白细胞介素-12 p35沉默的树突状细胞联合CD40L单克隆抗体对大鼠小肠移植免疫耐受的诱导作用

目的研究供体骨髓来源的白细胞介素12 p35(IL-12p35)基因沉默的树突状细胞(IL-12 p35 silenced DC)联合大鼠CD40L单克隆抗体(CD40L mAb)对大鼠小肠移植免疫耐受的诱导。方法实验动物随机分为3组,每组8对,分别于手术前7天进行不同预处理后进行小肠移植。A组:受体注射生理盐水;B组:受体注射供体IL-12 p35 silenced DC,C组:受体静脉注射供体IL-12 p35 silenced DC,同时腹腔注射CD40L mAb。观察受体存活时间,移植小肠采用病理学检察及细胞凋亡检测,受体血清检测IL-2、干扰素(INF)-γ水平。结果C组受体动物存活时间为(23.3±6.0)d,显著长于A、B两组[(6.5±1.5)d,(15.1±4.9)d];C组移植小肠炎性细胞浸润、黏膜结构破坏程度和肠黏膜细胞的凋亡数目明显低于A、B组[(9.5±5.6)比(23.8±6.2)、(18.7±6.3),(P<0.01)];C组受体大鼠血清IL-2浓度为(185.8±18.2)ng/L,显著低于A、B两组[(294.1±15.6)ng/L、(225.8±14.5)ng/L,(P<0.01、P<0.05)];C组受体大鼠血清INF-γ浓度为(75.6±8.6)ng/L,显著低于A、B两组[(110.5±12.4)ng/L、(89.1±9.2)ng/L,(P<0.01、P<0.05)]。结论术前输注供体来源的IL-12 p35 silenced DC联合CD40L mAb,可在一定程度上抑制小肠移植的排斥反应,诱导受体产生免疫耐受。     

大鼠小肠移植早期应用趋化因子受体拮抗剂抑制移植物急性排斥反应

目的探讨趋化因子受体拮抗剂(MetRANTES)在小肠移植早期应用对排斥反应的免疫抑制作用及其与他克莫司的协同效应。方法SD大鼠为供者,Wistar大鼠为受者,建立异基因节段性异位小肠移植模型。移植后将大鼠分为4组,每组24只。第1组为对照组,移植前后不作任何处理;第2组为MetRANTES组,小肠移植后(0～7d)腹腔注射MetRANTES200μg/d;第3组为小剂量他克莫司(FK506)组,小肠移植后(0～7d)腹腔注射FK5060.5mg·kg-1·d-1;第4组为MetRANTES联合FK506组,2种药物的应用方法与第2、3组相同。观察移植大鼠的一般状况和存活时间以及免疫细胞浸润情况,并于移植术后3、5、7d分别取各组大鼠移植肠标本(n=6)进行组织病理学检查,采用免疫荧光染色和激光扫描共聚焦显微镜技术对移植肠RANTES和CD4 、CD8 、CD25 T淋巴细胞的表达进行连续定量测定。结果第1～4组大鼠平均存活时间分别为(7.37±1.19)d、(22.32±8.60)d、(23.64±6.58)d和(30.55±4.18)d,第2、3、4组大鼠与第1组相比,存活时间明显延长(P<0.01);第4组大鼠存活时间更长,与第2、3组比较,差异均有统计学意义(P<0.01)。第1组全部死于急性排斥反应及感染,组织病理学检查显示移植后第3、5、7d分别符合轻、中、重度排斥反应。第2、3、4组病理学检查无明显排斥反应征象。第1组大鼠的移植肠RANTES表达在术后各时段均显著高于其他3组(P<0.01),其动态变化与急性排斥反应的进程呈正相关;第2组和第4组大鼠移植肠RANTES、CD4 、CD8 和CD25 T细胞的表达均明显低于第1组(P<0.01)。结论MetRANTES能明显抑制小肠移植急性排斥反应,有效保护移植肠功能,显著延长移植物的存活时间,并可增强小剂量他克莫司的免疫抑制作用。     

