致敏树突状细胞联合射频消融治疗对荷瘤大鼠免疫机能的影响

目的探讨经冻融肿瘤抗原致敏的树突状细胞（DCs）联合应用于射频消融术（RFA）后治疗大鼠实体瘤对大鼠抗肿瘤免疫的影响。方法30只荷Walker256实体瘤SD大鼠随机分成三组，每组10只：对照组1仅行RFA治疗；对照组2行RFA＋未致敏DC治疗；实验组行RFA＋冻融抗原致敏DC治疗。治疗前及治疗后第4天经流式细胞仪检测大鼠外周血中CD4^＋、CD8^＋及CD4^＋／CD8^＋比值；同时，超声评价治疗前后各组肿瘤体积变化，记录大鼠的荷瘤生存期，各组间比较。结果治疗后，实验组大鼠外周血CD4^＋T细胞及CD4^＋／CD8^＋比值的升高较两对照组比明显，与对照组2比差异显著（P〈0．05）；实验组外周血CD8^＋T细胞的降低显著大于两对照组（P〈0．05）。实验组大鼠肿瘤生长速度显著慢于对照组，其荷瘤生存期较对照组明显延长（P〈0．05）。结论肿瘤抗原致敏树突状细胞联合射频消融治疗可有效改善大鼠的抗肿瘤免疫机能，从而延缓肿瘤生长、延长荷瘤大鼠寿命。     

肝癌无水乙醇灭活后瘤区注射高聚生前后免疫应答的研究

目的研究无水乙醇治疗肝癌后瘤区连续注射超抗原生物制剂高聚生局部的免疫应答。方法选取46例肝癌患者，均于无水乙醇注射治疗原位灭活后第3周、第4周和第7周瘤区局部注射高聚生，每次注射1600U。分析比较注射高聚生治疗前后治疗区CD3^＋、CD4^＋、CD8^＋、CD57^＋和CD68^＋浸润变化情况。结果CD3^＋、CD4^＋、CD57^＋和CD68^＋局部浸润较注射高聚生前有明显增高（P〈0．01），且持续至注射后第五周仍高于治疗前（P〈0．05）。结论注射高聚生能够有效地增强肝癌患者局部抗肿瘤细胞免疫，可能具有一定的抗肿瘤复发和降低肝癌复发率的作用。     

原发性肝癌射频治疗后局部免疫功能的变化及其临床意义

目的对原发性肝癌（hepatocellular carcinoma，HCC）射频消融治疗（radiofrequency ablation，RFA）前后肿瘤内部及边缘热休克蛋白70（heat shock protein，HSP70）的表达、CD8^＋T细胞数量的变化以及RFA治疗后，肿瘤边缘HSP70表达与肿瘤边缘CD8^＋T细胞数量之间的关系进行观察，探讨RFA治疗对原发性肝癌局部免疫功能状态的影响及其可能的临床意义。方法对17例HCC分别在RFA治疗前、后1个月，于肿瘤内部和肿瘤边缘超声引导下穿刺活检取样；采用PowerVision^TM二步染色法进行免疫组化分析，测定HSP70的表达、CD8^＋T细胞的数量；随访HCC复发／新生情况。结果RFA治疗后肿瘤边缘组织HSP70表达增强（Z=3．337，P=0．001）、CD8^＋T细胞数量增多（Z=1．996，P=0．049）；RFA治疗后，≤4cm肿瘤组的占位边缘CD8^＋T细胞数量高于〉4cm肿瘤组（Z=1．966，P=0．048）。RFA治疗后，边缘组织HSP70表达与CD8^＋T细胞数量之间呈正相关关系（r=0．489，P=0．046）；RFA治疗后，无复发或新生组的占位边缘组织HSP70表达和CD8^＋T细胞数量分别高于复发或新生组（Z=2．009，P=0．045；Z=2．007，P=0．045）。结论RFA治疗后边缘HSP70表达增强、CD8^＋T细胞数量增多，显示RFA治疗后局部免疫原性提高，抗肿瘤效应细胞浸润增加。     

