatment (10 nmol/L, 20 nmol/L, 50 nmol/L and 100 nmol/L) exerted a definite inhibitory effect on the proliferation (P<0.01). Moreover, the cell-cycle distribution analysis showed that apoptosis peak was observed in different concentrations of CPT treatment after 48 h cultured with Con A. CONCLUSION: CPT significantly inhibits the early stages of the Con A-induced T cell activation and proliferation, and detents the T lymphocytes in G₀/G₁ phase.     

Effect of TMB-8 on the activation,proliferation and cell-cycle distribution of the mouse T lymphocytes in vitro

AIM: To study the effects of ［8-(diethylamino) octyl-3, 4, 5 -trimethoxybenzoate］ (TMB-8), an intracellular Ca2+ antagonist, on the activation, proliferation and cell-cycle distribution of the mouse T lymphocytes stimulated by concanvalin A (Con A) in vitro. METHODS: After stimulated with Con A, T cells were treated with different concentrations of TMB-8 alone and its combination with cyclosporine A (CsA). The expression of CD69, the early marker of CD3+ T cell activation, was measured by FACS. The proliferation-related index was determined by carboxyl fluorescin diacetate succinmidyl ester (CFDA-SE) flow cytometry. The cell-cycle distribution was analyzed by propidium iodide staining.RESULTS: After 6 h culture, the activation rate of CD69+ T cell in Con A group was (74.88±1.88)%. 10, 20 and 40 μmol/L of TMB-8 inhibited the expression of CD69 (P<0.01), especially in 40 μmol/L (52.55%±1.54%). After 48 h and 72 h culture, the PI of Con A group was 1.24±0.01, 2.05±0.07, respectively. TMB-8 with the concentration up to 5 μmol/L exerted a definite inhibitory effect on the proliferation with a maximal inhibition in 40 μmol/L(P<0.01). In the combination of 10 μmol/L of TMB-8 with 25 μg/L of CsA, an evident synergistic effect was observed (P<0.01). Moreover, the cell-cycle distribution analysis showed that after 48 h culture, the concentration of TMB-8 over 10 μmol/L showed an evident suppression in S phase.CONCLUSION: TMB-8 significantly inhibites the early steps of the Con A-induced T cell activation and proliferation, as well as the progression of T lymphocytes in S phase.     

Influence of histone deacetylase inhibitor romidepsin on phenotype and function of T lymphocytes in vitro

AIM: To explore the effects of romidepsin (FK228), a novel histone deacetylase inhibitor, on the effector and regulatory T cells in vitro.METHODS: As the reactive cells, lymphocytes, CD4⁺ T cells and CD8⁺ T cells were labelled with CFSE, and stimulated with anti-CD3 and anti-CD28 mAbs in the presence and absence of different levels of romidepsin (experimental group and positive control group), or PBS (placebo group).After 72 h, the proliferation of the cells was detected in different groups. The lymphocytes were stimulated with anti-CD3 and anti-CD28 mAbs in the presence and absence of different levels of romidepsin (experimental group and positive control group),or PBS (placebo group). After 72 h, the percentage of CD4⁺ Foxp3⁺ T cells and the levels of related cytokines were detected in different groups. RESULTS: The proliferation of CFSE-labelled lymphocytes, CD4⁺ T cells and CD8⁺ T cells triggered by anti-CD3 and anti-CD28 mAbs all were inhibited when cultured with romidepsin at concentrations of 1 μmol/L, 3 μmol/L and 5 μmol/L in a dose-dependent manner (P<0.05). Compared with placebo group, in the presence of anti-CD3 and anti-CD28 mAbs, 1 μmol/L romidepsin did not increase the percentage of CD4⁺ Foxp3⁺ T cells (P>0.05). When cultured with romidepsin at concentrations of 3 μmol/L and 5 μmol/L, the percentage of CD4⁺ Foxp3⁺ T cells was enhanced markedly (P<0.05). The levels of IL-10 and TNF-α in the supernatant were markedly increased in positive control group and 3 experimental groups (P<0.05), and the levels of cytokines in different experimental groups were gradually decreased with the elevation of FK228 concentration (P<0.05). The level of TGF-β was slightly increased in positive control group with no significant difference compared with placebo group (P>0.05). With the increase in the concentration of FK228 in different experimental groups, the TGF-β level was increased in a dose-dependent manner and there were significant differences in the 3 experimental groups. Meanwhile, significant differences existed between experimental groups and placebo group and between experimental groups and positive control group (P<0.05). CONCLUSION: Romidepsin inhibits the proliferation of CD4⁺ and CD8⁺ effector T cells and increases the percentage of CD4⁺ Foxp3⁺ regulatory T cells. It may be related to the increased level of TGF-β, but independent of IL-10.     

