新一代多克隆抗体抑制异种细胞免疫反应的研究

目的 研究新一代兔抗人免疫细胞多克隆抗体(newRALG)对异种细胞免疫反应的抑制作用.方法 应用活化的淋巴细胞和单核细胞致敏新西兰兔后获得newRALG.将PKH-26标记的猪血管内皮细胞(PEC)和正常人单个核细胞(PBMC)建立混合培养体系,培养液中分别加入newRALG、正常兔IgG、Thymoglobulin和Scavenger受体(SR)阻断剂Poly G,培养后收集PBMC,然后分别加入异硫氰基荧光素(FITC)标记的鼠抗人CD14、CD40、CD80、CD86和HLA-DR单克隆抗体.通过流式细胞术检测单核细胞对PEC膜的吞噬作用;通过淋巴细胞与PEC的混合培养体系,加入newRALG,以观察淋巴细胞的增殖反应和newRALG对增殖反应的阻断作用.结果 PBMC与PKH-26标记的PEC共培养后,PBMC中的CD86+单核细胞表达PKH-26,进一步研究发现PKH-26阳性的CD86+单核细胞不但表达CD14、CD86和HLA-DR,同时上调共刺激分子CD40和CD80的表达水平.正常兔IgG(作为阴性对照)对单核细胞吞噬PEC膜无阻断作用,Thymoglobulin具有较低的阻断效果,newRALG与Poly G相似,均具有较高的阻断作用.正常未经刺激的人淋巴细胞不具备增殖能力,经灭活的PEC刺激后,人淋巴细胞具有高水平的免疫增殖反应.正常兔IgG不能阻断PEC对人淋巴细胞的免疫增殖反应;Thymoglobulin的抑制作用随浓度的降低而增强;高浓度的newRALG不能抑制人淋巴细胞的免疫增殖反应,但低浓度的newRALG则显示出强大地抑制人淋巴细胞免疫增殖反应的作用.结论 单核细胞在异种免疫反应过程中发挥重要作用;newRALG可有效抑制单核细胞的吞噬功能和淋巴细胞的免疫增殖反应,从而有效的抑制异种移植排斥反应.     

T淋巴细胞共刺激分子调节单核细胞与内皮细胞相互作用中CD80表达的研究

目的 研究CD4+T淋巴细胞在调节单核细胞与血管内皮细胞相互作用中CDS0的表达水平及其意义.方法 建立单核细胞一血管内皮细胞(EC)共培养和单核细胞-CD4+T淋巴细胞-EC共培养体系,于培养72 h时收集细胞.采用流式细胞术(FACS)检测CD80在CD14+单核细胞表面的表达;通过实时逆转录聚合酶链反应(RT-PCR)技术检测CDS0基因转录的表达变化.建立含有或不含抗CD86、CD28和CD154单克隆抗体的单核白细胞(PBMC)-EC混合培养体系,通过FACS检测CD14+单核细胞表面CDSO的表达,采用混合淋巴细胞-EC反应(MLER)来研究CD54和CD28封闭在抑制淋巴细胞对同种异体EC增殖中的作用.结果 经FACS分析确定,无论是活化还是未活化的EC膜均缺乏CD80、CD86和CD14的表达.RT-PCR表明在缺乏CD4+T淋巴细胞时,经同种EC刺激的单核细胞可上调CD80基因转录水平的表达.但这些CD14+单核细胞膜表面CD80的表达并未上调,当CD4+T淋巴细胞加入到共培养中时,CD80的表达上调.用抗CD28和抗CD86抗体封闭CD28和0986并不能阻止CD14+单核细胞表面CDSO表达的上调.此外,阻断CD154也不能下调CD80的高表达.MLER证明淋巴细胞对EC的增殖反应可部分的被抗CD28和抗CD154抗体所阻断;阻断CD80可抑制经EC刺激的单核细胞诱导T淋巴细胞增殖反应.结论 在缺乏T淋巴细胞的条件下,经同种EC刺激的单核细胞可在基因转录水平上调CD80,而不是在其细胞膜表面的表达.当T淋巴细胞存在时,经EC刺激的CD14+单核细胞可在其膜表面上调CD80的表达.CD14+单核细胞膜表面CD80表达的上调并不能被CD154和CD28封闭所阻止,显示其上调是通过CD154和CD86非依赖性通道.     