结肠癌患者外周血 CD4+ CD25+调节性 T细胞检测的临床意义

目的 探讨结肠癌患者外周血CD4^＋CD25^＋调节性T细胞检测的临床意义。方法采用流式细胞技术对51例结肠癌患者和30例健康体检者外周血淋巴细胞CD4及CD25进行了检测。结果（1）结肠癌组CD4^＋CD25^＋细胞明显高于正常对照组（P〈0．05），CD4^＋CD25^＋细胞则显著低于正常对照组（P〈0．05）；（2）结肠癌组手术后患者CD4^＋CD25^＋、CD4^-CD25^＋细胞与手术前相比显著下降（P〈0．05）；（3）结肠癌组淋巴结转移患者CD4^＋CD25^＋细胞明显高于未转移组（P〈0．001）。结论CD4^＋CD25^＋调节性T细胞数可以反映肿瘤患者免疫系统存在抑制状态，且可能与肿瘤的免疫耐受有关，并可反映肿瘤淋巴结转移状况。     

Fas/FasL在强直性脊柱炎患者外周血T细胞亚群上的表达

目的检测强直性脊柱炎（As）患者外周血T细胞亚群上的Fas／FasL的表达水平，探讨Fas／FasL诱导的细胞凋亡在As免疫学发病机制中的作用。方法以临床确诊的60例As患者作为研究对象，同时选择30例正常对照，运用流式细胞仪（FCM）检测其外周血CD3^＋CD4^＋、CD3^＋CD8^＋T细胞亚群上的Fas／FasL表达水平。结果早、晚期AS患者外周血CD3^＋CD4^＋T细胞上的FasL的表达率分别为（0．59％、0．93％），CD3^＋CD8^＋T细胞上的FasL的表达率分别为（2．93％、4．32％），与健康对照组（0．48％、1．14％）比较，其表达率差异具有统计学意义（P〈0．05）；与健康对照组外周血CD3^＋CD4’、CD3^＋CD8’T细胞上的Fas表达率（58．25％、59．91％）比较，早期AS患者外周血CD3^＋CD4^＋T细胞上的Fas的表达率（64．75％）明显升高（P〈0．05），而CD3^＋CD8^＋T细胞上的Fas的表达率（48．64％）明显降低（P〈0．05），Fas在晚期AS患者外周血CD3^＋CD4^＋、CD3^＋CD8^＋T细胞上的表达率（57．63％、56．32％）无明显变化。结论Fas、FasL在外周血T细胞亚群上的表达水平与AS的病情发展阶段相关；Fas、FasL的异常表达所导致T细胞凋亡功能紊乱可能是AS发病的重要机制之一。     

Ag85A及Ag85B-DNA疫苗对移植性小鼠膀胱肿瘤免疫治疗的实验研究

目的：比较研究Ag85A-DNA疫苗和Ag85B-DNA疫苗对移植性小鼠膀胱肿瘤的免疫作用。方法：采用小鼠膀胱癌BTT739细胞株,构建可移植性T739小鼠膀胱肿瘤模型。随机分为生理盐水组、卡介苗（BCG）组、空白质粒（pcDNA3．1）组、pcDNA—Ag85A组、pcDNAAg85B组,荷瘤后第7天、14天和21天各肌注1次,末次注射后7天检测各组小鼠脾脏的CD4^＋和CD8^＋T细胞亚群数量及CD4^＋／CD8^＋比值、小鼠血清IFN-γ、肿瘤体积重量并作组织病理学检查。结果：Ag85B-DNA疫苗能够显著提高荷瘤小鼠CD4^＋T细胞亚群数量、CD8^＋T细胞亚群数量和CD4^＋T细胞与CD8^＋T细胞的比值,明显抑制肿瘤生长,促进IFN-γ分泌。肿瘤组织细胞坏死明显,瘤体周围及瘤体内有大量炎性细胞浸润,与各组相比差异有显著统计学意义（P〈0．01）,但低于BCG组;Ag85A—DNA疫苗组与生理盐水组和空白质粒组比较,差异无统计学意义（P〉0．05）。结论：单独应用Ag85B-DNA疫苗可以诱导Th1免疫反应,增加IFN-γ等细胞因子的分泌,提高荷瘤小鼠的细胞免疫功能,抑制肿瘤生长,但效果不及BCG。尚不能认为单独应用Ag85ADNA疫苗会诱发有效的抗膀胱肿瘤免疫反应。     