Analysis of T cell activation and regulatory T cell derived from murine Peyer's patches

AIM: To explore the characteristics of T cell activation and regulatory T cells derived from murine Peyer's patches through comparative studies on Peyer's patches, mesenteric lymph nodes and inguinal lymph nodes. METHODS: Signal cell suspendsions were prepared from murine mesenteric lymph nodes (MLNs), the Peyer's patches (PPs) and inguinal lymph nodes (ILNs), respectively. The percentage of cell subpopulations such as CD3+ T cells, CD3+CD4+ helper T cells and regulatory T cells (Treg, CD4+CD25+) were analyzed. Lymphocytes were activated by polyclonal stimulators such as concanavalin (Con A), phorbol 12, 13-dibutyrate (PDB) only, and PDB plus ionomycin (Ion). The expression of CD69 (the early marker of CD3+ T cell activation) was measured by FACS. RESULTS: A lower ratio of CD3+ T cells was seen in PPs than those in MLNs and ILNs. The ratios of CD3+ CD4+ T cells to CD3+ T cells in PPs, MLNs and ILNs were almost the same. A higher rate of Treg was seen in CD4+ T cells from the PPs as compared with those from MLNs and ILNs. A higher percentage of activated CD3+ T cells derived from the PPs cultured without polyclonal stimulators were detected as compared to MLNs and ILNs, while lower responsiveness of CD3+ T cells from the PPs stimulated by Con A was seen as compared with those from MLNs and ILNs. CONCLUSIONS: The lower rate of CD3+ T cells as well as higher rate of Treg in PPs was due to its desensitization. The higher rate of basic activated state in CD3+ T cells from the PPs indicated that the T cells were activated by enteric antigens in physiological conditions. The lower responsiveness of activation to some polyclonal stimulators probably reveals that the T cells are in a state of anergy. All the characteristics mentioned above contribute to prevent pathological inflammations and maintain tolerance to enteric antigens such as food proteins and commensal bacteria but simultaneously retain proper immune responses to pathogenic microbes.     

Mechanisms underlying the induction of IL-2 secretion by PDB pl us ionomycin in CD4+CD25+ T cells from cord blood and adult peripheral blood

AIM:To confirm that CD4+CD25+ regulato ry T cells don't have an instinctive defection in IL-2 secretion,and to have an insight into the maturation state of CD4+CD25+ T cells in cord blood.METHODS:CD4+CD25+ and CD4+CD25- T cells were purified f rom cord blood of term infants (CB) and adult peripheral blood (PB) by autoMACS,and stimulated with PDB plus ionomycin.After 45 hours of culture,cells were d etected for expression of CD69 and CD25 by flow cytometry,and the supernatants were measured for 7 kinds of cytokines by Luminex.RESULTS:CD4+CD25+ T cells from both CB and PB proliferated comparably with CD4+CD25- T cells when stimulated with PDB plus ionomycin.A fter 45 hours of culture,however,the CD4+CD25+ T cells underwent a tendenc y of cell death.Expression of CD25 was further upregulated when CD25+ cells w ere activated.Under stimulation of PDB plus ionomycin,both CD4+CD25+ and C D4+CD25- T cells in PB secreted high levels of IFN-γ,IL-2 and TNF-α,with CD25+ cells secreted much higher level of IL-5,IL-4 and IL-10 than those in CD25- cells;CD4+CD25+ and CD4+CD25- T cells in CB also secreted high level of IL-2 and TNF-α but much lower level of IFN-γ than those in PB,and no secretion of IL-5,IL-4 and IL-10 was observed.CONCLUSION:CD4+CD25+ regulatory T cells don't have an i nstinctive defection in IL-2 secretion,otherwise there may be a different TCR signaling pattern in CD4+CD25+ T cells from traditional T cells.The CD4+C D25+ T cells in cord blood have not fully matured in function.     

