Flt3L免疫扩增恒河猴外周血树突状细胞前体(pDC1/pDC2)的研究

目的 探讨Flt3L对恒河猴外周血树突状细胞前体(pDC1和pDC2)的免疫扩增作用。方法 健康猴艾滋病毒SIV阴性的恒河猴每日给予rhFlt3L，100μg／kg体重皮下注射，于第11天在氯胺硐全麻下采集静脉血，用Ficoll-Hypaque梯度离心法提取外周血单核细胞，利用与恒河猴有交叉反应的人单克隆抗体以及三色流式细胞仪分离pDC1和pDC2,并与未经Flt3L作用的pDC1和pDC2在数量和细胞表现型上进行对比。结果 经Flt3L扩增的恒河猴外周血pDC1和pDC2数量分别增加7倍和4倍，且细胞表现型和形态不发生改变。结论Flt3L对恒河猴外周血pDCl和pDC2具有免疫扩增作用。     

恒河猴外周血pDC1/pDC2的分离和细胞表现型的研究

目的 研究恒河猴外周血树突状细胞（DC）的亚群pDC1和pDC2的分离方法。并探讨其细胞表现型。方法 采集健康，SIV阴性的恒河猴外周血，用Ficoll-Hypaque梯度离心法提取外周血单核细胞（PBMC），利用与恒河猴有交叉反应的人单克隆抗体以及三色流式细胞仪分离pDC1和pDC2，并对其进行细胞表现型的鉴定。结果 首次分离出恒河猴pDC1和pDC2细胞；并保持其活性。pDC1细胞表现型为Lineage^-,HLA-DR^ 和BDCA1^ ;pDC2的细胞表现型为Lineage^-,HLA-DR^ 和IL-3Rα^ 。结论 成功分离和鉴定了与人相似的恒河猴pDC1和pDC2细胞，对今后异种移植排斥反应的研究具有一定的意义。     

pDC1抑制T细胞增殖的实验研究

目的　研究 pDC1体外对T细胞增殖的抑制作用。方法　采集健康恒河猴外周血 ,用Ficoll Hypaque梯度离心法和三色流式细胞仪分离 pDC1,经混合淋巴细胞培养 (MLR)研究其免疫调整功能。结果　新鲜分离的 pDC1具有较弱的刺激T细胞增殖能力 ;在经CD40L培养后 pDC1即成熟DC1成为有效的T细胞刺激增殖者。结论　本实验用MLR方法成功地研究了恒河猴外周血pDC1体外抑制T细胞增殖的作用 ,为阐明pDC1诱导免疫耐受的作用机制奠定了基础。     

人重症肌无力胸腺树突状细胞的分离和培养

目的　探讨人重症肌无力胸腺树突状细胞 (TDC)的分离方法和诱导培养的最佳条件 ,为研究胸腺树突状细胞的生理功能及其在重症肌无力抗原提呈中的作用奠定基础。方法　新鲜人胸腺组织经机械破碎 ,梯度离心获得胸腺低密度细胞 ,经粒 巨噬细胞集落刺激因子 (GM CSF)和干细胞因子 (SCF)诱导 ,所获得细胞通过透射电镜、扫描电镜观察 ,并用FITC标记的抗人S10 0b、HLA DR、CD11c抗体进行鉴定。结果　从胸腺组织可获取足够数量胸腺树突状细胞 ,体外培养 10～ 18d细胞生长较为活跃 ,第 14天左右达到最高值 ,维持 3～ 4d开始下降 ,2 0d内细胞数仍能维持在 (1.0～ 2 .5 )× 10 5个 /ml。结论　梯度离心法能成功分离胸腺树突状前体细胞 ,GM CSF和SCF体外诱导可提供胸腺树突状细胞原代培养较为满意的生长条件。     