胸腺切除对重症肌无力病人外周血T细胞的远期影响及意义

目的 探讨胸腺切除对重症肌无力（MG）病人外周血T细胞的远期影响及意义。方法 应用流式细胞术检测25例胸腺切除（手术组）、21例胸腺未切除（非手术组）MG病人和25名健康人（对照组）的外周血T细胞亚群CD4^＋T、CD8^＋T、CD4^＋／CD8^＋、CD4^＋CD25^＋T细胞的变化，应用ELISA法检测外周血IFN-γ、IL-4水平。结果 MG病人胸腺切除后完全缓解9例（36％），部分缓解13例（52％）。手术组与对照组比较CD4^＋CD25^＋T％显著降低（t=2．917，P=0．005）。手术组与非手术组比较CD4^＋CD25^＋T％、CD8^＋T％显著增高（t=7．935，P=0．000；t=2．619，P=0．012），CD4^＋CD8^＋显著降低（t=3．060，P=0．004）。手术组外周血IFN-γ水平显著低于非手术组（t=5．060，P：0．000），但显著高于对照组（t=3．709，P=0．001）。胸腺切除后部分缓解者CD4^＋CD25^＋T％显著低于完全缓解者（t=2．292，P=0．033），但高于无效者（t=5．225，P=0．000）。结论 MG病人外周血T细胞紊乱在胸腺切除后远期有一定程度的改善，但未完全恢复正常。CD4^＋CD25^＋调节性T细胞可能与MG发生、发展及预后有关。     

转染肿瘤mRNA的树突状细胞疫苗诱导抗肝癌免疫研究

目的探讨转染原发性肝癌(HCC)mRNA的树突状细胞(DC)能否诱导抗肿瘤特异性细胞毒性T淋巴细胞(CTL)。方法采用HCC患者外周血单核细胞(PBMC)体外刺激分化为DC细胞；从人肝癌HepG-2细胞和3例HCC患者的肝癌组织中体外扩增mRNA。以mRNA转染DC细胞，并与PBMC混合培养诱导扩增CTL。流式细胞计数仪检测培养细胞中CD3^ 、CD4^ 、CD8^ 细胞的比例。^51Cr释放法测定CTL的杀瘤活性。结果经扩增人肝癌HepG-2mRNA和2例AFP( )患者的AFP( )HCCmRNA诱导3周后，CD3^ 、CD8^ 细胞占淋巴细胞总数由诱导前的27．8％、26．5％、29．6％升高至89．3％、73．6％、86．8％；而经扩增AFP(-)HCCmRNA诱导3周后，CD3^ 、CD8^ 细胞占淋巴细胞总数由诱导前的25．4％升高至53．6％。转染HepG-2细胞和AFP( )的患者HCCmRNA的DC诱导的CTL对HepG-2细胞杀瘤活性明显高于AFP(-)的患者，其杀瘤特性由MHC-I限制的CD8^ T细胞所介导。结论HCCmRNA体外转染DC能诱导肿瘤特异性CTL，可为肝癌的免疫治疗提供新的有效手段。     

树突状细胞疫苗联合胸腺肽α1对人源化免疫重建裸鼠结肠癌生长的抑制效应

目的观察胸腺肽αl（Ted）联合结肠癌细胞裂解物致敏树突状细胞（LyDCs）对人源化免疫重建裸鼠结肠癌的免疫治疗效应。方法常规DCs负载结肠癌细胞裂解物制备LyDCs疫苗，流式细胞仪（FCM）检测Tαl体外刺激前、后的LyDCs表型。HT-29结肠癌裸鼠模型成瘤后，经尾静脉注射人外周血T淋巴细胞6×10^6个／只，2d后，FCM检测裸鼠外周血人源性CD4^＋、CD8^＋T细胞；将人源化免疫重建裸鼠分为3组，分别用LyDCs＋Tctl、LyDCs和生理盐水皮下免疫注射治疗；治疗后7d，体外观察各组裸鼠脾脏淋巴细胞的肿瘤杀伤作用及IFN-γ、IL4分泌水平，实验结束时，观察LyDCs联合Tctl对荷瘤裸鼠的体内抑瘤作用。结果LyDCs的表型HLA．DR、CD80、CD86、CD83较刺激前明显上调；免疫重建裸鼠均检测到人源性CD4^＋、CD8^＋T细胞；LyDCs＋Tctl组裸鼠脾脏T淋巴细胞的肿瘤杀伤作用与LyDCs组比较差异有统计学意义（P〈0．01），LyDCs＋Ted组T细胞的IFN-γ分泌水平与LyDCs组比较差异有统计学意义（P〈0．01）；接种HT-29细胞58d后，LyDCs＋Tαl组、LyDCs组抑瘤率分别为60．41％、37．20％，两组之间抑瘤效应比较差异有统计学意义（P〈0．01），两组的抑瘤效应与对照组比较分别（P〈0．01）。结论Tαl能增强LyDCs诱导的CD4^＋，Th1细胞反应和CTLs杀伤效应，对DCs疫苗的抗癌免疫治疗功效具有明显的放大作用。     

