Extracorporeal photochemotherapy is accompanied by increasing levels of circulating CD4+CD25+GITR+Foxp3+CD62L+ functional regulatory T-cells in patients with graft-versus-host disease

BACKGROUND: Extracorporeal photochemotherapy (ECP) produces clinical improvements in refractory/resistant graft-versus-host disease (GvHD). Immunological mechanisms of ECP are still under investigation. METHODS: We have evaluated the changes in frequency and immunophenotype of circulating regulatory T cells (T-regs) in 10 patients undergoing allogeneic hematopoietic stem cell transplantation, receiving ECP for acute (n=4) or chronic (n=6) GvHD. T-regs were monitored for expression of surface CD4, CD25, GITR, CD45RO, CD62L and intracytoplasmic Foxp3. T-regs were sorted by fluorescence-activated cell sorting to perform functional assays by interferon (IFN)-gamma enzyme-linked immunospot and real-time quantitative polymerase chain reaction (RQ-PCR) to measure Foxp3, transforming growth factor (TGF)-beta, and interleukin (IL)-10 mRNA. RESULTS: ECP was accompanied by a significant increase of CD4+CD25+ T-regs after six procedures, increasing from 8.9% to 29.1% of total CD4 (P<0.05), with a simultaneous increase of glucocorticoid induced tumor necrosis factor receptor expression on CD4+CD25+ cells (from 15% to 40.8%, P<0.05). This increase was sustained after 12 procedures. T-regs expressed high levels of CD62L, CD45RO, and Foxp3. Sorted CD4+CD25+ T-regs were potently inhibitory toward the CD4+CD25- fraction, when matched with an allogeneic target (IFN-gamma secretion was reduced by 79%). Trans-well experiments showed that cell-to-cell contact was necessary to exert inhibitory activity. RQ-PCR revealed a significant expression of Foxp3 in CD4+CD25+ T-regs, but there was virtually no detection of TGF-beta and IL-10. GvHD improved in all patients, allowing tapering or discontinuation of immunosuppressive drugs. CONCLUSION: Our study shows a time correlation between ECP and increasing percentages of circulating functional T-regs. Albeit suggestive, our results need to be confirmed on larger series to determine the actual role of T-reg in mediating the clinical effect of ECP.     

胃癌中CXCR4和CD133的表达及其对淋巴转移的影响

目的:探讨CXCR4和CD133在胃癌原发灶中的表达及其对淋巴转移的影响。方法:对50例原发性胃癌原发灶和癌旁胃黏膜组织行免疫组化染色法定位检测CXCR4和CD133蛋白;选用半定量RT-PCR及Western blot法测定CXCR4和CD133 mRNA与蛋白表达量,分析两者的相关性及其与淋巴管浸润和淋巴结转移的关系。结果:CXCR4和CD133分子均定位于肿瘤细胞膜表面,极少数CXCR4位于细胞核内。胃癌组织中CXCR4和CD133表达阳性率及mRNA和蛋白的表达量均明显高于癌旁胃黏膜组织(均P<0.05);CXCR4和CD133 mRNA相对灰度值在淋巴结转移组高于无淋巴结转移组(P=0.011,P=0.038);N1组CXCR4蛋白相对灰度值明显高于N0组(P=0.023),而低于N2+N3组(P=0.008),N1组和N2+N3组CD133蛋白灰度值明显高于N0组(P=0.04,P=0.01),但N1组与N2+N3组之间无明显差异;淋巴管浸润组中的CXCR4和CD133蛋白相对灰度值均高于淋巴管无浸润组(P<0.05);在淋巴转移患者中,CXCR4和CD133蛋白相对灰度值分别与转移淋巴结数(r=0.480,r=0.426)及转移性淋巴结比率(r=0.502,r=0.489)呈正相关。结论:CXCR4和CD133在胃癌原发灶中高表达,两者呈正相关,其联合表达与转移淋巴结比率和转移淋巴结数呈正相关,推测胃癌CD133阳性细胞亚群可能在CXCR4介导下更易导致淋巴管浸润和淋巴结转移。     

