Post-thymectomy autoimmune gastritis: fine specificity and pathogenicity of anti-H/K ATPase-reactive T cells

Thymectomy at day 3 of life (d3Tx) results in the development of organ-specific autoimmunity. We have recently shown that d3Tx BALB/c mice which develop autoimmune gastritis contain CD4+ T cells specific for the gastric parietal cell proton pump, H/K ATPase. Here, we demonstrate that freshly explanted gastric lymph node (LN) cells from d3Tx mice react significantly to the H/K ATPase alpha chain, but only marginally to the beta chain. Two H/K ATPase-reactive T cell lines were derived from the gastric LN of d3Tx mice. Both are CD4+, TCR alpha/beta-, and I-Ad restricted, and recognize distinct peptides from the H/K ATPase alpha chain. One cell line secretes Th1 and the other Th2 cytokines, but both are equally potent in inducing gastritis with distinct profiles of cellular infiltration in nu/nu recipient animals. Neither of the cell lines induced disease in normal BALB/c recipients and transfer of disease to nu/nu recipients was blocked by co-transfer of normal BALB/c spleen cells containing CD4+ CD25+ cells. Although CD4+ CD25+ T cells are thought to emigrate from the thymus after day 3 of life, they could be identified in LN of 2-day-old animals. The capacity of CD4+ CD25+ T cells to abrogate the pathogenic activity in vivo of both activated Th1/Th2 lines strongly suggests that this suppressor T cell population may have a therapeutic role in other models of established autoimmunity. The availability of well-characterized lines of autoantigen-specific T cells should greatly facilitate the analysis of the mechanism of action and target of the CD4+ CD25+ immunoregulatory cells.     

Wild-type and interleukin-10-deficient regulatory T cells reduce effector T-cell-mediated gastroduodenitis in Rag2-/- mice, but only wild-type regulatory T cells suppress Helicobacter pylori gastritis

CD4(+) CD45RB(hi) CD25(-) effector T cells (T(E)) promote Helicobacter pylori gastritis in mice, and CD4(+) CD45RB(lo) CD25(+) regulatory T cells (T(R)) are anti-inflammatory. Using adoptive transfer into H. pylori-infected Rag2(-/-) mice, we evaluated effects of wild-type (wt) C57BL/6 or congenic interleukin-10-deficient (IL-10(-/-)) T(R) cells on gastritis, gastric cytokines, and H. pylori colonization. Infected Rag2(-/-) mice colonized in the corpus and antrum with 10(5) to 10(6) H. pylori CFU/gram without associated gastritis. T(E) cell transfer caused morbidity and an H. pylori-independent pangastritis and duodenitis (gastroduodenitis) associated with increased expression of gamma interferon (IFN-gamma) and tumor necrosis factor alpha. T(E) cell transfer to H. pylori-infected mice led to additive corpus gastritis associated with inflammatory cytokine expression and reduced colonization. wt T(R) cells reduced morbidity, H. pylori corpus gastritis, gastroduodenitis, and inflammatory cytokine expression and reversed the decline in H. pylori colonization attributable to T(E) cells. Although less effective than wt T(R) cells, IL-10(-/-) T(R) cells also reduced morbidity and gastroduodenitis but did not reduce H. pylori corpus gastritis or impact T(E) cell inhibition of colonization. Gastric tissues from mice receiving wt T(R) cells expressed higher levels of Foxp3 compared to recipients of IL-10(-/-) T(R) cells, consistent with lower regulatory activity of IL-10(-/-) T(R) cells. These results demonstrate that wt T(R) cells suppressed T(E)-cell-mediated H. pylori-independent gastroduodenitis and H. pylori-dependent corpus gastritis more effectively than IL-10(-/-) T(R) cells. Compartmental differences in T(E)-cell- and H. pylori-mediated inflammation and in regulatory effects between wt T(R) and IL-10(-/-) T(R) cells suggest that IL-10 expression by wt T(R) cells is important to regulatory suppression of gastric inflammation.     