恒河猴外周血调节性T淋巴细胞的分离纯化及鉴定

目的建立恒河猴外周血CD4+CD25+调节性T淋巴细胞(regulatory T cells,Tregs)快速有效的分离纯化方法。方法 4～5岁健康恒河猴10只,雌性4只,雄性6只,体重5～8 kg;于大隐静脉抽取外周血,每只抽取8 mL。密度梯度离心法分离外周血单个核细胞(peripheral blood mononuclear cell,PBMC),分别采用非人灵长类Tregs分离试剂盒的生物素标记的混合抗体和磁珠标记的抗生物素抗体阴性分选,以及大鼠抗人CD4-活化蛋白C(activatedprotein C,APC)和抗APC多选试剂盒中的磁珠标记的抗APC抗体阳性分选出CD4+T淋巴细胞,比较两种方法获得细胞的得率、活性及纯度;选择得率、活性和纯度较高的分选方法获得的CD4+T淋巴细胞,用磁珠标记的抗人CD25抗体进行阳性分选,获得CD4+CD25+Tregs,流式细胞仪检测纯度、活性及FoxP3表达水平,以及Tregs对刀豆蛋白(concanavalinA,ConA)刺激的自体CD4+CD25-效应性T淋巴细胞(effective T cells,Teffs)增殖的抑制功能。结果 CD4+T淋巴细胞阳性分选和阴性分选后,细胞活性均达95%左右,差异无统计学意义(P>0.05),但阳性分选后CD4+T淋巴细胞得率和纯度均显著高于阴性分选(P<0.05)。后续CD4+CD25+Tregs分选采用阳性分选法获得的CD4+T淋巴细胞。经双阳性分选收集的目的细胞中CD4+CD25+Tregs占76.2%±8.6%,活细胞比例为93.3%±4.7%,FoxP3阳性细胞比例为74.2%±6.9%。混合培养后Tregs均对ConA刺激的Teffs的增殖有抑制作用。结论免疫磁珠双阳性分选法能有效分选出恒河猴外周血中有功能的CD4+CD25+Tregs。     

Therapeutic Effect of Chinese Herbal Compound Formula Aikeqing on Simian Immunodeficiency Syndrome Model

[目的]应用猴艾滋病模型评价艾可清治疗艾滋病的效果.[方法]以猴免疫缺陷病毒SIVmac239感染12只恒河猴,复制猴艾滋病模型后分为3组:SIV对照组,艾可清高、低剂量组(剂量分别为445.5、148.5 mg·kg-1·d-1),感染l0周后开始给药.给药8周后停药观察8周.每4周采集外周静脉血,进行外周血CD4+、CD8+T淋巴细胞计数检测,分离血浆进行外周血SIV病毒载量检测.每8周行浅表淋巴结活检,进行苏木素—伊红(HE)染色,光镜下分析浅表淋巴结病理表现.[结果]艾可清高剂量组时猴艾滋病模型外周血CD4+T细胞具有一定升高作用,但停药后会下降;艾可清高剂量组血浆病毒载量均数在治疗第4、第8周时分别下降0.18806l0g和0.806 60 log,艾可清低剂量组治疗8周时则下降0.8142log;艾可清高、低剂量组均使浅表淋巴结病变保持稳定.[结论]艾可清对猴艾滋病模型外周血CD4+T淋巴细胞计数有一定的升高作用,时病毒栽量有一定的抑制作用,使浅表淋巴结病理变化保持稳定.     

调节性树突状细胞在免疫反应中的作用

树突状细胞(dendritic cells,DC)是专职的抗原提呈细胞(APC),起源于骨髓CD34~+造血祖细胞,在各种细胞因子的刺激下分化为前体DC(pDC)、未成熟DC(iDC)和成熟DC(mDC)。DC广泛分布于脑以外的全身组织和脏器,数量较少,仅占人外周血单个核细胞的1%。DC是一类高度异     

树突状细胞与Foxp3+细胞在瘢痕疙瘩中的免疫调节作用

目的 探讨树突状细胞与Foxp3+细胞(T调节细胞,regulatory T cell,Treg)在瘢痕疙瘩发病机制中的相互关系和免疫调节作用.方法 应用磁珠分离、流式细胞术和ELISA法检测瘢痕疙瘩患者(K组,15例)和正常人(N组,15例)外周血中Foxp3+细胞和成熟树突状细胞表面分子MHCII、CD83的表达和功能,分离外周血中树突状细胞分别与CD44 CD25-细胞共培养,测定Foxp3+细胞生成和IL-10的表达.结果 ①K组Foxp3+细胞占CD4+CD25+细胞的(1.45±0.22)%,细胞培养上清中IL-10的浓度为(一),明显低于N组[(5.63±0.95)%,(137±12)ng/L],P<0.05;②K组MHCⅡ+CD83+细胞占(85.47±4.13)%,培养上清中IL-12 p70的浓度为(263±21)ng/L,明显高于N组[(12.79±6.84)%,(一)],P<0.05;③K组DC与CD4+CD25-细胞共培养,3 d后诱导的Foxp3+细胞占CD4+细胞的(0.27±0.18)%,分泌的IL-10浓度为(一),明显低于N组DC与CD4+CD25-细胞共培养诱导的Foxp3+细胞含量和IL-10浓度[(2.53±0.72)%,(79.6±3.24)ng/L],P<0.05.结论 ①外周血中Foxp3+细胞的表达减少,功能降低提示瘢痕疙瘩患者可能存在外周的主动免疫抑制功能减弱;②瘢痕疙瘩患者中树突状细胞与Foxp3+细胞之间存在免疫调节作用;③Foxp3+细胞与瘢痕疙瘩的发病关系密切.     