外源性IL-2对小鼠CD4^＋CD25^＋调节性T细胞影响的实验研究

目的：探讨IL-2对CD4＋CD25＋调节性T细胞（Tregs）的增殖及功能的影响。方法：提取B6小鼠脾脏细胞,流式细胞仪分离CD4＋CD25＋Tregs,将新鲜分离的CD4＋CD25＋Tregs与抗CD3单克隆抗体、同种同系抗原递呈细胞（APCs）及外源性IL-2共同培养,测定其增殖活性;并检测体外扩增后的CD4＋CD25＋Tregs的免疫抑制活性及其Foxp3的表达。结果：与外源性IL-2共同培养的CD4＋CD25＋Tregs增殖程度强烈,与对照组比较,差异有统计学意义（P〈0.05）;体外扩增的CD4＋CD25＋Tregs抑制CD4＋CD25-T细胞增殖活性的能力与新鲜分离的CD4＋CD25＋Tregs相似（P〉0.05）。体外扩增的CD4＋CD25＋Tregs的Foxp3表达与新鲜分离的CD4＋CD25＋Tregs亦相似（P〉0.05）。结论：外源性IL-2能够消除CD4＋CD25＋Tregs的无反应状态,且体外扩增的CD4＋CD25＋Tregs保持了其抑制活性。     

CD4+、CD8+和NK在结直肠癌中的表达及其与淋巴结微转移的关系

目的 检测不同Dukes分期结直肠癌患者淋巴结细胞角蛋白20(CK20)mRNA的表达及外周血CD4+、CD8+、CD4+/CD8+与NK细胞活性表达,探讨两者间关系.方法 采用常规苏木素.伊红(HE)染色病理切片检测21例结直肠癌患者281枚淋巴结转移癌灶及逆转录-聚合酶链反应(RT-PCR)方法检测患者淋巴结CK20 mRNA表达;采用流式细胞仪检测患者外周血CD4+、CD8+、CD4+/CD8+及NK细胞活性表达.结果 HE染色法检出有淋巴结转移者为16枚(6%,16/281),RT-PCR法检出有淋巴结转移者140枚(50%,140/281);按有淋巴结微转移重新Dukes分期后,手术前,A、B期患者血CD4+、CD4+/CD8+高于c期(P<0.05);A期患者血CD8+低于C期(P<0.05);B期患者血NK细胞活性高于C期(P<0.05).新Dukes分期与血CD4+呈负相关(r=-0.497,P<0.01);与CD4+/CD8+呈负相关(r=-0.714,P<0.01);与CD8+呈正相关(r=0.945,P<0.01).结论 RT-PCR方法对淋巴结微转移的检出率明显优于HE染色切片法,结直肠癌淋巴结微转移的发生与患者免疫功能明显低下密切相关.     

胰腺癌荷瘤鼠吲哚胺2,3-双加氧酶表达和CD4~+CD25~+Treg细胞数的变化

目的 观察吲哚胺2,3-双加氧酶(IDO)的表达以及调节性T细胞(Treg)数量在胰腺癌荷瘤鼠中的变化,并探讨胰腺肿瘤细胞免疫逃逸的机制.方法 建立小鼠胰腺癌模型,荧光定量聚合酶链反应(PCR)、Western blot分析肿瘤组织周嗣引流淋巴结(TDLNs)IDO表达水平;并利用流式细胞术检测荷瘤鼠TDLNs及脾脏中CD4~+CD25~+T细胞占CD4~+T比例;荧光定虽PCR测量Foxp3在TDLNs及脾脏mRNA水平.结果 胰腺癌荷瘤鼠TDLNs中IDO在mRNA(1.670±0.099比1.123±0.243),蛋白表达水平(1.401±0.079比0.872±0.193),高于正常对照组.差异均有统计学意义(P<0.05);荷瘤鼠和脾细胞中CD4~+CD25~+T细胞占CD4~+T细胞的比例明显高于对照鼠[TDLNs分别为(25.00±2.16)%和(11.02±1.31)%,P<0.05;脾脏分别为(22.14±2.33)%和(12.11±0.93)%,P<0.05];荷瘤鼠Foxp3 mRNA表达水平显著高于对照鼠,荷瘤鼠分别为:TDLNs 3.427±0.164、脾细胞2.098±0.112,而对照鼠分为:0.933±0.253、1.137±0.343(P<0.05).结论 胰腺癌荷瘤鼠癌组织周围引流淋巴结IDO表达明显增高,且TDLNs及脾脏CD4~+CD25~+Treg细胞的数量增加.     