主要组织相容性复合体基因胸腺转移减轻异种移植排斥反应的实验研究

目的探讨主要组织相容性复合体(MHC)基因胸腺转移减轻异种移植排斥反应的可行性。方法通过分子克隆技术,提取、扩增供体小鼠(Balb/c)的H-2Kd基因(小鼠MHC-Ⅰ类基因),构建表达型载体质粒pCI-H-2Kd。受体选用20只SD大鼠,采用随机数字表法分为实验组和对照组,每组10只。采用超声引导下穿刺和脂质体转染的方法,实验组受体胸腺注射pCI-H-2Kd;对照组受体胸腺注射空pCI-neo载体质粒。两组随即均行小鼠→大鼠耳后心肌移植,观察异种移植排斥反应。结果实验组异种移植心肌的存活时间较对照组明显延长,差异有统计学意义(14.61±2.98dvs.6.40±1.58d,t=-7.619,P0.05);移植心肌病理切片显示:对照组有较多淋巴细胞浸润,移植心肌大部分坏死;实验组无淋巴细胞浸润,尚有较多心肌存活;混合淋巴细胞反应:实验组对供体小鼠反应明显低于对照组(t=4.758,P=0.000);流式细胞学检测:大鼠胸腺细胞表面表达异种MHC分子,转染效率达60.7%。结论 MHC基因胸腺转移可以减轻异种移植排斥反应,为异种移植免疫耐受的实现提供理论和实验依据。     

输血诱导免疫耐受对大鼠小肠移植急性排斥反应的影响

目的 观察供体特异性输血(DST)诱导免疫耐受对大鼠小肠移植后急性排斥反应的影响.方法 采用SD至Wistar大鼠异位小肠移植模型,实验组分别予以DST,环孢素(CsA)及DST联合CsA干预,Wistar大鼠同系间移植作为对照,于3、5、7 d观察移植肠管病理变化及受体血清中肿瘤坏死因子(TNF)-α和干扰素(IFN)-γ表达程度.结果 病理显示CsA干预组在7 d出现轻度移植排斥反应;DST联合CsA干预组与对照组结果相似,在3、5、7 d均无明显的移植排斥反应发生.并且DST联合CsA干预组TNF-α表达程度低于单独CsA干预组,在第7天时与对照组比较差异无统计学意义(P＞0.05);IFN-γ表达程度低于单独CsA干预组,在第5、7天时与对照组比较差异无统计学意义(P＞0.05).结论 异基因大鼠间小肠移植在CsA配合应用的情况下,进行DST可诱导其产生一定的免疫耐受,预防及减轻大鼠小肠移植急性排斥反应的发生及反应程度.     

RNA干扰技术阻断CD40/CD40L共刺激通路对小鼠移植心存活时间的影响

目的 探讨慢病毒介导RNA干扰(RNAi)技术阻断CD40/CD40L共刺激通路对小鼠移植心存活时间的影响.方法 以针对小鼠CD40基因的RNAi慢病毒载体在体外感染供者骨髓来源的树突状细胞(DC),制备低表达CD40的耐受性DC(Tol-DC).荧光实时定量聚合酶链反应及流式细胞术检测DC感染前后CD40 mRNA及DC表面抗原CD40、CD11c和MHCⅡ的表达.建立小鼠异位腹腔心脏移植模型.在小鼠异位心脏移植前7 d,经静脉给受者输注体外制备的低表达CD40的Tol-EC(慢病毒感染DC注射组),并设置单纯移植对照组和未感染DC注射组作为对照.观察各组移植心的存活时间,评定各组术后第7天移植心排斥反应的病理分级.结果 CD40-RNAi慢病毒载体在体外感染DC 48 h后,CD40 mRNA表达明显受到抑制,抑制率为80.9%;CD40表达明显下降,由(74.37±4.08)%降至(40.07±4.03)%(P＜0.05).单纯移植对照组、未感染DC注射组和慢病毒感染DC注射组移植心存活时间分别为:(8±2)d、(9±1)d和(14±4)d,慢病毒感染DC注射组移植心存活时间明显延长,并且移植心排斥反应病理分级显著降低,差异有统计学意义(P＜0.05).结论 阻断CD40/C40L共刺激通路可抑制异系T淋巴细胞的活化,从而抑制急性排斥反应,延长小鼠移植心的存活时间.     