核转录因子-κB通路在CD40-CD154诱导血管内皮细胞活化中的作用

目的 探讨血管内皮细胞(VEC)内核转录因子-κB(NF-κB)通路在CD40-CD154相互作用诱导VEC活化中的作用.方法 应用重组人肿瘤坏死因子α(rhTNF-α)和重组人γ干扰素(rhIFN-γ)刺激人主动脉VEC,通过流式细胞仪(FACS)检测CD40和粘附因子表达水平;通过表达CD154的细胞株D1.1和VEC共同培养后,观察VEC粘附因子的表达水平;通过抗CD154单克隆抗体阻断CD40-CD154间的反应;应用NF-κB阻断剂BAY11-7082观察阻断NF-κB通路后对CD40-CD154诱导VEC活化的影响.结果 未活化的VEC膜表达低水平的CD40、CD54和CD106,但不表达CD62E,经白细胞介素刺激后可上调这些蛋白分子的表达水平.D1.1细胞株和VEC共同培养后可诱导VEC活化并上调CD62E、CD54和CD106的表达水平;抗CD154抗体可阻断CD40-CD154间的反应所诱导的VEC活化;NF-κB阻断剂可阻断rhTNF-α诱导的VEC活化以及CD40-CD154间的反应所诱导的VEC活化.结论 VEC中CD40和T淋巴细胞中CD154之间的相互作用在诱导VEC活化过程中起重要作用;CD40-CD154相互作用并通过NF-κB通路诱导VEC活化;NF-κB通路阻断剂可抑制VEC活化.     

阻断OX40和CD40协同共刺激信号延长小鼠胰岛移植物存活时间

目的 研究阻断OX40/OX40L和CD40/CD154L协同共刺激通路对小鼠胰岛移植物存活的影响及其机制.方法 以DBA/2小鼠为供者,C57BL/6小鼠为受者,制作胰岛移植模型.受鼠分为4组.(1)对照组,注射IgG; (2)抗OX40组,注射抗OX40L单克隆抗体;(3)抗CD154组,注射抗CD154单克隆抗体;(4)联合治疗组,注射抗OX40L单克隆抗体和抗CD1 54单克隆抗体.记录各组胰岛移植物平均存活时间(MST).将CD154敲除小鼠处死,取其脾脏T淋巴细胞,体外检测活化T淋巴细胞表面OX40的表达;在活化T淋巴细胞中加入不同浓度的抗OX40L单克隆抗体,体外检测T淋巴细胞增殖情况.结果 对照组胰岛移植物MST为19 d,抗CD154组胰岛移植物MST为48 d(P＜0.05);抗OX40组胰岛移植物MST为22 d,与前两组相比较,差异无统计学意义(P＞0.05);联合治疗组胰岛移植物MST＞ 150 d,高于另外3组(P＜0.05).66％的胞表达OX40,较初始T淋巴细胞的表达率高(2％,P＜0.05);加入抗OX40L单克隆抗体后,T淋巴细胞增殖受抑制且呈剂量依赖性.结论 阻断OX40/OX40L和CD40/CD154L双通路可诱导小鼠胰岛移植物长期存活, 其发挥作用的关键机制是抑制了T淋巴细胞的增殖.     

西罗莫司和环孢素A对Th17细胞和调节性T淋巴细胞分化影响的比较

目的 比较西罗莫司(SRL)和环孢素A(CsA)对体外诱导CD4+T淋巴细胞向Th17和调节性T淋巴细胞分化的影响.方法 使用抗CD3和抗CD28单克隆抗体(简称抗CD3单抗和抗CD28单抗)刺激CD4+T淋巴细胞,并在培养体系中分别加入转化生长因子β(TGF-β);TGF-β+白细胞介素6(IL-6);TGF-β+IL-6+SRL;TGF-β+IL-6+CsA.72 h后用流式细胞术检测细胞内Foxp3和IL-17的表达水平,观察CD4+T淋巴细胞的分化情况及SRL和CsA对CD4+T淋巴细胞体外分化的影响.结果 在经抗CD3单抗和抗CD28单抗刺激后,TGF-β可诱导Foxp3+细胞(调节性T淋巴细胞,Treg)的增殖与分化,而TGF-β+ID6可诱导Th17细胞的增殖与分化.在培养体系中加人SRL和CsA,均可使Th17细胞的增殖与分化减少;SRL可促进Treg的增殖与分化,而CsA抑制Treg的增殖与分化.结论 SRL可以促进CD4+T淋巴细胞向Treg增殖与分化,而抑制Th17细胞的增殖与分化;CsA既抑制Treg的增殖与分化,又抑制Th17细胞的增殖与分化.     