非小细胞肺癌患者围术期细胞免疫功能的变化及免疫治疗

目的观察围术期非小细胞肺癌患者细胞免疫功能的变化及应用胸腺肽α1后对机体免疫功能的影响,为临床应用免疫增强剂联合手术治疗非小细胞肺癌患者提供依据。方法将97例行肺叶或右全肺切除术的非小细胞肺癌患者分为两组,组1：围术期给予胸腺肽α1治疗;组2：围术期未给予胸腺肽α1治疗;对照组：另选择19例同期非肺癌而采取手术治疗的肺部疾病患者作为对照。采用间接免疫荧光法（IFCA）测定3组围术期T细胞亚群的百分率变化。结果术后第1d组1 CD4^＋T、CD4^＋T／CD8^＋T高于组2（CD4^＋T 36．92％±2．10％ vs．31．18％±7．64％;CD4^＋T／CD8^＋T 1．31±0．36 vs．1．09±0．32;P〈0．05）,术后第3d组1 CD4^＋T和CD4^＋T／CD8^＋T高于组2（CD4^＋T 45．66％±3．77％ vs．34．70％±8．42％;CD4^＋T／CD8^＋T 1．42±0．11 vs．1．14±0．20;P〈0．05）;术后第9d CD4^＋T、CD4^＋T／CD8^＋T高于组2（CD4^＋T 47．28％±1．96％ vs．39．12％±3．10％;CD4^＋T／CD8^＋T 1．46±0．14 vs．1．22±0．36;P〈0．05）;术后第16d组1 CD4^＋T、CD4^＋T／CD8^＋T与组2和对照组比较差异无统计学意义（P〉0．05）。结论非小细胞肺癌患者的免疫功能低下,应用胸腺肽α1后细胞免疫功能较快恢复至正常状态,对非小细胞肺癌患者早期采用手术、化疗／放疗的综合治疗有助于提高治疗效果。     

微波消融灭瘤联合瘤内接种树突状细胞诱导特异性抗肝癌免疫

目的 探讨微波消融(MWA)灭瘤联合瘤内接种树突状细胞(DCs)诱导特异性抗肝癌免疫的效能.方法 采用GM-CSF联合IL-4体外培养C57BL/6小鼠骨髓来源的DCs,于第6天收集使用.建立C57BL/6小鼠皮下Hepa1-6肝癌模型,随机分为对照组、瘤内接种DCs组(DC组)、肿瘤微波消融组(MWA组)及肿瘤微波消融+瘤内接种DCs组(MWA+DC组).免疫组织化学法检测肿瘤组织内CD4+和CD8+T细胞的浸润,MTT法检测小鼠脾脏细胞对Hepa1-6的特异性杀伤活性,观测各组小鼠肿瘤生长情况.结果 免疫组织化学法检测显示MWA+DC组肿瘤组织内有大量的CD4+和CD8+T淋巴细胞浸润,显著高于其它组(P<0.05).MwA+DC组脾细胞对Hepa1-6细胞有特异性杀伤效能,在E/T=40和100时,MWA+DC组脾细胞对Hepa1-6细胞的特异性杀伤力显著高于对照组、DC组及MWA组(P<0.05).MWA+DC组小鼠肿瘤完全消退率显著高于其它各组(P<0.05).结论 MWA联合瘤内接种DCs可有效诱导机体产生特异性抗肝癌免疫,是预防MwA治疗后肝瘤复发的一种有效方法 .     