CD4+ and CD8+ T cell responses in Helicobacter pylori-infected individuals

In order to characterize T cell responses in human Helicobacter pylori infection, we have examined proliferative responses and cytokine production by CD4+ and CD8+ T cells isolated from duodenal ulcer patients and asymptomatic H. pylori carriers, after activation with some H. pylori antigens that may be important in disease development. For control purposes, T cells from uninfected volunteers were also examined. The different H. pylori antigens induced only modest proliferative responses in circulating CD4+ and CD8+ T cells from both H. pylori-infected and uninfected individuals. However, circulating T cells from H. pylori-infected subjects produced larger amounts of interferon-gamma (IFN-gamma) in response to the Helicobacter antigens than did T cells from uninfected volunteers. Furthermore, CD8+ T cells produced larger amounts of IFN-gamma than did CD4+ T cells, on a per cell basis. Most IFN-gamma-producing cells from both infected and uninfected volunteers appeared to be naive T cells expressing CD45RA. Increased production of IL-4 and IL-5 was, on the other hand, only seen in a few instances after stimulation of isolated CD4+ and CD8+ T cells. Stimulation of freshly isolated gastric T cells with the different H. pylori antigens did not result in increased proliferation or cytokine production. In conclusion, our results show that several different purified H. pylori antigens induce production of IFN-gamma, preferentially by CD8+ cells. Therefore, they suggest that IFN-gamma-secreting CD8+ cells contribute significantly to the cytokine response induced by H. pylori infection.     

CD4+ CD25+ regulatory T cells modulate the T-cell and antibody responses in helicobacter-infected BALB/c mice

Gastric Helicobacter spp. induce chronic gastritis that may lead to ulceration and dysplasia. The host elicits a T helper 1 (Th1) response that is fundamental to the pathogenesis of these bacteria. We analyzed immune responses in Helicobacter-infected, normal mice depleted of CD4+ CD25+ T cells to investigate the in vivo role of regulatory T cells (Tregs) in the modulation of Helicobacter immunopathology. BALB/c and transgenic mice were depleted of CD4+ CD25+ T cells by administration of an anti-CD25 antibody either at the time of infection with Helicobacter or during chronic infection and gastritis. Depletion of CD25+ Tregs prior to and during infection of mice with Helicobacter spp. did not affect either bacterial colonization or severity of gastritis. Depletion of CD25+ Tregs was associated with increased Helicobacter-specific antibody levels and an altered isotype distribution. Paragastric lymph node cells from CD25+ Treg-depleted and control infected mice showed similar proliferation to Helicobacter antigens, but only cells from anti-CD25-treated animals secreted Th2 cytokines. CD25+ Tregs do not control the level of gastritis induced by gastric Helicobacter spp. in normal, thymus-intact BALB/c mice. However, CD25+ Tregs influence the cytokine and antibody responses induced by infection. Autoimmune gastritis is not induced in Helicobacter-infected mice depleted of CD25+ Tregs but is induced in CD25+ Treg-depleted mice, which have a higher frequency of autoreactive T cells.     

Cure of innate intestinal immune pathology by CD4+CD25+ regulatory T cells

CD4+CD25+ regulatory T (T(R)) cells are a naturally occurring population of T cells that suppress the development of a variety of pathological immune responses. However, as human inflammatory diseases are usually not diagnosed until after the onset of clinical symptoms, it is of great interest to determine whether CD4+CD25+ T(R) cells can reverse established pathology. To examine this question we have utilized a murine model of human inflammatory bowel disease (IBD), where pathology is triggered by infection of immune deficient RAG-/- mice with the pathogenic bacterium Helicobacter hepaticus. Here we demonstrate that adoptively transferred CD4+CD25+ T(R) cells can cure established intestinal inflammation that is mediated by innate immune activation in H. hepaticus-infected RAG-/- mice. CD4+CD25+ T(R) cell-mediated amelioration of innate intestinal pathology was accompanied by a reversal in systemic innate immune activation, but did not involve any detectable anti-bacterial effects, as bacterial colonization levels were unchanged. Cure of established pathology was not achieved using subpopulations of CD4+CD25- T cells, further emphasizing the enhanced regulatory activity of CD4+CD25+ T(R) cells.     

CD4+ T cells, but not CD8+ T cells, are required for the development of experimental autoimmune gastritis.