腺相关病毒载体介导结缔组织生长因子对体外转染恒河猴和人腰椎间盘细胞蛋白多糖和Ⅱ型胶原影响的比较

目的比较腺相关病毒载体介导结缔组织生长因子（connective tissue growth factor,CTGF）对体外培养的恒河猴和人腰椎间盘细胞转染后蛋白多糖含量和Ⅱ型胶原的影响。方法将恒河猴及成人腰椎间盘髓核细胞进行体外培养,应用rAAV2-CTGF体外转染细胞,分别通过^35S标记蛋白多糖的方法和Ⅱ型胶原的SP-ABC免疫组化法检测腺相关病毒载体介导生长因子对椎间盘细胞蛋白多糖合成和Ⅱ型胶原的影响。结果恒河猴椎间盘细胞能进行体外培养并传代,其形态学特性与培养的成人椎间盘细胞相似,rAAV2-CTGF与对照组相比可促进恒河猴和人椎间盘髓核细胞的蛋白多糖生物合成（P〈0．01）,恒河猴和成人相比生长因子对于蛋白多糖的促进作用无明显差异（P〉0．05）。结论成功培养恒河猴及成人椎间盘细胞,腺相关病毒载体介导CTGF能显著促进髓核细胞蛋白多糖的合成,生长因子对恒河猴和人椎间盘细胞蛋白多糖含量的影响具有很大的相似性。     

创伤早期患者外周血树突状细胞的变化及临床意义

目的　探讨创伤后早期患者外周血树突状细胞 (DC)变化及临床意义。方法　分离创伤患者 ( 2 7例 ,创伤组 )和健康人 ( 12例 ,对照组 )外周树突状细胞 ;通过流式细胞仪检测各组的DC数量 (CMRF 44标记法 )及DC表面HLA DR、CD80、CD86表达水平以及DC诱导的T细胞反应性增殖。检测各组外周血上清中白细胞介素 6(IL 6)、IL 10的浓度。结果　创伤组DC细胞数( 7.9± 3 .2 )× 10 6/L明显低于对照组DC ( 14 .9± 5 .1)× 10 6/L(P <0 .0 1)。创伤组DC表面HLA DR及CD80、CD86的表达水平与对照组相比明显下调 (P <0 .0 1)。DC诱导的T细胞增殖能力对照组明显强于创伤组 (P <0 .0 1)。在创伤组中血清IL 6、IL 10的浓度 ( 2 .42± 0 .3 3 ) μg/L和( 1.49± 0 .2 7) μg/L显著升高 ,与对照组比较差异有非常显著性 (P <0 .0 1)。 结论　创伤早期患者外周血DC数量少 ,功能低下 ,与创伤后的免疫功能低下关系密切。     

Dendritic Cell Subset Ratio in Peripheral Blood Correlates with Successful Withdrawal of Immunosuppression in Liver Transplant Patients

Human dendritic cell (DC) subsets appear to play distinct roles in the induction and regulation of immune responses. While monocytoid DC (DC1) induce T-helper (Th) 1-type responses, plasmacytoid DC (DC2) have been reported to selectively induce Th2 responses. In blood, their precursors (p) can be identified as HLA-DR+ lineage- cells that are further characterized as CD11c+ CD123-/lo (IL-3Ralpha-/lo) (pDC1) or as CD11c- CD123hi (pDC2) by rare event, flow cytometric analysis. We compared the incidences of pDC1 and pDC2 in peripheral blood mononuclear cell populations isolated from normal healthy controls and from 3 groups of clinically stable liver transplant patients. Group A had been successfully withdrawn from immunosuppression, whereas group B were undergoing prospective drug weaning and on minimal anti-rejection therapy. In group C, drug withdrawal had either failed or never been attempted and patients were on maintenance immunosuppression. Assessment of DC subsets and the pDC2 : pDC1 ratio showed good intra-and interassay reproducibility. Compared with patients in group C, those in groups A and B demonstrated a significantly higher relative incidence of pDC2 and a lower incidence of pDC1 - similar to those values observed in normal healthy controls. Moreover, the pDC2 : pDC1 ratio was significantly higher in patients undergoing (successful) weaning and in those off immunosuppression compared with patients on maintenance immunosuppression.     