胸腺修饰诱导同种异体静脉移植的免疫耐受研究

目的 观察大鼠胸腺内注射异基因抗原在同种异体异基因股静脉移植免疫耐受中的作用.方法 将48只SD大鼠随机分为4组:自体股静脉移植组(A组)、异体股静脉移植组(B组)、异体股静脉移植免疫抑制剂组(C组)、胸腺内注射供体组织相容性(MHC)抗原后移植组(D组).于2周后进行影像学、组织学、免疫学检测.结果 组织学检测结果显示:D组、C组急性排斥反应损伤较轻,B组血管壁的各层结构破坏最重,可见大量炎性细胞浸润.B组受体大鼠血清干扰素(IFN)-γ浓度为(86.707±10.928)ng/L,显著高于A、C、D组[(29.328±4.170)、(69.076±8.059)、(63.355±4.895)ng/L,P<0.05];B组受体大鼠血清白细胞介素(IL)-4浓度为(23.656±3.369)ng/L,显著低于C、D组[(29.425±4.174)、(31.000±4.659)ng/L,P<0.05].结论 胸腺内注射异基因MHC抗原可诱导大鼠对同种异体血管移植的特异性免疫耐受.     

CD4+CD25+T细胞联合CD154单抗抑制大鼠肝移植急性排斥反应的研究

目的探讨CD4 CD25 T细胞联合应用CD154单抗在抑制大鼠肝移植急性排斥反应中的作用。方法分离Lewis大鼠脾脏CD4 _CD25 T细胞后与DA大鼠脾细胞单向混合淋巴细胞反应行体外激活。用"二袖套法"行DA到Lewis的原位肝移植48例。A组为对照组;B、C组单独术前回输体外激活的CD4 CD25 T细胞或术后腹腔注射抗CD154单抗;D组联合应用CD4 CD25 T细胞和CD154单抗。每组大鼠12对。术后7 d各组处死6只受体,检测移植肝内T细胞亚群和细胞因子水平。余大鼠观察生存情况,死亡大鼠观察移植肝病理变化。结果D组受体生存期(52.00±10.64)d明显长于B、C组(P<0.01);移植肝内CD4 CD25 T细胞比例(16.43±4.28)%明显高于B、C组(P<0.05、P<0.01),而淋巴细胞浸润数量[(3.47±1.21)%×106]和(CD8 T细胞百分比(14.19±3.02)%明显低于B、C组(P<0.05、P<0.01);移植肝内白细胞介素- 2(IL-2)水平(6.44±1.83)ng/L低于B、C组(P<0.05),IL-10(43.72±7.55)ng/L和转化生长因子-β1(TGF-β1)(270.06±46.91)ng/L明显高于B、C组(P<0.05、P<0.01)。结论联合应用CD154单抗能明显增强CD4 CD25 调节性T细胞对大鼠肝移植急性排斥反应的抑制作用。     