第三方未成熟树突状细胞负载异体抗原诱导免疫耐受的实验研究

目的观察第三方未成熟树突状细胞（imDC）负载同种异体抗原后对其免疫特性的影响。方法从健康足月新生儿脐血中分离单核细胞，采用重组人粒细胞巨噬细胞集落刺激因子（rhGM-SCg）和重组人白细胞介素（rhIL）4联合培养7d，诱导其分化成imDC，并通过光学显微镜和扫描电镜观察细胞形态、检测细胞表型及混合淋巴细胞反应（MLR）；将培养的细胞与异体淋巴细胞抗原共同孵育，并给予共刺激分子阻断剂细胞毒性T淋巴细胞相关抗原4免疫球蛋白（CTLA-4Ig）处理，检测抗原负载前后的细胞表型变化，通过MLR比较致敏前后T淋巴细胞增殖的能力。结果（1）培养后细胞具有典型的imDC特征，CD1α、CD83、CD80、CD86等成熟标志呈低表达，分别为21．42％、0．59％、5．39％、3．85％；人类白细胞DR抗原（HLA-DR）的表达率为60．66％，MLR结果提示其不能刺激同种异体T淋巴细胞增殖。（2）负载抗原后的DC表现出成熟特性，CD1α、CD83、CD80、CD86及HLA-DR表达明显增高，分别为65．51％、42．20％、56．45％、38．52％、76．44％（P〈0．05）；能够刺激T淋巴细胞增殖[刺激指数（SI）〉2．00]。（3）给予共刺激阻断剂CTLA-4Ig致耐处理后，SI由2．51下降到0．39。可明显抑制T淋巴细胞增殖。结论第三方imDC负载抗原后能表现出成熟特性；经CTLA-4Ig致耐处理，不能刺激未致敏T淋巴细胞增殖。有望诱导受体针对供者抗原的特异性免疫耐受。     

转化生长因子-β1差异性诱导CD4+和CD8+T细胞表达转录因子FOXP3的研究

目的 研究CD4+T细胞和CD8+T细胞被转化生长因子-β1(TGF-β1)诱导表达转录因子FOXP3的差异性.方法 给予CD4+T细胞和CD8+T细胞抗T细胞受体(TCR)刺激的同时,加入TGF-β1培养4d后,通过胞内细胞因子染色,分析二者被诱导表达FOXP3的情况.同时通过CFSE标记实验检测被TGF-β1诱导表达FOXP3的淋巴细胞的增殖情况;通过annexin V流式染色检测TGF-β1对于T淋巴细胞凋亡的影响.结果 相对于CD8+T淋巴细胞,TGF-β1主要诱导CD4+T淋巴细胞阳性表达FOXP3[外周血单个核细胞(PBMC):29.66±3.624比7.430±0.643;癌旁组织浸润淋巴细胞(NIL):31.74±2.612比8.637±1.146].被TGF-β1诱导表达FOXP3的淋巴细胞增殖活跃(94.39±1.179),受到活化刺激后依旧能够有效分泌干扰素-γ(IFN-γ)效应细胞因子(39.58±1.611).TGF-β1能够有效降低CD8+T淋巴细胞活化后的细胞凋亡率(25.39±2.158比9.320±0.3219).结论 与肝癌患者FOXP3表达阳性的淋巴细胞＞95％(97.15±0.3807,n=10)集中在CD4+T淋巴细胞的结果一致,TGF-β1优先选择性诱导CD4+T细胞表达FOXP3.与天然Treg(调节性T细胞)低增殖活性和低细胞因子分泌活性不同,被TGF β1阳性诱导表达FOXP3的T细胞依旧具有活跃的细胞增殖和分泌IFN γ效应细胞因子的功能.CD8+T淋巴细胞相对于CD4+T淋巴细胞,更易发生活化后细胞凋亡,而TGF-β1能够有效降低这种细胞凋亡率.     