Murine autoimmune gastritis, induced by neonatal thymectomy, is characterized by a mononuclear infiltrate within the gastric mucosa, loss of parietal and zymogenic cells and circulating autoantibodies to the gastric H/K ATPase. The infiltrate contains both CD4+ and CD8+ T cells. Here we have investigated the roles of CD4+ and CD8+ T cells in the development of gastritis by in vivo treatment with depleting rat anti-CD4 and anti-CD8 monoclonal antibodies. Depletion of CD4+ T cells decreased the incidence of gastric mononuclear infiltrates from 63% (5/8), observed in normal rat immunoglobulin G (IgG)-injected mice, to 8% (1/12) and also abolished the production of antigastric autoantibodies. In contrast, depletion of CD8+ T cells did not reduce the incidence of gastritis. The absence of CD8+ T cells in the infiltrate of the stomach of anti-CD8(+)-treated mice was confirmed by immunocytochemistry. These results argue that neonatal thymectomy-induced autoimmune gastritis is mediated by CD4+ T cells and that CD8+ T cells do not play a significant role in the development of the gastric lesion.     

Antigen-specific in vitro suppression of murine Helicobacter pylori-reactive immunopathological T cells by CD4CD25 regulatory T cells

A Helicobacter pylori-specific in vitro coculture system was established and used to study the role of CD4+CD25+ regulatory T cells (Treg) in gastritis development in mice with H. pylori infection. Effects of therapeutic immunization against H. pylori infection on the Treg function were also studied to better understand the mechanisms leading to postimmunization gastritis in these mice. Depletion of Treg led to extensive proliferation to H. pylori antigens of CD4+ T cells isolated from either na?ve, H. pylori-infected or H. pylori-immunized mice. Using the Treg-depleted CD4+ T cells from immunized mice as effector cells, we compared the suppressive efficacy of Treg isolated from na?ve, infected or immunized mice and found that Treg from na?ve mice, and slightly less efficiently from infected mice, suppressed the CD25- effector T-cell response and in most cases were distinctly more efficacious than Treg isolated from immunized mice. The suppressive efficacy of Treg isolated from the differently treated mice correlated closely with production of interleukin-5 (IL-5) by the Treg and suppression of interferon-gamma and IL-2 production by the CD25- effector T cells. Our study is the first to demonstrate in H. pylori-induced chronic infection, antigen-specific Treg with differential efficacy in suppressing H. pylori proinflammatory T effector cells.     

Resistance of primary murine CD4+ T cells to Helicobacter pylori vacuolating cytotoxin

Persistent colonization of the human stomach by Helicobacter pylori is a risk factor for the development of gastric cancer and peptic ulcer disease. H. pylori secretes a toxin, VacA, that targets human gastric epithelial cells and T lymphocytes and enhances the ability of H. pylori to colonize the stomach in a mouse model. To examine how VacA contributes to H. pylori colonization of the mouse stomach, we investigated whether murine T lymphocytes were susceptible to VacA activity. VacA inhibited interleukin-2 (IL-2) production by a murine T-cell line (LBRM-33), similar to its effects on a human T-cell line (Jurkat), but did not inhibit IL-2 production by primary murine splenocytes or CD4+ T cells. VacA inhibited activation-induced proliferation of primary human CD4+ T cells but did not inhibit the proliferation of primary murine CD4+ T cells. Flow cytometry studies indicated that the levels of VacA binding to primary murine CD4+ T cells were significantly lower than levels of VacA binding to human CD4+ T cells. This suggests that the resistance of primary murine CD4+ T cells to VacA is attributable, at least in part, to impaired VacA binding to these cells.     