大鼠原位和异位小肠移植慢性排斥反应模型的制作与比较

目的比较大鼠原位和异位小肠移植慢性排斥反应模型的建模效果。方法采用F344(RT11vr)大鼠作为供体,Lewis(RT11)大鼠作为受体,构建异系异位和原位大鼠小肠移植模型(各8只),术后0~14 d给予皮下注射环孢素。观察术后受体的体质量变化及存活时间。采用苏木素-伊红(HE)染色观察肠组织病理学变化,酒精苏木素染色后观察肠组织胶原纤维和弹性纤维的变化。计算两组受体大鼠的成模率。结果异位和原位小肠移植大鼠均能长期存活,大部分超过90 d。原位小肠移植组大鼠术后第3日恢复正常饮食,于术后14 d左右体质量可恢复至术前水平,之后缓慢增长,但大部分原位小肠移植大鼠在术后150 d出现持续的体质量下降,且不能被环孢素逆转。异位小肠移植组大鼠术后第1日恢复进食,于术后25~30 d才能恢复至术前的体质量水平,术后30~90 d期间,体质量逐渐上升并保持在较高水平。原位小肠移植组大鼠术后90 d的小肠组织未出现慢性排斥反应的病理学改变且未见明显纤维化,术后163 d和术后200 d的小肠组织出现慢性排斥反应的病理学改变,且出现移植小肠系膜纤维化。异位小肠移植组大鼠术后90 d和术后200 d的小肠组织均出现典型的慢性排斥反应的病理学改变和移植小肠系膜纤维化。异位小肠移植组全部出现特征性病理改变,成模率为100%,与原位小肠移植组成模率75%比较,差异无统计学意义(P0.05)。结论应用F344→Lewis大鼠组合建立原位和异位小肠移植模型,术后给予小剂量环孢素,均可在术后不同时间点出现慢性排斥反应。与原位大鼠小肠移植模型相比,大鼠异位小肠移植模型建模操作简单,慢性排斥反应成模时间较短,病理改变程度相对一致,更适合用于实验研究。     

TGF-β1基因修饰的未成熟树突状细胞对小肠移植大鼠外周血及移植肠浸润T细胞的影响

目的探讨经转化生长因子-β1（TGF-β1）基因修饰的未成熟树突状细胞（imDC）预处理大鼠小肠移植受体后的外周血及移植肠浸润T细胞的变化及意义。方法选用近交系F344／N和BN大鼠建立全小肠异位移植模型,实验分4组（每组24只）：同基因移植组（BN-BN组）、异基因移植组（F344／N-BN组）、异基因移植＋TGF-β1基因转染imDC组（F344／N-BN＋TGF-β1组）和异基因移植＋TGF-β1基因转染imDC＋FK506组（F344／N-BN＋TGF-β1＋FK506组）。各组大鼠分别于术后3、5、7d各处死6只,获取大鼠静脉血和移植肠。应用免疫组化SABC法检测受体鼠外周血及移植肠CD4^＋、CD8^＋、CD25^＋细胞和IL-4的表达。同时行移植肠组织病理学检查并观察大鼠生存情况。结果TGF-β1修饰的DC细胞能显著抑制外周血及移植肠浸润淋巴细胞CD4^＋、CD8^＋及CD25^＋的表达,并提高IL-4的表达;显著延长受体大鼠的生存时间,但移植肠仍有排斥反应的病理组织学征象。结论TGF-β1修饰的DC通过影响受体外周血及移植肠浸润T细胞对大鼠小肠移植发挥免疫抑制作用。     

异基因大鼠胰十二指肠移植模型建立的手术技巧

目的 探讨异基因大鼠全胰十二指肠移植(WPDT)模型手术过程的操作要点.方法 将供、受体腹主动脉端侧吻合,供体门静脉与受体左肾静脉袖套吻合,供体十二指肠与受体小肠端侧吻合,成功建立Lewis→Wistar糖尿病大鼠异基因WPDT模型.结果 50只糖尿病大鼠行WPDT术式,44只移植成功.其中8只于术后3 d内死亡,其余36只受体大鼠存活时间为6～16 d,平均为(10.45±3.30) d,术后7～10 d为死亡高峰,移植物病理改变呈典型的急性排斥反应. 结论 熟练的显微技术和重视操作细节是成功建立WPDT模型的关键因素.     

Lymphocyte subtypes CD3+, CD19+, CD16+CD56+, CD4+, CD8+, and CD3+HLA-DR+ in peripheral blood obtained from patients after thoracic organ transplantation

Introduction

The aim of this study was to assess peripheral blood lymphocyte subtypes (CD3⁺, CD19⁺, CD16⁺CD56⁺, CD4⁺, CD8⁺, and CD3⁺HLA-DR⁺) obtained from thoracic organ recipients at various periods after transplantation.