Kinetics of leukocyte and myeloid cell traffic in the murine corneal allograft response

BACKGROUND: Little information exists on the trafficking of myeloid and lymphoid cells between the transplanted cornea and the secondary lymphoid tissue. This study reports on changes in the cornea and the draining lymph node (DLN) from the time of graft emplacement. METHODS: Using a mouse corneal graft model (C57BL/10Sn to BALB/c), eyes and submandibular DLN were examined by immunohistochemistry and three-color flow cytometry for evidence of T cell activation and dendritic cell (DC) conditioning (up-regulation of costimulatory molecules) at various times (15 min to 24 days; n=4 for each time). RESULTS: In the DLN, early (2 hr) DC conditioning was sustained throughout allograft rejection whereas a remarkable drop in percentage of activated CD4+ and CD8+ T cells (P <0.001) was followed by a biphasic rise in activated CD4+ and, to a lesser extent, CD8+ T cells (24 hr, P <0.001 and 6 days, P <0.01). CD11b+ and MOMA-2+ macrophages, MHC Class II+ cells, CD86+ DC, and neutrophils were the earliest cells infiltrating the cornea (at 24 hr), whereas T cells appeared after 2 days, with CD4+ T cells being confined largely to the graft recipient border. CONCLUSIONS: Immediate and rapid changes in T cell and DC populations in the DLN correlate with the type of cellular infiltration in the corneal graft. The data are consistent with a model in which CD4+ T cell help for CD8+ cytotoxic T cells could be provided by sequential two-way activation of T cells and DC in the DLN. The majority of cells infiltrating the graft were macrophages and neutrophils, with fewer DC and T cells.     

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目的 研究乳腺癌腋窝淋巴结发生癌转移和未发生癌转移时的免疫功能。方法2004年8月至2005年7月采用流式细胞技术检测乳腺癌病人乳腺癌前哨淋巴结（SLN）与乳腺癌非前哨淋巴结（NSLN）中免疫细胞CD3^＋T、CD4^＋T、CD8^＋T、CD16^＋NK、CD^19^＋B的比例，并相互比较。结果淋巴结未发生癌转移时，SLN与NSLN的免疫细胞CD3^＋T、CD4^＋T、CD8^＋T、CD16^＋NK、CD^19^＋B的比例差异无统计学意义（P〉0．05）；而且当SLN与NSLN发生癌转移后，它们的免疫细胞的比例也无差异。但SLN发生癌转移与未发生癌转移时相比，其CD4^＋T、CD8^＋T、CD16^＋NK的比例发生显著改变（P〈0．05），CD3^＋T、CD19^＋B的比例改变无统计学意义（P〉0．05）。结论 当腋窝淋巴结未发生癌转移时，它仍有正常的免疫功能。当腋窝淋巴结发生癌转移后，它的免疫微环境发生了改变，免疫功能呈现一种抑制状态。     

A clinical study of immunological status in gastric cancer patients--with special reference to T-cell subsets in the lymphocytes of regional lymph nodes

Non-metastatic regional lymph node lymphocytes of 41 patients with gastric cancer were studied by using different monoclonal antibodies and flow cytometry. Used monoclonal antibodies were OKT3 (total T; CD3), OKT4 (helper/inducer T; CD4), OKT8 (suppressor/cytotoxic T; CD8) and Leu11 (NK/K cell; CD16). The results were as follows: 1. The percentage of CD3 cells and CD4 cells were about ten point fewer in lymph nodes than in peripheral blood. 2. CD8 cells were found to be one half or one third lesser in lymph nodes than in peripheral blood. 3. CD16 cells were found to be rare in lymph nodes. 4. The percentage of CD3, CD4 and CD8 cells were higher in distal lymph nodes than proximal ones. 5. The percentage of CD3, CD4 and CD8 cells were not different with progression of the cancer, whereas CD3 cells and CD8 cells were decreased in lymph nodes of stage IV. 6. The percentage of CD8 cells was higher in distal nodes of stage III. Regional lymph nodes are necessary to protect against cancer metastasis, and killer T cells and cytotoxic T cells were fewer in lymph nodes. These results suggested that killer activity and cytotoxicity of the lymph node lymphocytes are inactive and anergy.     