CD154 on the surface of CD4+CD25+ regulatory T cells contributes to skin transplant tolerance

BACKGROUND: It is known that the infusion of whole blood from donors (donor-specific transfusion) into recipients combined with anti-CD154 therapy can prolong allograft survival. It has generally been agreed that the effectiveness of anti-CD154 therapy is caused by the inactivation of alloreactive CD4+ and CD8+ effector T cells. The recent literature has implicated CD4+CD25+ regulatory T cells in the suppression of autoimmunity and graft rejection, and we therefore examined whether CD154 blockade is effective because of its blockade of inflammatory T-cell activation or because of a direct impact on the regulatory T cells. METHODS: RAG(-/-) mice were adoptively transfused with CD4+ T cells or a subset of the population (CD4+CD25+ or CD4+CD25- T cells) alone or in combination with donor-specific transfusion and anti-CD154 and given an allo-skin transplant. The longevity of the transplant was determined over time. CD154(-/-)CD4+ T cells were used to assess the importance of CD154 in graft rejection and acceptance. RESULTS: CD154 blockade (or loss of CD154) on CD4+CD25+ regulatory T cells enhanced their immunosuppressive activities and was a contributing factor to anti-CD154-induced immune suppression in vivo. In a model of allograft tolerance, suppression was elicited by antigen and anti-CD154 or antigen alone if the CD4+CD25+ regulatory T cells were deficient in CD154 expression. CONCLUSIONS: Neutralizing the function of CD154 on regulatory T cells upon antigen exposure induces heightened levels of suppressive activities and is likely a contributing factor to the long-lived therapeutic effects of anti-CD154 treatment.     

The type and frequency of immunoregulatory CD4+ T-cells govern the efficacy of antigen-specific immunotherapy in nonobese diabetic mice

Antigen-specific immunotherapy, an approach to selectively block autoimmune diabetes, generally declines in nonobese diabetic (NOD) mice as disease progresses. To define the parameters influencing the efficacy of antigen-specific immunotherapy once diabetes is established, plasmid DNA (pDNA) vaccination was used to suppress autoimmune-mediated destruction of syngeneic islet grafts in diabetic NOD recipients. pDNAs encoding a glutamic acid decarboxylase 65 (GAD65)-Ig molecule (pGAD65), interleukin (IL)-4 (pIL4), and IL-10 (pIL10) significantly delayed the onset of recurrent diabetes compared with pGAD65+pIL10-vaccinated recipients. Despite differences in efficacy, a similar frequency of GAD65-specific CD4(+) T-cells secreting IL-4, IL-10, or interferon-gamma were detected in mice treated with pGAD65+pIL4+pIL10 and pGAD65+pIL10. However, the frequency of FoxP3-expressing CD4(+)CD25(+)CD62L(hi) T-cells was increased in the renal and pancreatic lymph nodes of diabetic recipients vaccinated with pGAD65+pIL4+pIL10. These immunoregulatory CD4(+)CD25(+) T-cells (CD4(+)CD25(+) Treg) exhibited enhanced in vivo and in vitro suppressor activity that partially was transforming growth factor-beta dependent. Furthermore, duration of islet graft protection in pGAD65+pIL4+pIL10-vaccinated diabetic recipients correlated with the persistence of CD4(+)CD25(+) Treg. These data demonstrate that the frequency and maintenance of FoxP3-expressing CD4(+)CD25(+) Treg influence antigen-induced suppression of ongoing beta-cell autoimmunity in diabetic recipients.     