B cells pulsed with Helicobacter pylori antigen efficiently activate memory CD8+ T cells from H. pylori-infected individuals

Helicobacter pylori infection causes chronic gastritis that may progress to peptic ulcers or gastric adenocarcinoma and thereby cause major world-wide health problems. Previous studies have shown that CD4+ T cells are important in the immune response to H. pylori in humans, but the role of CD8+ T cells is less clear. In order to study the CD8+ T cell response to H. pylori in greater detail, we have evaluated efficient conditions for activation of CD8+ T cells in vitro. We show that H. pylori-reactive CD8+ T cells can be activated most efficiently by B cells or dendritic cells pulsed with H. pylori antigens. We further show that the majority of CD8+ T cells in H. pylori-infected gastric mucosa are memory cells, and that memory CD8+ T cells sorted from peripheral blood of H. pylori-infected individuals respond 15-fold more to H. pylori urease compared to memory cells from uninfected subjects. We conclude that CD8+ T cells do participate in the immune response to H. pylori, and this may have implications for the development of more severe disease outcomes in H. pylori-infected subjects.     

Differential cytokine requirements for regulation of autoimmune gastritis and colitis by CD4(+)CD25(+) T cells

Murine autoimmune gastritis, induced by neonatal thymectomy or the injection of CD25-depleted lymphocytes into nu/nu recipients, is characterized by an inflammatory infiltrate into the gastric mucosa, parietal cell destruction and circulating anti-parietal cell antibodies. Using RAG-2(-/-)mice as recipients, we determined that the induction of disease relies on CD4(+)CD25(-)effector cells and prevention relies on CD4(+)CD25(+)regulatory cells; neither requires participation of CD8 cells or B cells. The severity of gastritis was dependent on the cytokine repertoire of CD4(+)CD25(-)effector T cells. Recipients of IL-4(-/-)T cells developed more severe gastritis and recipients of INF-gamma(-/-)T cells developed milder disease than recipients of wildtype or IL-10(-/-)effector T cells. Gastritis did not develop in the absence of IL-12. Protection from gastritis does not require either IL-4 or IL-10 because CD4(+)CD25(+)cells from IL-4(-/-)or IL-10(-/-)mice completely abrogated the disease process. CD4(+)CD25(+)cells also protected RAG-2(-/-)recipients from colitis and inhibitory activity was partially dependent on IL-10 expression. These findings highlight the critical role of CD4(+)CD25(+)regulatory T cells in protection from several autoimmune syndromes and delineate the differential contribution of IL-10 to CD4(+)CD25(+)Treg activity in the settings of gastritis and colitis.     

Helicobacter pylori-specific CD4+ CD25high regulatory T cells suppress memory T-cell responses to H. pylori in infected individuals

Helicobacter pylori colonizes the gastric and duodenal mucosa. The infection normally persists for life and causes peptic ulcers and gastric cancer in a subset of infected individuals. We hypothesized that the inability to clear the infection may be a consequence of H. pylori-specific regulatory T cells that actively suppress T-cell responses. Therefore, we characterized the T-cell responses to H. pylori in H. pylori-infected individuals without any subjective symptoms and in uninfected control subjects and investigated the role of regulatory CD4+ CD25(high) T cells during infection. The stimulation of CD4+ peripheral blood T cells with monocyte-derived dendritic cells pulsed with a membrane preparation of H. pylori resulted in proliferation and gamma interferon production in both infected and uninfected individuals. Sorted memory cells from infected individuals responded less than cells from uninfected subjects, and the unresponsiveness could be abolished by depletion of CD4+ CD25(high) regulatory T cells or the addition of interleukin 2. Furthermore, CD4+ CD25(high) T cells suppressed H. pylori-induced responses in cocultures with CD25(low/-) cells. Tetanus toxoid induced comparable responses in memory cells from infected and uninfected individuals in both the presence and the absence of regulatory T cells, suggesting that the suppression was H. pylori specific. In conclusion, we have shown that H. pylori-infected individuals have impaired memory CD4+ T-cell responses to H. pylori that are linked to the presence of H. pylori-specific regulatory T cells that actively suppress the responses.     