1-甲基色氨酸对胰腺癌荷瘤鼠中调节性T细胞数量变化的影响

目的 观察1-甲基色氨酸(1-MT)对胰腺癌荷瘤鼠中调节性T细胞(Treg)数量变化的影响,比较树突状细胞(DC)疫苗与1-MT联合应用前后抗肿瘤作用的强弱.方法 建立小鼠胰腺癌模型;利用流式细胞术检测荷瘤鼠应用1-MT前后肿瘤组织周围引流淋巴结(TDLNs)及脾脏中CD4~+ CD25~+T细胞占CD4~+T比例;荧光定量聚合酶链反应(PCR)测量Foxp3在TDLNs及脾脏mRNA水平;利用肿瘤细胞裂解物冲击DC制备DC疫苗,并根据是否与1-MT联合应用分组(各组均为n=8);观测各组肿瘤体积的差异.结果 应用1-MT后,荷瘤鼠CD4~+ CD25~+ T细胞占CD~+T细胞的比例明显低于未应用组(TDLNs)分别为(16.01±2.21)%和(25.00±2.16)%(P<0.05);脾脏分别为(13.11±1.93)%和(22.14±2.33)%(P<0.05,P<0.01);应用1-MT组Foxp3 mRNA表达水平显著低于未应用组,应用1-MT组相对表达值:TDLNs0.947±0.216、脾细胞1.198±0.347,而未应用组分别为:1.927±0.256、1.798±0.237(P<0.05);1-MT+DC疫苗组肿瘤生长显著受到抑制,第36天肿瘤体积为(789.0±111.0)mm~3;显著小于DC疫苗组、1-MT组及对照组,肿瘤体积分别为:(1768.0±251.3)、(1854.0±192.1)、(1899.0±201.2)mm~3(P<0.01).结论 1-MT可以有效抑制胰腺癌荷瘤鼠癌组织周围引流淋巴结及脾脏CD4~+ CD25~+ Treg细胞的数量增加,从而增强DC疫苗抗肿瘤作用.     

Infiltration of activated dendritic cells and T cells in renal cell carcinoma following combined cytokine immunotherapy

OBJECTIVES: In a phase I study the feasibility, toxicity and immunological effects of peri-operative cytokine immunotherapy of renal cell carcinoma were studied. Main goals were to determine the maximal tolerable dose and detailed in situ analysis of tumor infiltrates. METHODS: Fifteen patients with renal cell carcinoma, undergoing nephrectomy, received subcutaneous immunotherapy, consisting of low-dose IL-2, IFNalpha and GM-CSF, from day -3 prior, until day +5 following surgery in a dose escalation study. Infiltrates from resected tumor tissues from patients undergoing immunotherapy or control patients that underwent nephrectomy only, were examined using quantitative immunohistological analysis and 3-color immunofluorescence staining and confocal laser scanning microscope analysis. RESULTS: Toxicity was limited and the maximal tolerable dose was established. In peripheral blood an increase was found in total lymphocytes, (activated) T cells, NK cells and monocytes. Quantitative immunohistological analysis of tumor infiltrates showed enhanced numbers of CD3+ T cells, S100+ DC, CD83+ DC and IL-2 receptor positive cells (4-fold, 2-fold, 10-fold and 20-fold, respectively, compared to controls). In treated patients preferential invasion was observed of TNFalpha positive CD8+ T cells and DC, positive for DC-SIGN (CD209), CD83, CD80, IL-12 and the DC specific chemokine, DC-CK1 (CCL18). CONCLUSIONS: These findings show increased infiltration of activated, mature DC and functionally active CD8+ T cells in renal tumors, which may suggest clinical potential of cytokine immunotherapy.     