CD4+CD25+调节性T细胞对小鼠肝脏移植自发性免疫耐受的影响

目的 研究CD4+CD25+调节性T细胞在诱导自发性肝脏免疫耐受中的作用.方法 向受体和供体注射抗CD25抗体(PC61)后进行小鼠原位肝脏移植,观测其生存时间.术后20～30 d切取移植肝脏行HE染色,同时观察CD4+CD25+T细胞对CD4+T细胞和CD8+T细胞功能的影响.结果 去除受体而不是供体小鼠的CD4+CD25+T细胞可以导致肝移植排斥反应.而且,去除CD4+CD25+T细胞使移植物的白细胞浸润明显增多,组织损伤加重.同时,去除CD4+CD25+T细胞导致CD4+T细胞的增殖活性和CD8+T细胞的细胞毒活性明显增强.结论 受体来源的CD4+CD25+调节性T细胞在小鼠肝脏移植免疫耐受诱导中起重要作用.

Abstract：

Objective To examine the contribution of CD4+ CD25+ regulatory T cells to liver transplant tolerance. Methods After injection of anti-CD25 monoclonal antibody (mAb, PC61), mouse orthotopic liver transplantation was performed and survivals were determined. The paraffin-embedded sections of hepatic allografts were cut and stained with hematoxylin and eosin (HE). Furthermore, the effect of CD4+ CD25+ regulatory T cells on proliferative response of CD4+ T cells and cytotoxicity of CD8+ T cells was examined by depleting these regulatory T cells. Results Depletion of these cells in the recipients but not in the donors before liver transplantation caused rejection. Histological analyses of hepatic allografts with PC61 treatment showed extensive leukocyte infiltration and tissue destruction, whereas those in the control group showed minimal changes. Moreover, elimination of CD4+CD25+ T cells resulted in the enhancement of both proliferative response of CD4+ T cells and cytotoxicity of CD8+ T cells against donor-type alloantigen. Conclusions These results suggest that CD4+CD25+ regulatory T cells were important for tolerance induction to hepatic allografts.     

CD8+T细胞在胃癌组织中的表达及其临床意义

目的 观察胃癌组织中浸润性淋巴细胞CD8+T的表达及临床作用.方法 应用免疫组织化学染色检测102例胃癌患者手术标本中浸润淋巴细胞CD8+T的表达.结果 在胃癌、胃炎及胃息肉组织中均可见浸润淋巴细胞CD8+T的表达,但胃癌组浸润淋巴细胞CD8+T的表达[(29.53±14.71)个/HPF]明显高于慢性胃炎组[(19.54±8.10)个/HPF]和胃息肉组[(14.26±6.83)个/HPF],差异有统计学意义(P＜0.05).CD8+T细胞的表达与胃癌患者的性别、年龄、肿瘤部位、肿瘤大小、淋巴结转移、复发、病理分级和TNM分期无明显相关(P＞0.05);但与组织学类型和浸润深度有关,其中分化型胃癌患者CD8+T细胞的表达[(33.9±18.4)个/HPF]高于低分化型患者的表达[(27.6±12.0)个/HPF],差异有统计学意义(t=2.04,P＜0.05);未侵入肌层组的CD8+T细胞表达[(38.8±13.8)个/HPF]明显高于侵入肌层组[(27.7±14.1)个/HPF],差异有统计学意义(t=2.45,P＜0.05).与CD8+T细胞低表达组比较,CD8+T细胞高表达组胃癌患者中位生存时间延长5个月,但差异无统计学意义(χ2=1.0274,P＞0.05).多因素COX模型分析显示,与浸润淋巴细胞CD8+T低表达组比较,高表达组有降低胃癌死亡风险的趋势(RR=0.82,95%CI=0.41～1.65).结论 浸润性淋巴细胞CD8+T的表达与胃癌的预后有关.