Rare-event analysis of circulating human dendritic cell subsets and their presumptive mouse counterparts.

BACKGROUND: Considerable interest has focused recently on murine CD8alpha- and CD8alpha+ dendritic cell (DC) subsets, because of their roles in initiating and regulating immune responses. Attention has also centered on their presumed human counterparts, DC1 and DC2, respectively, and their precursors. Identification and quantification of these subsets in the blood may be crucial to understanding and monitoring of their immunologic significance, particularly in humans, where blood may be the only tissue readily or routinely available. METHODS: Leukocytes were isolated from anticoagulated human or mouse (C57BL/10J) blood using conventional procedures. Four-color, rare-event, flow cytometric analysis was used to identify DC1 precursors (pDC1; lineage [lin]- CD4+ CD11c+ HLA-DR+) or DC2 precursors (pDC2; lin- CD4+ CD11c- CD123(hi) [IL-3Ralpha(hi)] HLA-DR+) in normal humans. In mice, CD8alpha+ (CD11b(lo), CD11c+) and CD8alpha- (CD11b(hi), CD11c+) DC subsets were identified both in normal animals and after administration of the potent DC growth factor, fms-like tyrosine kinase 3 ligand (Flt3L). RESULTS: All human subjects examined had discrete populations of pDC1 and pDC2 comprising approximately 0.6% and 0.1% of blood mononuclear cells. CD8alpha- and CD8alpha+ DC constituted approximately 0.75% and 0.2%, respectively, of blood mononuclear cells in normal mice, and 12% and 0.5%, respectively, in Flt3L-treated animals. Flt3L administration substantially increased the absolute numbers of circulating CD11c+ DC by approximately 200-fold. CONCLUSIONS: In addition to pDC1 and CD8alpha- DC, pDC2 and CD8alpha+ DC can be identified in normal human or mouse blood, respectively. Monitoring and isolation or characterization of these cells may provide novel insights into their functional significance in transplantation and other clinical conditions.     

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.     

猕猴组织工程化骨异体植入修复骨缺损T淋巴细胞亚群的检测

目的　通过对T淋巴细胞亚群的检测 ,从免疫学方面探讨同种异体细胞来源的组织工程化骨植入猕猴体内修复长段骨缺损的可行性。方法　用骨髓基质干细胞 (MSCs)经诱导分化为成骨样细胞后与生物衍生骨材料复合培养 ,体外构建组织工程化骨 ,植入 15只异体猕猴体内桥接桡骨 2 .5cm节段骨缺损作为实验组 ;用单纯生物衍生骨材料桥接对侧同样大小骨缺损作为对照组 ;分别于术后 1、2、3、6、12周抽取静脉血和作双侧局部组织取材 ,样本应用流式细胞术检测T淋巴细胞亚群CD3 /CD4 /CD45 、CD3 /CD8 /CD45 和CD2 8 阳性率。结果　术后第 1、2、3、6、12周实验组和对照组T淋巴细胞亚群CD3 /CD4 /CD45 、CD3 /CD8 /CD45 和CD2 8 阳性率两者差异均无显著性 (P >0 .0 5 )。结论　生物衍生骨材料和猕猴MSCs复合构建组织工程化骨植入异体猕猴体内其术后 12周内免疫反应水平低 ,可用以修复猕猴节段骨缺损。     