Tim-3在小鼠心脏移植受者体内不同部位T淋巴细胞上表达的变化

目的 检测激活的T_H1类淋巴细胞标记分子"T淋巴细胞免疫球蛋白域及粘蛋白域蛋白-3(Tim-3)"在受者体内不同部位T淋巴细胞上表达的变化,探讨其与急性排斥反应的关系.方法 建立小鼠同基因和异基因心脏移植模型(简称:同基因组和异基因组);移植术后第3和第6天,分离和制备两组受者外周血、脾脏、引流淋巴结和移植心内淋巴细胞悬液,采用流式细胞仪检测Tim-3阳性细胞在CD4~+ 和CD8~+ T淋细胞中的比值.结果 两组受者术后外周血和脾脏内Tim-3~+/CD4~+以及Tim-3~+/CD8~+ 的比值比较,差异均无统计学意义(P>O.05).与同基因组比较,异基因组引流淋巴结内Tim-3~+/CD4~+ 比值轻度升高(P<0.05);但异基因组术后第6天与第3天比较,差异无统计学意义(P>0.05).与同基因组比较,移植心内Tim-3~+/CD4~+和TiM-3~+/CD8~+比值均显著升高(P<0.01);异基因组术后第6天与第3天比较.移植心TiM-3~+/CD4~+和Tim-3~+/CD8~+比值也显著升高(P<0.01).结论 小鼠异基因心脏移植受者引流淋巴结和移植心内T淋巴细胞上Tim-3的表达升高与急性排斥反应的进展动态相关.     

Direct versus Indirect Allorecognition: Visualization of Dendritic Cell Distribution and Interactions During Rejection and Tolerization

Interactions of donor and recipient dendritic cells (DCs) with CD4+ T cells determine the alloantigenic response in organ transplantation, where recipient T cells respond either directly to donor MHC, or indirectly to processed donor MHC allopeptides in the context of recipient MHC molecules. The present study evaluates donor and recipient alloantigen-presenting DC trafficking and their interactions with CD4+ T cells in the lymph nodes (LNs) and the spleen under tolerogenic treatment with anti-CD2 plus anti-CD3 mAb compared with untreated rejecting conditions. CX3CR1(GFP) BALB/c (I-A(d)) donor hearts were transplanted into C57BL/6 (I-A(b)) mice and quantification of donor DC direct (GFP+ or I-A(d+)) and recipient DC indirect (YAe+) trafficking and interactions with host CD4+ T cells was performed by fluorescent microscopy. Our data indicate that although both direct and indirect interactions between CD4+ T cells and donor and recipient DCs occur shortly after engraftment, only indirect presentation persists in the LN, but not the spleen, of tolerized recipients. These data suggest that distinct anatomic lymphoid compartments play a critical role in peripheral tolerance induction and maintenance, and persistent indirect presentation to CD4+ T cells within the LNs is an important process during tolerization.     

CD4+CD25+T细胞对同种异体小鼠心脏移植的免疫调节作用

目的 观察CD4+CD25+T细胞(Treg)对小鼠同种异体心脏移植的免疫调节作用.方法 流式细胞仪检测正常小鼠和胸腺切除+PC61小鼠淋巴结、脾脏和外周血的Treg的比例.将供体鼠BALB/C心脏移植到受体鼠B6腹腔内,观察对照组(n=6)、胸腺切除组(THY,n=8)、hCTLA4Ig组(n=8)、DST+hCTLA4Ig组(n=8)和THY+PC61+DST+hCTLA4Ig组(n=6)小鼠心脏移植后生存时间和移植心脏病理学检查.结果 正常B6小鼠淋巴结、脾脏和外周血的Treg的比例分别为5.1%、4.5%和1. 7%,明显高于胸腺切除+PC61处理组(1. 8%、1.7%、0.7%).移植心脏平均存活时间在对照组和胸腺切除组分别为(8.2±2.9)d和(7.6±3.0)d,两组间差异无统计学意义(P＞0.05);而在hCTLA4Ig组和DST+hCTLA4Ig组分别为(43.0±11.8)d和(135.0±29.7)d,均较对照组或胸腺切除组明显延长(P＜0.01);THY+PC61+DST+hCTLA4Ig组移植心脏平均存活时间(25.8±8.9)d,也明显较对照组明显延长,但短于hCTLA4Ig组和DST+hCTLA4Ig组(P＜0.01).DST+hCTLA4Ig组移植的心脏存活时间(135.0±29.7)d明显高于其他各组(P＜0.01),其病理组织学表现为间质内有较多的淋巴细胞浸润,伴毛细血管增生,管壁增厚,间质纤维化.结论 CD4+CD25+T细胞水平对同种异体心脏移植的免疫耐受具有免疫调节作用.