Abstract：

Objective To study the expression and clinical significance of infiltrating lymphocytes CD8 + T in gastric carcinoma tissues.Methods The expression of infiltrating lymphocytes CD8 + T in 102surgically resected specimens of gastric cancer tissues was detected by using immunohistochemistry.Results CD8+ T cells were expressed both in gastric carcinoma,gastritis and gastric polyp tissues,but the number of CD8 + T cells in gastric carcinoma tissues [(29.53 ± 14.71 )/high powered field (HPF)]wassignificantly greater than in gastritis tissues [( 19.54 ±8.10)/HPF]and polyp tissues [( 14.26 ±6.83)/HPF](P ＜0.05).No correlation was found between CD8 + T lymphocyte count and gender,age,tumor location,tumor size,lymph node metastasis,tumor relapse,pathological grade or TNM stage ( P ＞ 0.05 ).However,there was a statistically significant correlation between the number of CD8 + T cells and the histological type,invasive depth.The CD8 + T cell count was obviously higher in the differentiated group [(33.9 ± 18.4)/HPF]than in the undifferentiated group [(27.6 ± 12.0)/HPF]( t = 2.04,P＜0.05 ).And the number of CD8 + T cells was greater in the non-invasive group [(38.8 ± 13.8)/HPF]than in the invasive group [(27.7 ± 14.1 )/HPF]( t = 2.45,P＜0.05 ).The median survival time of gastric cancer patients with high expression of CD8 + T cells was 5 months longer than in those with low expression,but there was no significant difference.Multi-factor COX model analysis demonstrated that the risk of death in gastric cancer patients was lower in the CD8 + T cells high expression group than in the low expression group ( RR = 0.82,95%,CI = 0.41-1.65 ).Conclusion Detection of infiltrating lymphocytes CD8 + T expression in gastric carcinoma tissues will be beneficial to the judgment of the prognosis.     

Interleukin-2 blocks the antitumour activity caused by depletion of CD25 cells in a murine renal adenocarcinoma model

OBJECTIVE: To test the effectiveness of antimouse CD25 monoclonal antibody (mAb) against murine renal adenocarcinoma (RENCA) cells, as immunoregulatory/suppressor cells are known to be involved in tumour development in vivo, but the functions of these cells are not yet clear, and eliminating naive CD25 (interleukin-2 receptor alpha)-positive T cells elicits potent immune responses to syngeneic tumours in vivo. MATERIALS AND METHODS: Aliquots of 1 x 10(4) or 1 x 10(5) RENCA cells were implanted into the subcapsule of the left kidney of syngeneic male Balb/c mice. Mice were injected with 125 micro g of antimouse CD25 mAb to deplete CD25(+) cells before RENCA implantation. Then 10(4) units of recombinant human interleukin-2 (rhIL-2) were subcutaneously injected twice daily for 7 days. Fourteen or 25 days later the tumour size was determined by laparotomy, and cells sorted using two-colour flow cytometry. RESULTS: Depletion of naive CD25(+) cells with anti-CD25 mAb and rhIL-2 administration effectively induced anti-RENCA tumour activity in Balb/c hosts. However, co-administration of anti-CD25 mAb and rhIL-2 abrogated this significant suppression of RENCA tumour growth. RENCA implantation reduced the proportion of CD4(+) cells among splenocytes, whereas anti-CD25 mAb treatment increased it. The proportion of CD25(+)CD8(+) cells among splenocytes and that of CD25(+) cells among CD8(+) cells were markedly reduced by co-administration of anti-CD25 mAb and rhIL-2 with RENCA implantation. Both CD4(+) and CD8(+) cells were stained around the remnant microscopic RENCA tumour after anti-CD25 mAb treatment. CONCLUSION: Either depletion of naive CD25(+) cells or rhIL-2 administration suppressed RENCA tumour growth in murine hosts. However, co-administration of anti-CD25 mAb and rhIL-2 abrogated this significant suppression of RENCA tumour growth.     