肾移植受者术后早期外周血树突状细胞亚群的动态变化及意义

目的 分析肾移植受者手术前后外周血中树突状细胞(DC)及其亚群骨髓源性DC(mDC)和浆细胞源性DC(pDC)的动态变化,探讨其与排斥反应的关系.方法 检测28例肾移植受者术前,术后1、7和28 d外周血中白细胞总数和单个核细胞数(PBMNC);应用流式细胞术测定DC及其亚群的数量和pDC/mDC.应用酶联免疫吸附试验法测定手术前后血清白细胞介素(IL)-10和IL-12水平.15名健康志愿者作为正常对照.结果 移植组术前外周血DC总数、pDC和mDC数量均低于对照组(P＜0.05),但两组pDC/mDC的差异无统计学意义(P＞0.05).移植组受者术后第1天外周血DC数量骤然降低,然后缓慢上升,第28天恢复至手术前的73.7％;mDC和pDC术后也降低,但mDC恢复较快,pDC恢复缓慢,至术后28 d分别达到术前水平的80.1％和50.1％(P＜0.05).术后第7天,移植组发生排斥反应者mDC数量高于未发生排斥反应者(P＜0.01).受者手术前后IL-10和IL-12的水平变化不明显.结论 DC及其亚群的变化与肾移植受者免疫状态有关,其变化异常提示受者免疫状态不稳定,在受者发生急性排斥反应时,可以作为诊断的参考指标.     

CD40L, CD28, and CTLA-4 expression on CD4+ T cells in kidney graft recipients: a relationship with post-transplantation clinical course

BACKGROUND: Experimental studies have demonstrated that the intensity of alloreactivity against a transplanted organ results from an interaction of positive (CD40/CD40L and B.7/CD28) and inhibitory (B.7/CTLA-4) signals between antigen-presenting cells (APCs) and T lymphocytes. METHODS: We examined the CD40L, CD28, and both surface (s) and intracellular (i) CTLA-4 expressions on freshly drawn and anti-CD3+rIL-2-stimulated peripheral blood CD4+ T cells in groups of kidney transplant recipients in relation to distinct clinical course using the tri-color immunofluorescence method. RESULTS: The median proportions of freshly isolated CD3+/CD4+/CTLA-4+ and CD3+/CD4+/CD40L+ cells in all groups of graft recipients were higher than in control subjects. In patients with stable graft function (SGF), non-significantly higher sCTLA-4, significantly higher iCTLA-4 expression, and significantly lower CD40L expression on freshly drawn CD4+ T cells compared with recipients with chronic allograft nephropathy (CAN) were found. Moreover, CD4+ T cells from SGF patients showed a higher potential to express sCTLA-4 and CD40L molecules and to down-regulate the CD28 molecule in response to ex vivo stimulation than those from patients with CAN. In patients without acute graft rejection (NAGR), a markedly higher proportion of freshly drawn CD3+/CD4+/iCTLA-4+ cells compared with patients with acute graft rejection (AGR) and an up-regulation of the median percentage of CD3+/CD4+/CD40L+ cells after ex vivo stimulation was found. CONCLUSIONS: In patients with SGF, peripheral blood CD4+ T cells exhibited a higher potential to express surface CTLA-4 and CD40L and to down-regulate CD28 costimulatory molecules in response to ex vivo stimulation, indicating a relationship between the expression patterns of both costimulatory and inhibitory molecules in CD4+ T cells and clinical course after renal transplantation.     

腺病毒介导G250基因转染树突状细胞激活免疫效应细胞治疗肾癌的实验研究

目的 探讨以腺病毒(Ad)载体介导肾癌相关抗原G250基因转染制备树突状细胞(DC)瘤苗.体外诱导自体T淋巴细胞特异性抗肾癌免疫效应. 方法 自健康人外周血中提取单核细胞,将贴壁细胞分为3组(Ad-G250基因转染组、G250蛋白致敏组、未致敏组),用粒细胞-巨噬细胞集落刺激因子和诱导活化;3组DC细胞中分别加入自体T淋巴细胞,获得细胞毒性T淋巴细胞(CTL).RT-PCR检测G250在DC细胞内的转录情况;流式细胞仪检测DC表面标志分子和G250抗原蛋白的表达情况;四甲基偶氮唑盐法检测3组CTL对肾癌细胞株786-0和肺癌细胞株A549的杀伤活性. 结果 Ad-G250高效转染DC,G250阳性细胞率为(52.2±1.5)%,G250蛋白在DC:内成功表达:基因转染组DC中成功扩增出G250产物;Ad-G250转染的DC表面标志CD_(80)、CD_(83)、CD_(86)、CD_(1a)、HLA-DR表达高于其他2组.差异均有统计学意义(P<0.05).Ad-G250基因转染组、G250蛋白致敏组、未致敏组诱导的3组CTL对786-0靶细胞杀伤活性分别为(83.4±2.8)%、(79.6±2.4)%、(77.3±2.1)%,组间比较差异有统计学意义(F=69.172,P=0.000);3组CTL对A549靶细胞杀伤活性差异无统计学意义(F=0.373,P=0.693). 结论 以Ad为载体介导抗原基因转染DC,并诱导特异的CTL,技术上可行,所诱导的CTL杀伤活性强,有望成为一种肿瘤免疫治疗方法.     