CTLA4Ig与CD40Ig局部共转染诱导异种大鼠移植肾免疫耐受的影响

CD28-B7和CD40-CD40L共刺激分子系统是T细胞依赖的免疫反应启动和维持所必不可少的基本信号系统.缺乏或阻断共刺激信号,将会引起T细胞的无反应性或细胞凋亡.我们用CTLA4Ig和CD40Ig双基因局部转染大鼠肾脏后进行异种移植,观察双基因局部共转染能否诱导移植肾免疫耐受.     

RATG对CD4+细胞和CD8+细胞共刺激分子基因表达和细胞因子分泌的影响

目的 探讨兔抗人胸腺细胞多克隆抗体(RATG)在体外对CD4+细胞和CD8+细胞共刺激分子基因表达和细胞因子分泌的影响.方法 从正常成人外周血单个核细胞中分离和纯化CD4+细胞和CD8+细胞,加入RATG,37℃下培养.分别于24、48和72 h收集培养上清液和细胞,以不处理的细胞为正常对照,正常兔Ig处理的细胞作为阴性对照.采用实时聚合酶链反应技术检测培养细胞的细胞毒性T淋巴细胞相关抗原4(CTLA4)、CD154、Foxp3、OX40、γ干扰素(IFN-γ)、白细胞介素2(IL-2)、IL-10和IL-2受体(CD25)的基因表达水平,应用Multiplex检测技术测定培养上清液中IFIN-γ、IL-2、IL_4和IL-10的水平.结果 与正常CD4+细胞比较,加入RATG的CD4+细胞培养24 h,其CTLA-4、CD154、Foxp3、OX40、IFN-γ、IL-2、IL-10和CD25基因转录表达均上调,阴性对照CD4+细胞则无这些基因转录表达的上调.处理48 h后,CD4+细胞的CD154和IL-2的基因转录表达呈现下调现象,而CTLA4、Foxp3、0X40、IFN-γ、IL-10和CD25的基因转录水平均较24 h时明显降低.处理72 h后,CD4+细胞的CTLA4、Foxp3、OX40和CD25的基因转录表达再次呈现高水平,CD154和IFN-γ的基因转录表达上调表达不明显,而IL-2和IL-10的基因转录表达则呈现明显的下调.RATG处理的CD4+细胞培养上清液中的IFN-γ、IL-2、IL-4和IL-10浓度显著增加,以处理24 h的水平最高,而阴性对照者未测出.与正常CD8+细胞相比较,加入RATG处理的CD8+细胞培养24 h,其CTLA4、Foxp3、OX40、IFN-γ,、IL-2、IL-10和CD25的基因转录表达呈现明显上调,而CD154基因转录表达稍有下调.RATG处理48 h,CD8+细胞的CTLA4、Foxp3、OX40、IFN-γ和CD25基因转录表达仍维持在高水平,CD154基因转录表达仅仅呈现低水平的上调,IL-10基因转录表达水平显著下降,而IL-2的基因转录表达则明显下调.处理72 h,CD8+细胞的CTLA4、Foxp3、OX40、IFN-γ、IL-10和CD25的基因转录表达仍维持在高水平,CD154基因转录表达则呈现下调,而IL-2基因转录表达的下调更为显著.阴性对照CD8+细胞则未呈现这些基因转录的上调现象.RATG处理的CD8+细胞培养上清液中IFN-γ、IL-2和IL-10显著增多,以处理24 h的浓度最高,IL-4浓度升高的幅度较小,而正常CD8+细胞和阴性对照CD8+细胞几乎检测不到IFN-γ、IL-2、IL-4和IL-10.结论 在体外,RATG可以刺激CD4+细胞和CD8+细胞上调多种促进免疫抑制的共刺激分子基因表达,促进其分泌与免疫调节相关的IFN-γ、IL-2、IL-4和IL-10.     