Characterization of naturally occurring CD4+CD25+ regulatory T cells in rhesus monkeys

BACKGROUND: Translational research in a relevant preclinical model is recommended before Treg-inducing protocols can be implemented in humans. We have characterized rhesus monkey CD25 cells phenotypically and functionally. METHODS: The phenotype of CD4(+)CD25(high) cells was determined by FACS, focusing on established markers of mouse and human Treg cells. Percentages of cells positive for CD45RA, CD62L, and intracellular CTLA-4 and FOXP3 were compared between CD4(+)CD25(high) and CD4(+)CD25(-) cells. CD25 cells stimulated with anti-CD3, ConA, and/or allogeneic peripheral blood mononuclear cells were mixed with freshly isolated CD25 cells. The suppressive activity of the CD25 cells in vitro was assessed using several experimental conditions. RESULTS: Rhesus monkey CD4(+)CD25(high) cells expressed high intracellular levels of CTLA-4 and FOXP3, whereas expression was negligible in CD4(+)CD25(-) cells. The CD25(high) population was mostly CD45RA(-), indicative of a memory phenotype. The CD25(+) cells were anergic, because they showed low proliferative responses, no interleukin-2 production, and some interferon-gamma and interleukin-10 production. Proliferation of CD4(+)CD25(-) cells stimulated by anti-CD3 or allogeneic cells was decreased when CD4(+)CD25(-) cells were added at a 1:1 ratio. In addition, we found that CD25 cells inhibited the interleukin-2 and interferon-gamma production by anti-CD3-stimulated CD25 cells in a dose-dependent fashion, through a cell-cell contact-dependent mechanism. CONCLUSIONS: Rhesus monkey CD4(+)CD25(+) cells have similar phenotypic and functional characteristics as natural Tregs in humans. These findings allow testing of Treg expansion and induction protocols in a relevant preclinical model, the rhesus monkey.     

Analysis of regional lymph node T-lymphocyte subsets in patients with gastric cancer

The proportion of T-lymphocyte subsets of the regional lymph node lymphocytes of 36 gastric cancer patients was analysed using single and two color flow cytometry technique. The antibodies against human T-lymphocyte were anti-CD3, CD4, CD8, CD4 X Leu8, CD4 X 4B4 and CD8 X CD11. The results were as follows. 1) Using single color analysis, the proportion of CD3+ and CD4+ cells in the metastatic lymph node was decreased and that of CD8+ cells was not changed compared with the non-metastatic lymph node. Therefore, the ratio of CD4 to CD8 was reduced. Using two color analysis, proportion of CD4+ Leu8- cells and CD4+ 4B4- cells was reduced. On the contrary, that of CD8+ CD11+ cells was increased in the metastatic lymph node compared with the non-metastatic lymph node. 2) The proportion of CD3+, CD4+ and CD4+ 4B4- cells was decreased in the non-metastatic lymph node of patients with lymph node metastasis compared with that of patients without lymph node metastasis. 3) The proportion of T-lymphocyte subsets was not changed in the metastatic lymph node, whether the volume including cancer cell is much or not. These results suggest that the metastatic lymph node was less defensive against lymph node metastasis in the gastric cancer.     

Functional defects and the influence of age on the frequency of CD4+ CD25+ T-cells in type 1 diabetes

CD4+ CD25+ T-cells appear to play a crucial role in regulating the immune response. Therefore, we evaluated the peripheral blood frequency and function of CD4+ CD25+ T-cells in 70 type 1 diabetic patients and 37 healthy individuals. Interestingly, a positive correlation was observed between increasing age and CD4+ CD25+ T-cell frequency in both subject groups. In contrast to previous studies of nonobese diabetic mice and type 1 diabetic patients, similar frequencies of CD4+ CD25+ and CD4+ CD25(+Bright) T-cells were observed in healthy control subjects and type 1 diabetic patients of similar age. There was no difference between type 1 diabetic subjects of recent-onset versus those with established disease in terms of their CD4+ CD25+ or CD4+ CD25(+Bright) T-cell frequency. However, type 1 diabetic patients were markedly defective in their ability to suppress the proliferation of autologous effector T-cells in vitro. This type 1 diabetes-associated defect in suppression was associated with reduced production of interleukin (IL)-2, gamma-interferon, and transforming growth factor-beta, whereas other cytokines including those of adaptive and innate immunity (IL-10, IL-1beta, IL-6, IL-8, IL-12p70, and tumor necrosis factor-alpha) were similar in control subjects and type 1 diabetic patients. These data suggest that age strongly influences the frequency of CD4+ CD25+ T-cells and that function, rather than frequency, may represent the means by which these cells associate with type 1 diabetes in humans.     