Abstract：

Objective To investigate the immunoregulation effects of CD4 + CD25 + T cells in mice heart allograft transplantation. Methods Flow cytometry was used to analyze the contents of CD4 + CD25 +T regulatory cells (Tregs) of the lymph nodes, spleen and blood in the normal mice group and the thymusectomy (THY) + PC61 group. BALB/C mice served as the donors and C57BL/6 (B6) mice as the recipients. Five groups were established, including control group ( n = 6 ), THY group ( n = 8 ), hCTLA4Ig group ( n = 8 ), DST ( donor-specific T-depleted spleen cells) + hCTLA4Ig group ( n = 8) and THY + PC61+ DST + hCTLA4Ig group (n = 6). The survival time after heart allograft transplantation was observed and pathological examination was done in different groups. Results In control group, the rate of Tregs in lymph nodes, spleen and blood was 5. 1%, 4. 5% and 1.7% respectively, which was significantly higher than in THY + PC61 group ( 1. 8% , 1. 7% and 0. 7% respectively). The average survival time of control and THY groups was 8. 2 ± 2.9 and 7.6 ± 3. 0 days respectively ( P ＞ 0. 05 ). The average survival time of hCTLA4Ig and DST + hCTLA4Ig groups was 43.0 ± 11.8 and 135.0 ± 29. 7 days respectively, which was significantly longer than in control group or THY group ( P ＜0. 01 ). The average survival time of THY +PC61 + DST + hCTLA4Ig group was 25.8 ± 8.9 days, which was significantly longer than in control group,but shorter than in hCTLA4Ig group or DST + hCTLA4Ig group ( P ＜ 0. 01 ). The survival time in DST +hCTLA4Ig group was 135.0 ± 29. 7 days, which was significantly longer than other groups ( P ＜ 0. 01 ).The pathological examination revealed that there were more lymphocytes infiltration and capillary vessel proliferation in the desmohemoblast in the transplanted heart of DST + hCTLA4Ig group. Conclusion CD4 +CD25 +T cells regulate the immune tolerance in the allograft transplantation.     

Effect of Vitamin E Supplementation on Lymphocyte Distribution in Gut‐Associated Lymphoid Tissues Obtained from Weaned Piglets

Fifteen piglets were used to determine the effect of vitamin E supplementation on the number of CD4‐immunoreactive (CD4+) T‐lymphocytes, CD8‐immunoreactive (CD8+) T‐lymphocytes and IgA‐immunoreactive (IgA+) B‐lymphocytes per follicle in the Peyer's patch of distal ileum and the mesenteric lymph nodes of weaned piglets. Piglets, following a 3‐day adaptation period after weaning, were assigned to one of three experimental groups: control (no vitamin E supplementation), vitamin E supplementation of 100 mg/kg of diet and vitamin E supplementation of 300 mg/kg of diet. Supplementation of vitamin E lasted for a period of 36 days. The basal diet contained 80 mg α‐tocopherol/kg of diet. All piglets were killed at day 39 after weaning and samples of the distal ileum and adjacent mesenteric lymph nodes were collected. The number of cells for each lymphocyte subset was counted in the Peyer's patch and the mesenteric lymph nodes follicles, in cryostat sections processed for immunohistochemistry. Results showed that vitamin E supplementation (300 mg/kg diet) of piglets caused an increase (P < 0.05) in the number of IgA+ B‐lymphocytes in the Peyer's patch, but not in the mesenteric lymph nodes, compared with the corresponding values in control animals. Vitamin E supplementation had no effect (P > 0.05) on the number of CD4+ and CD8+ T‐lymphocytes in the follicles of the Peyer's patch and the adjacent mesenteric lymph nodes. Thus, vitamin E had relatively minor effects on distribution of the major immunocompetent cells in the gut. The numbers of CD4+ and CD8+ T‐lymphocytes as well as IgA+ B‐lymphocytes per follicle were higher by 26–77% (P < 0.05) in the mesenteric lymph nodes than the corresponding values in the Peyer's patch.