Critical role of the liver CD8+ CD122+ T cells in the generalized Shwartzman reaction of mice

We examined the role of mouse CD8+ CD122+ T cells, which increase in number with age, in the generalized Shwartzman reaction. This reaction was induced by IL-12 priming and subsequent LPS challenge (after 24 h) in mice of various ages (4-50 weeks of age). Although most young mice (4 or 6 weeks of age) survived, mortality essentially increased with increasing age of the mice, and all mice of 20 weeks of age or older died within 48 h. Serum TNF-alpha levels after LPS challenge also increased age dependently. The neutralization of either IL-12-induced IFN-gamma or LPS-induced TNF-alpha improved the survival of middle-aged (25-week-old) mice. Both IFN-gamma production after IL-12 priming and TNF-alpha production from the liver mononuclear cells after LPS challenge were also prominent in the middle-aged mice. CD8+CD122+ T cells cultured with IL-12 produced a much larger amount of IFN-gamma than CD8+CD122- T cells. Although the depletion of NK/NK T cells did not decrease the IFN-gamma or TNF-alpha production in the Shwartzman reaction of the middle-aged mice, an additional depletion of CD8+CD122+ T cells did decrease such production and also improved mouse survival. Furthermore, young mice transferred with CD8+CD122+ T cells from aged B6 nude mice showed an enhanced Shwartzman reaction.     

Regulatory function of CD4+CD25+ T cells from Class II MHC-deficient mice in contact hypersensitivity responses

Contact hypersensitivity (CHS) is a CD8+ T cell-mediated, inflammatory response to hapten sensitization and challenge of the skin. During sensitization, the magnitude and duration of hapten-specific CD8+ T cell expansion in the skin-draining lymph nodes (LN) are restricted by CD4+CD25+ T regulatory cells (Treg). The regulation of hapten-specific CD8+ T cell priming in Class II MHC-deficient (MHC-/-) mice was investigated. Although hapten-specific CD8+ T cell priming and CHS responses were elevated in Class II MHC-/- versus wild-type mice, presensitization depletion of CD4+ or CD25+ cells in Class II MHC-/- mice further increased CD8+ T cell priming and the elicited CHS response. Flow cytometry analyses of LN cells from Class II MHC-/- mice revealed a population of CD4+ T cells with a majority expressing CD25. Forkhead box p3 mRNA was expressed in LN cells from Class II MHC-/- and was reduced to background levels by depletion of CD4+ or CD25+ cells. Isolated CD4+CD25+ T cells from wild-type and Class II MHC-/- mice limited in vitro proliferation of alloantigen- and hapten-specific T cells to antigen-presenting stimulator cells. These results identify functional CD4+CD25+ Treg in Class II MHC-/- mice, which restrict hapten-specific CD8+ T cell priming and the magnitude of CHS responses.     

Adoptive transfer of CD4+ T cells specific for subunit A of Helicobacter pylori urease reduces H. pylori stomach colonization in mice in the absence of interleukin-4 (IL-4)/IL-13 receptor signaling

Protection in the murine model of Helicobacter pylori infection may be mediated by CD4+ T cells, but the mechanism remains unclear. To better understand how protection occurs in this model, we generated and characterized H. pylori urease-specific CD4+ T cells from BALB/c mice immunized with Salmonella enterica serovar Typhimurium expressing H. pylori urease (subunits A and B). The CD4+ T cells were found to be specific for subunit A (UreA). Upon antigen-specific stimulation, expression of interleukin 4 (IL-4), IL-10, gamma interferon (IFN-gamma), and tumor necrosis factor alpha was induced. Immunocytochemical analysis showed that the majority of cells produced IFN-gamma and IL-10. Adoptive transfer of the UreA-specific CD4+ T cells into naive syngeneic recipients led to a threefold reduction in the number of bacteria in the recipient group when compared to that in the nonrecipient group. Stomach colonization was also reduced significantly after transfer of these cells into patently infected mice. Adoptive transfer of UreA-specific CD4+ T cells into IL-4 receptor alpha chain-deficient BALB/c mice indicated that IL-4 and IL-13 were not critical in the control of bacterial load. In addition, synthetic peptides were used to identify three functional T-cell epitopes present in subunit A which were recognized by the UreA-specific T cells. Analysis of H. pylori-specific cellular immune responses in recipient challenged and nonrecipient infected mice indicated a strong local restriction of the response in infected animals. The implications of these findings for the mechanism of protection and the development of peptide-based vaccination are discussed.     