抑制黏附分子CD44S阻断胃癌腹膜转移的实验研究

目的：评价细胞表面黏附分子CD44被单克隆抗体封闭后,胃癌细胞黏附、迁移能力及其腹膜种植潜能的变化,为将阻断CD44作为抑制胃癌腹膜转移的治疗策略提供实验依据.方法：用细胞黏附试验及迁移试验（Boyden小室法）评价经抗CD44S单克隆抗体作用后,体外培养的MKN45胃癌细胞与间皮细胞发生黏附的能力及其穿越间皮细胞发生迁移的能力变化.通过建立裸鼠腹膜种植瘤模型,评价抗CD44S抗体处理对胃癌细胞腹膜肿瘤生成能力及荷瘤裸鼠生存时间的影响.结果：经抗CD44S抗体作用后,胃癌细胞MKN45与间皮细胞间的黏附能力明显减弱;经较高浓度抗体处理后,MKN45细胞的迁移能力也有明显下降.体内实验结果提示,接种经抗CD44S抗体处理MKN45胃癌细胞的裸鼠生存时间明显长于对照组（P＜0.01）.结论：黏附分子CD44参与胃癌腹膜转移过程,与肿瘤细胞-间皮细胞间的黏附和肿瘤细胞迁移等步骤密切相关;阻断CD44可作为抑制胃癌腹膜转移的潜在治疗策略.     

小鼠CD4+CD25+T调节细胞的分离、纯化及其功能检测

目的 探讨小鼠CD4+CD25+T调节细胞(Treg)的分离培养、纯化及其部分功能检测.方法 采用免疫磁珠分离法(MACS)对分离小鼠的脾淋巴细胞进行分选CD4+CD25+Treg细胞,锥虫蓝细胞染色检测其活性,流式细胞仪检测分选所得活性细胞的纯度,酶联免疫吸附试验(ELISA)检测培养上清液中白细胞介素(IL)-2、IL-10水平的浓度.结果 MACS分离的CD4+CD25+Treg细胞的纯度达83%～96%.体外培养中Treg组、T组和混合组IL-2和IL-10的平均水平分别为:(10.25±2.31)、(40.32±8.05)ng/L;(5 8.21±13.05)、(11.52±3.01)ng/L;(39.54±12.82)、(31.25±4.36)ng/L,数据差异有统计学意义(P＜0.05,P＜0.01).结论 采用MACS系统两步法,可获得高纯度、具有免疫抑制功能的Treg细胞,该细胞对CD4+CD25-T细胞的免疫抑制作用可能是通过IL-10对IL-2的调节作用实现的.     

胃癌组织中HIF-1α,CD68及CD206在胃癌及癌旁组织中的表达及临床意义

目的 分析低氧诱导分子-1α（HIF-1α）及肿瘤相关巨噬细胞（TAM）相关抗原CD68、CD206在胃癌及癌旁组织中的表达情况。方法 利用免疫组化技术检测43例胃癌和癌旁组织中HIF-1α、CD68、CD206的表达状态,计算三种蛋白表达的阳性率及阳性细胞数,揭示其与临床因素的相关性。结果 HIF-1α、CD68、CD206的阳性表达率分别为58.1%、69.8%、51.2%,均高于癌旁组织（P<0.05）。胃癌、癌旁组织中每个视野下CD68⁺细胞数分别为（39.7±7.6）、（8.5±2.8）个;CD206⁺阳性细胞数分别为（32.0±9.2）、（3.4±1.8）个;HIF-1α⁺细胞数（22.9±5.6）、（2.1±1.2）个;组间比较差异均有统计学意义（P<0.01）。胃癌组织中CD206⁺细胞数与HIF-1α⁺细胞数表达呈正相关（P<0.01,R²=0.641）。三种蛋白的表达与胃癌病理分期、淋巴结转移明显相关（P<0.05）。结论 胃癌组织中CD68、CD206、HIF-1α表达率及阳性细胞数明显增加,且CD206与HIF-1α表达呈正相关。胃癌缺氧区域对M2型巨噬细胞有趋化作用,促进胃癌发生发展。     

The sialoadhesin (CD169) expressing a macrophage subset in human proliferative glomerulonephritis.