Immunosupressive mechanism of cornus officinalis glycosides on the corneal allograft rejection in rats

AIM: To investigate the influence of the cornus officinalis glycosides (COG) on immunological function of corneal transplantation model of rats, and to clarify the immunosuppressive mechanism of COG through observing the activation of lymphocytes in blood. METHODS: Wister rats were used as recipients and SD rats were used as corneal graft donors, then the corneal allografts transplantation model on the closed colony rats were set up. Splenocytes proliferation and mixed lymphocyte reaction of Wister rats activated by ConA were observed. The phenotype change of CD4, CD8, CD25 in blood in different time postoperatively were observed by the di-sign flow cytometry, and the rate of CD4/CD8 was calculated. RESULTS: 1. The COG suppressed the proliferation of T lymphocytes and one-way mixed lymphocyte reaction on the corneal allografting. 2. The phenotype change of lymphocytes in boold was as follows: there was no significant difference between the different time of the CD4, CD8 expression and the CD4/CD8 rate in blood of the control group. The CD4 positive cells expressed CD25 postoperatively increased obviously. The CD4/CD8 rate of medicine group had the tendency to decrease. The CD4 positive cells expressed CD25 postoperation in the medicine group were less than that in the control group obviously. CONCLUSION: The suppression of the T lymphocyte proliferation, mixed lymphocyte reaction, CD molecule expressed by the activated T lymphocytes and the IL-2 receptor expression may be the main immunosuppressive mechanisms of Cornus officinalis glycosides on the cell-mediated immunity.     

Comparative analysis of cultured endothelial progenitor cells in vitro from PBMCs and enriched CD133+ cells

AIM: To compare the methods of two currently employed isolation methods for endothelial progenitor cells (EPCs): from total peripheral blood mononuclear cells (PBMCs) and from enriched CD133+ cells, by defining the cell morphology, phenotype, reproductive activities and function in vitro, providing a reference for clinic application. METHODS: PBMCs from the healthy subjects were used for CD133+ sorting or not. The two groups of isolated cells were suspended in complete medium M199 for 7 d to 14 d. EPCs phenotype were characterized by FACS. The proliferation of differentiated EPCs was studied by MTT assay, and VEGF concentration was measured using an ELISA kit. Matrigel experiment and migration assay were imitated vascularization in vivo. RESULTS: PBMCs produced more colony-forming units (CFU) than CD133+ cells from the same volume of blood (P<0.01). From 7 d to 14 d, the two groups show decreased expression of hematopoietic stem cell markers and increased level of endothelial markers, but CD144+ cells in CD133+ group were lower than those in PBMCs groups (P<0.01). Cells in PBMCs group secreted more VEGF than that in CD133+ group on 7 d (P<0.01). Compared to CD133+ group, PBMCs group showed more potential of proliferation and vascularization in vitro. CONCLUSION: CD133+ sorted cells show a lower capacity of differentiation, secretion, proliferation and vascularization in vitro, which is unable to differentiate to mature endothelial cells, indicating that its not a preferential way to obtain EPCs for clinic therapy.     

Roles of dendritic cells treated with 17β-estradiol in immune tolerance induction in skin allograft

AIM: To study the roles of bone marrow-derived dendritic cells from donor mouse treated with 17β-estradiol (E₂) in immune tolerance induction in skin allograft. METHODS: Bone marrow-derived dendritic cells from C57 mouse as donor were cultured respectively treated with E₂ (E₂ group). BALB/c mouse as recipient received respectively one injection of dendritic cells of E₂ group, mature dendritic cell group and immature dendritic cell group intravenously. Skin transplantation was performed in the absence of immunosupression after 7 d. Mice that received PBS were served as control. The time of skin survival was observed after transplantation. Flow cytometry was used to analyze the percentage of CD4+CD25+ T cells in peripheral blood respectively before and after transplantation. RESULTS: Compared with immature dendritic cells and control group, the time of skin survival in E₂ group was significantly longer (P<0.01), especially, the time of skin survival still prolonged 10.6 d after skin rejection in immature dendritic group. The percentage of CD4+CD25+ regulatory T cells in E₂ group was significantly higher than that in immature dendritic cell group and control group (P<0.01). CONCLUSION: In skin allograft model, dendritic cells treated with E₂ prolong the allograft survival time.     

Correlation between CD4+CD28-T cells and lymphocytic apoptosis in rheumatoid arthritis patients

AIM: To investigate the fraction of CD4⁺CD28^-T cells and its correlation with lymphocytic apoptosis in peripheral blood of rheumatoid arthritis (RA) patients.METHODS: The RA patients and age-matched health controls were selected in the study. The lymphocytes were isolated from peripheral blood. CD4⁺ T cells without CD28 expression (CD4⁺ CD28^-) were analyzed by flow cytometry. The incidence of apoptosis in the cells cultured with or without PHA for 24 h was determined by flow cytometry with Annexin V-FITC/PI di-staining. The correlation between the fraction of CD4⁺CD28^-T cells and lymphocytic apoptosis was also observed.RESULTS: The fraction of CD4⁺CD28^-T cells was significantly higher in RA group than that in healthy control group (7.79%±3.52% vs 1.89%±1.78%, P<0.05). The apoptotic level in PHA cultured lymphocytes was significantly lower in RA group than that in healthy controls (11.38%±5.73% vs 19.46%±6.32％, P<0.05). Spearman analysis showed that there was a negative correlation between the fraction of CD4⁺CD28^-T cells and apoptotic level of activated lymphocytes (r=-0.433,P<0.01).CONCLUSION: The increased CD4⁺CD28^-T cells contribute to prolong the lifespan of activated lymphocytes in peripheral blood of RA patients, and the persistence of activated lymphocytes may contribute to the pathogenesis of RA.     