小鼠补体调节蛋白对CD4+T淋巴细胞的调控作用及其机制

目的 研究小鼠补体调节蛋白Crry对CD4+T淋巴细胞的调控作用及诱导同种移植免疫低反应性的机制.方法 分离C57BL/6小鼠脾淋巴细胞,用免疫磁珠法分选出CD4+T淋巴细胞后,将CD4+T淋巴细胞分为A、B、C、D、E和F组,分别用抗小鼠CD3、CD28、Crry、CD3/CD28、CD3/Crry和CD3/CD28/Crry抗体共刺激通路与CD4+T淋巴细胞进行反应,采用噻唑蓝(MTT)法检测各组CD4+T淋巴细胞的增殖情况,并采用酶联免疫吸附试验检测CD4+T淋巴细胞培养上清中白细胞介素2(IL-2)、γ干扰素(γ-IFN)、IL-4和IL-10的水平;另外,以BALB/c小鼠和C57BL/6小鼠的脾细胞分别作为刺激细胞和反应细胞,建立同种混合淋巴细胞反应(MLR)体系并加入抗小鼠Crry抗体,通过岍法观察Crry对MLR的影响.结果 D、E、F组的CD4+T淋巴细胞均出现明显增殖,增殖活性显著高于A、B、C组(P<0.05),其中F组显著高于D组和E组(P<0.05),D组和E组间增殖活性的差异无统计学意义.D组CD4+T淋巴细胞经抗CD3/CD28抗体共刺激后,培养上清中γ-IFN和IL-2的水平显著升高,与A、B、C和E组比较,差异均有统计学意义(P<0.05),但与F组的差异无统计学意义;E组CD4+T淋巴细胞经抗CD3/Crry抗体共刺激后,IL-4的水平显著升高,与A、B、C、D组比较,差异均有统计学意义(P<0.05),但显著低于F组(P<0.05);各组间IL-10水平的差异无统计学意义.Crry可以明显抑制MLR中的细胞增殖(P<0.05).结论 补体调节蛋白Crry能刺激CD4+T淋巴细胞的增殖,并使其IL-4的表达升高及抑制IL-2和γ-IFN的表达,从而诱导同种移植免疫低反应性.     

CD4+ T-cell and monocyte interdependence during discordant xenoimmune responses

This study was designed to examine our hypothesis that human monocytes provide missing constimulatory signals to host CD4⁺ cells during interactions with porcine endothelial cells (PECs). PECs were isolated from the aorta. Human CD4⁺ T cells and monocytes were purified from peripheral blood mononuclear cells (PBMCs). A xenogeneic mixed lymphocyte-PEC reaction (xMLER) was performed to determine the proliferation of PBMCs or CD4⁺ cells in response to PEC. Monocyte-PEC cocultures with or without CD4⁺ cells were followed by analysis using fluorescence-activated cell scanning (FACS). We evaluated the CD4⁺ cells proliferation induced by PEC-conditioned monocytes with or without costimulation blockade. xMLER demonstrated strong lymphocyte proliferation in response to PECs. However, purified CD4⁺ cells showed reduced proliferative responses to PECs when compared with PBMCs. FACS analysis found that CD14⁺ monocytes up-regulated CD40 and CD80 expressions in the presence of CD4⁺ cells. PEC-activated but not resting monocytes induced CD4⁺-cell proliferation, which was inhibited by anti-CD154, anti-CD80, or anti-CD86 antibodies. In summary, human monocytes exposed to PECs are conditioned to up-regulate costimulatory molecules upon exposure to T cells. PEC-conditioned monocytes induced T-cell proliferation by indirect presentation. Costimulation blockade inhibited T-cell proliferation induced by PEC-conditioned monocytes. Our findings suggested that monocytes play an important role in indirect xenoantigen presentation, providing costimulation to T cells. This interaction can occur distant from the initial site of xenoantigen, but monocytes remaide void of costimulatory signals until their interaction with T cells.     

Rabbit anti-human leukocyte polyclonal antibody inhibits xenogeneic cell-mediated immune responses