BACKGROUND: Sialoadhesin (Sn; CD169) is a lectin-like receptor whose expression is restricted to subsets of tissue and inflammatory macrophages. We have previously identified accumulation of Sn+ macrophages as an important marker of disease progression versus remission in rat mesangial proliferative nephritis. The current study examined the significance of Sn+ macrophages in human proliferative glomerulonephritis. METHODS: Frozen kidney sections from normal adult human kidney (n = 4) and pediatric nephropathy (n = 40) were stained for total macrophages (CD68+ cells), Sn+ macrophages, CD3+ T-cells and collagen type I by immunofluorescence. Leukocyte infiltration and the severity of glomerular lesions and interstitial damage were scored. A second protocol biopsy was performed in 27 cases and clinical and biopsy-based data obtained. RESULTS: Sn+ macrophages were absent from glomeruli in normal adult human kidney and in thin basement membrane disease (n = 4), but were detected in 4 of 9 cases of purpura nephritis; 7 of 17 IgA nephropathy; 5 of 5 membranoproliferative glomerulonephritis, and 5 of 5 lupus nephritis. Sn+ macrophages were localized in areas of focal glomerular and interstitial damage. Two-colour immunostaining confirmed that Sn+ cells are a subset of total CD68+ macrophages. The number of glomerular Sn+ macrophages correlated with the degree of proteinuria and glomerular lesions (r = 0.44, P = 0.0045 and r = 0.82, P<0.0001; respectively), while interstitial Sn+ macrophages correlated with the degree of proteinuria and interstitial damage (r = 0.59, P<0.0001 and r = 0.75, P<0.0001; respectively). Combined immunostaining revealed that interstitial Sn+ macrophages and CD3+ T-cells co-localized in areas of tubulointerstitial damage with increased type I collagen deposition. There was significant correlation between the number of interstitial Sn+ macrophages and CD3+ T-cells (r = 0.74, P<0.0001). Most patients responded to a 2 year period of glucocorticoid therapy with a reduction in proteinuria and glomerular lesions and this correlated with the reduction in the number of glomerular Sn+ macrophages. CONCLUSION: This study has identified Sn+ cells as a macrophage subset whose accumulation in the kidney correlates with proteinuria and histologic damage. These results, together with recent findings from animal studies, suggest that Sn+ macrophages may play an important role in progressive renal disease.     

CD4+ CD25+ Tr细胞与大鼠肝移植自发免疫耐受关系的研究

目的研究CD4^＋CD25^＋Tr细胞及其相关基因Foxp3与大鼠肝移植自发免疫耐受的关系。方法双袖套法建立大鼠原位肝移植模型;密度梯度离心法分离肝内淋巴细胞;免疫磁性分离法（MACS）分选CD4^＋CD25^＋Tr细胞,流式细胞术（FCM）检测所得细胞纯度。体外细胞增殖试验研究CD4^＋CD25^＋Tr细胞的免疫抑制作用。Western蛋白印迹法检测CD4^＋CD25^＋Tr细胞Scurfin蛋白表达。结果自发耐受组大鼠移植肝内CD4^＋CD25^＋Tr细胞含量显著高于急性排斥组。混合淋巴细胞反应中,LEW大鼠的脾细胞比DA大鼠自身的脾细胞更能刺激CD4^＋CD25^＋T细胞的增殖。CD4^＋CD25^-T细胞能抑制CD4^＋CD25^-T细胞的增殖,当加入外源性IL-2（200U／ml）时,该抑制作用被逆转。结论转录因子Foxp3介导的CD4^＋CD25^＋Tr细胞的免疫抑制作用可能是诱导大鼠肝脏移植自发免疫耐受的机制之一。