The inflammatory response in CD1 mice shortly after infection with a CagA+/VacA+ Helicobacter pylori strain

To investigate the early events of Helicobacter pylori infection in a mouse model, CD1 mice were infected with a type I (CagA+/VacA+) H. pylori strain. Up to 4 weeks after infection the majority of gastric tissue biopsies were positive in culture. Immunohistochemical analysis showed that inflammatory changes started to occur after 3 weeks. Four weeks after infection a significant increase in T cells was observed in the cardia/corpus region of the stomachs of infected mice. These T cells were CD4+ and CD8+, and they were located in an area with increased expression of MHC class II antigens. In 50% of the infected mice also an increased number of mast cells was seen. Furthermore, aggregates of B and T cells were present in the submucosa. Characterization of cytokines by immunohistochemistry showed an increase in IL-5-secreting cells in the inflamed area of the infected stomach. No difference was observed between interferon-gamma (IFN-gamma)-, IL-4- and IL-10-secreting cells in control and infected mice. These results suggest that no polarized T-helper cell response was present at this early phase of infection. Infection with H. pylori also induced a serum response and especially IgG was increased after 4 weeks of infection. However, no particular increase in IgG1, IgG2a or IgG3 isotype was observed. Part of the serum antibodies was directed against lipopolysaccharide (LPS), but no evidence for anti-Lewis antibodies or antibodies against epitopes on the gastric mucosa was found.     

Murine Splenocytes Induce Severe Gastritis and Delayed-Type Hypersensitivity and Suppress Bacterial Colonization in Helicobacter pylori-Infected SCID Mice

The goal of this study was to evaluate the role of host immunity in gastritis and epithelial damage due to Helicobacter pylori. Splenocytes from H. pylori-infected and uninfected C57BL/6 mice were adoptively transferred to H. pylori-infected and uninfected severe combined immunodeficient (SCID) mice. Transfer was verified by flow cytometry, and all mice were evaluated for the presence of delayed-type hypersensitivity (DTH) by footpad inoculation with sterile H. pylori sonicate and for humoral immunity by enzyme-linked immunosorbent assay. The severity of gastritis and gastric epithelial damage was quantified histologically, epithelial proliferation was determined by proliferating cell nuclear antigen staining, and colonization was quantified by culture. C57BL/6 mice, but not nonrecipient SCID mice, developed moderate gastritis in response to H. pylori. In contrast, recipient SCID mice developed severe gastritis involving 50 to 100% of the gastric mucosa and strong DTH responses not present in C57BL/6 mice. DTH, but not serum anti-H. pylori immunoglobulin G, correlated with adoptive transfer, gastritis, and bacterial clearance. Severe gastritis, but not bacterial colonization, was associated with epithelial metaplasia, erosions, and an elevated labeling index. This study demonstrates that (i) adaptive immunity is essential for development of gastritis due to H. pylori in mice, (ii) T-cell-enriched lymphocytes in SCID mice induce DTH and gastritis, which is more severe than donor gastritis, and (iii) the host inflammatory response, not direct bacterial contact, causes epithelial damage. The greater severity of gastritis in recipient SCID mice than in donor C57BL/6 mice suggests that gastritis is due to specific T-cell subsets and/or the absence of regulatory cell subsets in the transferred splenocytes.     

Role of CD4+ lymphocytes in resistance to mucosal candidiasis.

The role of CD4+ lymphocytes in resistance of N:NIH(S) III bg/bg nu/+ mice to mucosal candidiasis was evaluated. Alimentary tract colonization with a pure culture of Candida albicans induced a population of lymphocytes in both the Peyer's patches and spleens of bg/bg nu/+ mice, but not bg/bg nu/nu mice, that proliferated and produced interleukin-2 (IL-2) in response to C. albicans antigens. The induction of candida-specific lymphocytes correlated with the clearance of C. albicans from the esophagus and tongue of resistant bg/bg nu/+ mice. Isogenic bg/bg nu/nu mice which do not develop candida-reactive lymphocytes were unable to clear C. albicans from their tongues and esophagi. Treatment of bg/bg nu/+ mice with anti-CD4+ monoclonal antibodies depleted their CD4+ lymphocytes and increased their susceptibility to mucosal candidiasis of the tongue and esophagus. In vivo treatment of bg/bg nu/+ mice with anti-IL-2, anti-gamma interferon (IFN-gamma), or both anti-IL-2 and anti-IFN-gamma monoclonal antibodies did not abrogate their resistance to mucosal candidiasis. Furthermore, treatment of C. albicans-susceptible bg/bg nu/nu mice with IFN-gamma and IL-2 did not protect them from mucosal candidiasis. Thus, CD4+ cells apparently play a critical role in resistance to mucosal candidiasis; however, we were unable to demonstrate a role for IL-2 and IFN-gamma in mediating resistance to mucosal candidiasis.     