We studied the interactions between human monocytes and porcine endothelial cells (PEC) as well as the effects of a new generation of rabbit anti-human leukocyte polyclonal antibody (newRALG) to inhibit xenogeneic cell-mediated immune responses. Human peripheral blood mononuclear cells (PBMC) were cocultured with the florescent dye PKH-26 labeled-PEC, which showed membrane uptake by monocytes detected by florescence activated cell scanning (FACS). Scavenger receptor (SR) ligand poly-(G) or the newRALG or Thymoglobulin was added into the cocultures followed by FACS. Lymphocyte proliferation upon exposure to PEC with or without newRALG or thymoglobulin was evaluated by a xenogeneic mixed-lymphocyte-endothelial cell reaction (xMLER). FACS analysis demonstrated that CD14⁺ monocytes became positive for PKH-26 following their interaction with PKH-26-labeled PEC. These PKH-26⁺ monocytes displayed up-regulated CD40 and CD80 expression during the PBMC-PEC interaction. Furthermore, SR blockade with poly-(G) prevented PEC membrane uptake by CD14⁺ monocytes. The newRALG from rabbits immunized with activated human monocytes and lymphocytes greatly reduced SR-mediated PEC membrane uptake. xMLER demonstrated strong lymphocyte proliferation in response to PEC. Lymphocyte proliferation was dramatically inhibited in dose-dependent manner by the newRALG. In summary, monocytes up-regulate costimulatory molecules during xenogeneic interactions, indicating that they may serve as a source of T-cell costimulation during xenogeneic reactions, enguling PEC membranes. This phenomenon was inhibited by poly (G), suggesting that PEC membrane uptake was via SR. The newRALG inhibited monocyte SR-mediated PEC membrane uptake and lymphocyte proliferation in response to PEC suggesting that this new polyclonal preparation may impair the initiation of xeno-specific immune responses.     

The interleukin-2 receptor α chain (CD25) plays an important role in regulating monocyte-derived CD40 expression during anti-porcine cellular responses

Long-term xenograft survival is limited by delayed xenograft rejection, and monocytes are thought to play an important role in this process. Although typically considered a T cell surface marker, interleukin 2 the receptor chain CD25 is also functional on monocytes. We hypothesized that CD25 expression on monocytes functions to augment monocyte activation in xeno-specific cellular responses. Xenogeneic mixed lymphocyte-endothelial cell reactions were used to study the role of CD25 in facilitating xenogeneic cell-mediated immune responses an in vitro. We also tested the effect of the anti-CD25 antibody daclizumab on monocyte-mediated T cell activation during xeno-specific cellular responses. Co-culture with porcine endothelial cells (PEC) elicited a pronounced proliferative response by human peripheral blood mononuclear cells (PBMC) that was accompanied by upregulation of CD25 and CD40 on CD14⁺ monocytes. CD4⁺ cells proliferated in response to PEC-conditioned monocytes, while blockade of CD25 with daclizumab reduced CD4⁺ cell proliferation in the presence of PEC-conditioned monocytes. In addition, daclizumab inhibited proliferation of PBMC in responses to PEC. Analysis of monocytes from PBMC-PEC cocultures by flow cytometry indicated that daclizumab inhibited CD40 upregulation on PEC-activated monocytes. These data demonstrate that CD25 blockade prevents xenogeneic cellular responses by directly blocking CD25 expression on both activated T cells and monocytes. CD25 blockade on T cells or monocytes may indirectly affect upregulation of CD40 on xenoreactive monocytes. Our data strengthen the rationale for incorporating CD25 directed therapy in discordant xenotransplantation.     

Stimulating PD-1-negative signals concurrent with blocking CD154 co-stimulation induces long-term islet allograft survival

BACKGROUND: A balanced network of positive and negative T-cell co-stimulatory signals is important in regulating T-cell activation. Blocking CD28, CD154 (CD40L), or both co-stimulatory molecules has been efficacious in preventing acute allograft rejection in certain but not all transplantation models. In the present study, the authors tested the hypothesis that stimulating programmed death 1 (PD-1)-triggered negative signals concurrent with blocking CD154 co-stimulatory signals would facilitate islet allograft tolerance. METHODS: The authors used a dimeric PD-L1 immunoglobulin (Ig) fusion protein to stimulate the inhibitory receptor PD-1, and a monoclonal antibody to block CD154. The effects of PD-1 engagement and CD154 blockade on lymphocyte activation were determined by cell proliferation, flow cytometry, and a model of islet transplantation. RESULTS: PD-L1Ig inhibited the proliferation of both CD4+ and CD8+ T cells stimulated by anti-CD3. The inhibitory effect of PD-L1Ig was enhanced by concurrent blockade of CD154 co-stimulatory signals, as demonstrated by T-cell proliferation and expression of cell surface activation markers. PD-L1Ig and anti-CD154 also synergistically blocked the activation and maturation of antigen-presenting cells. In an islet transplantation model, treatment of recipient C57BL/6 (H-2b) mice with PD-L1Ig and anti-CD154 induced long-term survival of DBA/2 (H-2d) islet allografts, whereas treatment with each reagent alone failed to prevent islet allograft rejection. CONCLUSIONS: These results suggest that engaging the negative receptor PD-1 exhibits critical immunoregulatory effects in the allograft response, and blocking positive co-stimulatory molecules with active delivery of inhibitory signals may represent a novel therapeutic strategy in transplantation.     