Peripheral blood dendritic cells in human end-stage heart failure and the early post-transplant period: evidence for systemic Th1 immune responses.

OBJECTIVES: Dendritic cells (DCs) are antigen presenting cells that play a central role in inflammation, allograft rejection and immune tolerance. Myeloid (mDC) and plasmacytoid (pDC) subsets regulate immune reactions by polarising naive T-helper cells into a Th1 or Th2 response, respectively. In this study we examined total peripheral blood DCs, mDC and pDC subsets in chronic heart failure (CHF) and clinical heart transplantation (HTx). METHODS: We compared 16 heart transplant patients before and after HTx to 14 healthy controls. Whole blood was collected pre-HTx and 1-week post-HTx from patients and at corresponding time-points from controls. All patients received induction and maintenance immunosuppression post-HTx. mDCs and pDCs were measured by flow cytometry and were further characterised for maturation and homing potential to the secondary lymphoid organs with CD83 and CCR7, respectively. Data were expressed as absolute numbers/microl whole blood, percentage (%) mDC or pDC of total blood DCs and % positive DCs for CD83 and CCR7. RESULTS: CHF patients had more peripheral blood DCs compared to controls (P<0.01) while only the mDC fraction was increased compared to controls (P=0.01). Percentage CD83(+) and CCR7(+) mDCs was also higher than control levels (P<0.05). One week post-HTx, total DCs, mDCs and pDCs decreased below controls (P<0.001). At the same time % mDCs in peripheral blood increased markedly compared to CHF and control levels (P<0.001). The %CD83(+) mDC, %CD83(+) pDC and %CCR7(+) mDC also returned to control levels and only %CCR7(+) pDC decreased below control levels (P=0.005). CONCLUSIONS: Total peripheral blood DCs are elevated during CHF due to an increase in the mature fraction of the mDC subset suggesting a possible Th1 response in end-stage heart failure. The decrease in total DCs and mature mDCs and pDCs seen post-HTx, probably reflects immunological quiescence through adequate immunosuppression. Peripheral blood DC monitoring may provide a new insight into mechanisms of heart failure and allograft rejection by safe weaning from immunosuppression after clinical HTx.     

Marked inhibition of transplant vascular sclerosis by in vivo-mobilized donor dendritic cells and anti-CD154 mAb

BACKGROUND: Combination of donor dendritic cells (DC) and anti-CD40 Ligand (L) (CD154) monoclonal antibody (mAb) markedly prolongs heart or skin allograft survival, but the influence of this strategy in models of chronic rejection is unknown. Our aim was to ascertain the influence of in vivo-mobilized immature donor DC plus anti-CD40L mAb on vascular sclerosis in functional murine aortic allografts. METHODS: C3H He/J (C3H;H2k) mice received 2 x 106 freshly isolated, immunobead-purified (>90%) fms-like tyrosine kinase 3 ligand-mobilized C57BL/10 (B10;H2b) CD11c+ DC intravenously (IV), together with 500 microg of anti-CD40L mAb (MR1) intraperitoneally (IP) on days -7, 0, 4, and 10. Controls received either no donor cells, no mAb, or were untreated. B10 aortic grafts were transplanted in the abdominal aorta on day 0. At day 30, antidonor T-cell proliferative and cytotoxic responses and both complement fixing and immunoglobulin (Ig)G alloantibody levels were determined. Grafts were harvested on days 30 and 60 and examined by histology and immunohistochemistry. RESULTS: DC infusion alone enhanced ex vivo antidonor proliferative and cytotoxic T-cell activity. By contrast, complement-fixing alloantibody levels were reduced. Anti-CD40L mAb alone strongly suppressed each of these responses. Graft inflammatory cell infiltration, intimal smooth muscle cell proliferation, fibrosis, and elastic lamina disruption observed in untreated animals were reduced in response to anti-CD40L mAb or donor DC alone. Antidonor immune reactivity, including IgG levels, and intimal proliferation were all markedly suppressed to an overall greater extent in mice given both treatments. CONCLUSION: Whereas blockade of the CD40-CD40L pathway ameliorated transplant vasculopathy, preservation of near-normal vessel architecture was achieved by simultaneous administration of donor DC. This strategy represents a novel application of DC for suppression of chronic rejection.