胃癌患者PBMCs中CD4+CD25+T细胞体外增殖及对CD4+CD25-T细胞增殖的影响

目的:探讨胃癌患者外周血单个核细胞（PBMCs）中的CD4+CD25+T细胞体外增殖及对CD4+CD25-T细胞增殖的影响。 方法:以免疫磁性分离方法 （MACS）分选出胃癌患者外周血单个核细胞中的CD4+CD25+T及CD4+CD25-T细胞后，用流式细胞仪分析细胞的纯度及活力；再以小鼠抗人CD3单抗、小鼠抗人CD28单抗及rh IL-2作为共刺激因子，观察与CD4+CD25-T细胞共培养时，CD4+CD25+T细胞对CD4+CD25-T细胞增殖的抑制效应。 结果:（1）分选后健康对照组及胃癌患者PBMC 中CD4+CD25+ T细胞纯度分别为83.8%±1.84%、84.13%±2.77％，两者相比，无显著差异（P＞0.05）;(2)经MACS 分选后正常对照组与胃癌患者CD4+CD25+ T细胞活力分别为98.52%±0.72%、97.80%±0.95%，两者相比，无显著差异（P＞0.05）；(3)无论是健康对照还是胃癌患者的CD4+CD25+T均具有明显抑制效应性T细胞如CD4+CD25-T细胞的增殖，随着CD4+CD25+T细胞数的增加，这种抑制增殖的能力也相应增加，当CD4+CD25+∶〖KG-*2〗CD4+CD25-T达 1∶〖KG-*2〗1时，抑制率最大达到50％。 结论:MACS分选法能够分选出高纯度及活力的CD4+CD25+T细胞，分选后CD4+CD25+T细胞在体外均能抑制CD4+CD25-T细胞增殖，且这种抑制效应呈一定效靶比关系。     

IL-10 is involved in the suppression of experimental autoimmune encephalomyelitis by CD25+CD4+ regulatory T cells

CD25(+)CD4(+) regulatory T cells inhibit the activation of autoreactive T cells in vitro and in vivo, and suppress organ-specific autoimmune diseases. The mechanism of CD25(+)CD4(+) T cells in the regulation of experimental autoimmune encephalomyelitis (EAE) is poorly understood. To assess the role of CD25(+)CD4(+) T cells in EAE, SJL mice were immunized with myelin proteolipid protein (PLP)(139-151) to develop EAE and were treated with anti-CD25 mAb. Treatment with anti-CD25 antibody following immunization resulted in a significant enhancement of EAE disease severity and mortality. There was increased inflammation in the central nervous system (CNS) of anti-CD25 mAb-treated mice. Anti-CD25 antibody treatment caused a decrease in the percentage of CD25(+)CD4(+) T cells in blood, peripheral lymph node (LN) and spleen associated with increased production of IFN-gamma and a decrease in IL-10 production by LN cells stimulated with PLP(130-151) in vitro. In addition, transfer of CD25(+)CD4(+) regulatory T cells from naive SJL mice decreased the severity of active EAE. In vitro, anti-CD3-stimulated CD25(+)CD4(+) T cells from naive SJL mice secreted IL-10 and IL-10 soluble receptor (sR) partially reversed the in vitro suppressive activity of CD25(+)CD4(+) T cells. CD25(+)CD4(+) T cells from IL-10-deficient mice were unable to suppress active EAE. These findings demonstrate that CD25(+)CD4(+) T cells suppress pathogenic autoreactive T cells in actively induced EAE and suggest they may play an important natural regulatory function in controlling CNS autoimmune disease through a mechanism that involves IL-10.