A nuclear factor-kappaB inhibitor BAY11-7082 inhibits interactions between human endothelial cells, T cells, and monocytes

Costimulatory molecules play critical roles during cell-mediated immune responses. We undertook this study to determine whether CD154-CD40 interactions induced human endothelial cell (EC) activation via the nuclear factor (NF)-κB pathway, and whether the upregulation of monocyte-derived CD40 and CD80 is NF-κB pathway dependent. A CD154-expressing D1.1 cell-EC coculture with or without the NF-κB inhibitor BAY11-7082 was established to examine EC activation as indicated by CD62E expression. Peripheral blood mononuclear cell (PBMC)-EC cocultures were performed in the presence or absence of BAY11-7082; the expression of CD40 and CD80 on monocytes was analyzed by FACS. Allogeneic mixed lymphocyte-EC reaction (MLER) was performed to determine the inhibitory effects of BAY11-7082 to prevent lymphocyte proliferation. FACS demonstrated upregulation of EC-derived CD62E expression induced by CD154 expressing D1.1 cells. BAY11-7082 pretreated EC failed to upregulate CD62E after interaction with D1.1 cells. Monocytes upregulated CD40 and CD80 expression during PBMC-HEC interaction, and BAY11-7082 suppressed monocyte-derived CD40 and CD80 expression in a dose-dependent manner. The monocyte-derived CD86 expression was downregulated by NF-κB inhibitor. BAY11-7082 demonstrated inhibition of lymphocyte proliferation of allogeneic MLER. This study demonstrated that the NF-κB inhibitor BAY11-7082 prevented CD154-CD40 interaction-induced EC activation, suggesting that the activation of EC by T-cell-derived CD154 is via NF-κB pathway. The NF-κB inhibitor suppressed upregulation of monocytederived CD40 and CD80. Additionally, BAY11-7082 suppressed lymphocyte proliferation in response to allogeneic EC. These data indicated that NF-κB plays an important role in regulating costimulatory molecules in allogeneic immune responses, and strengthens the rationale for the use of NF-κB-directed therapy in allotransplantation.     

Costimulatory molecules are active in the human xenoreactive T-cell response but not in natural killer-mediated cytotoxicity

BACKGROUND: T-cell costimulatory blocking agents inhibit allospecific T-cell responses in vitro and prevent allograft rejection in vivo. Costimulatory requirements for discordant xenospecific cellular responses remain undefined. We have evaluated costimulatory molecule expression by porcine endothelial cells (PEC) after interaction with human cells and tested agents known to inhibit allospecific responses for their ability to inhibit xenospecific responses in vitro. METHODS: Human-specific agents were screened for their ability to bind porcine costimulatory molecules by FACS. Up-regulation of B7 molecules on PEC was evaluated by FACS after exposure to human cells or supernatants. The effect of human and/or porcine costimulatory blockade was tested in xeno-mixed lymphocyte reactions (XMLRs) and in natural killer (NK) cell cytotoxicity assays. RESULTS: B7 expression was induced on PEC after exposure to human T and NK cells or T cell-conditioned medium. The human XMLR was attenuated by human CTLA4-Ig and anti-human CD154 (hu5C8), and the combination was synergistic. Anti-human CD80 and CD86 antibodies alone had minor effects in the XMLR, but in combination with hu5C8 were as effective as human CTLA4-Ig plus hu5C8. Anti-hCD80 and hCD86 antibodies that did not cross-react with porcine CD80 or CD86 were as effective in blocking the MLR as those that did cross-react, indicating that the predominant costimulation in vitro was derived from the responding cells. None of the agents affected the xeno-NK response. CONCLUSIONS: We conclude that the costimulation-modulating agents block human anti-porcine T-cell responses in vitro predominantly through interruption of costimulation derived from responding cells. They have no effect on NK cell-mediated cytotoxicity.