Effects of magnesium on apoptosis and expression of Foxp3 in peripheral CD4+CD25+ regulatory T cells from asthma patients

AIM:To observe the apoptosis and the expression of forkhead box protein 3(Foxp3) induced by magnesium in CD4⁺CD25⁺ regulatory T cells isolated from healthy and asthmatic human peripheral blood. METHODS:Peripheral blood from healthy volunteers and asthma patients was collected. CD4⁺CD25⁺ T cells were separated by Percoll centrifugation and magnetic separation. The cells were cultured for 72 h and treated with magnesium(10 mmol/L) or control solution. The apoptotic rate and the expression of Foxp3 in the cells were analyzed by flow cytometry. RESULTS:The purity of CD4⁺CD25⁺T cells was 77.4%~92.3% in health group, and was 75.2％~93.8％in asthma group. The proportion of CD4⁺CD25⁺ T cells in CD4⁺T cells was 4.12%~7.98% in healthy adults, and 4.51%~8.68% in asthma patients. No significant difference between the 2 groups was observed. Magnesium at concentration of 10 mmol/L up-regulated the apoptotic rate of CD4⁺CD25⁺ T cells(P<0.05) and did not affect the Foxp3 expression in the cells in both health and asthma groups. CONCLUSION:Magnesium plays therapeutic effects on asthma by inducing the apoptosis of peripheral CD4⁺CD25⁺ regulatory T cells.     

Distribution and identification of immunocytes in humanized SCID mouse model

AIM: Humanized-NOD/SCID(hu-NOD/SCID) mouse model was established and the level of immune reconstitution was assessed in this model. METHODS: Mononuclear cells (MNC) and CD34⁺ cells were isolated or sorted from cord blood(CB). Human CD45, CD19, CD3 markers on cells from NOD/SCID murine peripheral blood(PB), bone marrow(BM), thymus were detected by FCM from 4 to 10 weeks after hematopoietic stem cell transplantation. After 10 weeks, the gene expressions of the human β₂M and RAG2 were detected by RT-PCR in PB or bone marrow of mice model. RESULTS: Human CD45, CD19, CD3 cells populations in PB and BM were found by flow cytometry in mice model transplanted with CD34+ cells or CB MNC from 4 to 10 weeks. The highest positivity of human lymphocytes was at 8 week after transplantation. The levels of human cell engraftment in mice transplanted with CD34⁺ cells were higher than those in mice transplanted with CB MNC. The mRNA of human β₂M and RAG2 were found by RT-PCR in BM.CONCLUSION: The higher level of human lymphocyte engraftment is established in NOD/SCID mouse model transplanted with CD34⁺ compared with CB MNC. The maturation of T lymphocytes could be happened in bone marrow of mice model.     

Suppression of allergic airway inflammation in a mouse model by Der p2 recombined BCG-induced adaptive CD4+CD25+ Treg

AIM: The bacillus calmette-guerin (BCG) vaccine is the most widely used Th1-inducing vaccine. In recent years, some studies argued that mycobacterium vaccae can be used as adjuvant to induce regulatory T cells (Treg) and then suppress asthmatic airway inflammation. We previously have engineered recombined BCG that expressed Der p2 of house dust mites (Der p2 rBCG) on the cell wall. The aim of this study is to investigate the immune regulatory mechanisms of Der p2 rBCG. METHODS: Mice were vaccined with PS, BCG or rBCG. The relative proportion and the absolute numbers of related Tregs in spleen cells were analyzed. The suppressive activity of Der p2 rBCG-induced CD4+CD25+ T cells was detected both in vitro and in vivo. RESULTS: (1) Der p2 rBCG induced a CD4+CD25+Foxp3+ T cell subtype. (2) Der p2 rBCG-induced CD4+CD25+ T cells suppressed the proliferation of Th2 effector cells in vitro in an antigen-specific way. (3) Der p2 rBCG-induced CD4+CD25+ T cells mediated Der p2 specific suppression of airway allergy in vivo. CONCLUSION: Der p2 rBCG induces a CD4+CD25+Foxp3+ T cell subtype, which suppresses inflammation in allergic airway in a mouse model.