Material and Methods

Seventeen patients after lung transplantation (LT) and 5 patients after heart transplantation (HT) included 13 males (76.5%) and 4 females (23.5%) of overall mean age at the time of transplantation of 46.7 ± 11.55 years and mean body mass index of 21.1 ± 4. Lymphocyte phenotypes were estimated using Simultest IMK Plus.

Results

A significant decrease in lymphocytes of the majority of subtypes was observed at 1 year posttransplantation compared with normal ranges: CD19⁺ B lymphocytes in 56% of patients, CD8⁺ T cells among 48% and CD16⁺CD56⁺ natural killer elements, 56%. In contrast, there were increased numbers of activated lymphocytes (CD3⁺HLA-DR⁺). Beyond the 1-year observation, we observed a trend to normalize parameters among the majority of subjects.

Conclusion

A clear tendency to a decrease number of peripheral blood lymphocytes of various subtypes was observed among thoracic organ recipients in the first year posttransplantation with the exception of activated HLA-DR⁺ cells. After the first year, there was slow restoration of lymphocytes.     

Upregulation of CD40, CD80, CD83 or CD86 on alveolar macrophages after lung transplantation.

BACKGROUND: Alveolar macrophages (AMs) are known to be poor antigen-presenting cells, and lack the accessory molecules such as CD40, CD80 or CD86 to activate T cells. The question raised is about the potential changes in phenotypes after lung transplantation, particularly during acute rejection episodes. METHODS: The present study analyzed the phenotype of AMs longitudinally in 45 lung transplant patients, between August 1997 and April 2002, with a follow-up period of 27.2 +/- 2.5 (mean +/- SEM) months. There were 7.7 +/- 0.6 bronchoalveolar lavage (BAL) assessments performed per patient (i.e., 345 BALs), simultaneously with transbronchial biopsies. Transplantation was soon followed by a progressive upregulation of CD40 on 49.7 +/- 8% of AMs during the first month, and this marker remained elevated at 60 +/- 8% after 5 years. RESULTS: Both CD86 and CD80, as well as CD83, a marker of dendritic cells, were enhanced for most AMs during Grade A2 and A3 rejection episodes. A correlation was found between expression of CD83 and CD86, but not between CD1a and CD86. Immunohistology confirmed that CD40-positive cells in the alveoli corresponded to AMs and to some dendritic cells in the basal layers of the airways. In vitro studies showed that harvested AMs with these enhanced accessory molecules remained poor stimulators of allogeneic cells, a phenomenon that may be related to the ongoing immunosuppressive treatments. CONCLUSIONS: AM phenotypes showed marked changes during early or late acute rejection episodes, acquiring CD80, CD83 and CD86, while CD40 expression was further enhanced. This finding may provide clues on how to monitor the tolerance of transplanted lungs and may also provide new insights into the pathophysiology of lung transplantation.     

CD27/CD70, CD134/CD134 ligand, and CD30/CD153 pathways are independently essential for generation of regulatory cells after intratracheal delivery of alloantigen

BACKGROUND: We investigated whether blockade of tumor necrosis factor receptor-ligand pathways could generate regulatory cells induced by intratracheal delivery of alloantigen. METHODS: CBA (H-2k) mice were pretreated with intratracheal delivery of splenocytes (1x10(7)) from C57BL/10 (H-2b) mice and intraperitoneal administration of monoclonal antibody (mAb) specific for CD70, CD134 ligand (CD134L), CD153, or CD137L. Seven days later, C57BL/10 hearts were transplanted into pretreated CBA mice. Some naive CBA mice underwent adoptive transfer of splenocytes (5x10(7)) from pretreated CBA mice and transplantation of a C57BL/10 heart on the same day. RESULTS: Untreated CBA mice rejected C57BL/10 cardiac grafts acutely (median survival time [MST] 12 days). Pretreatment with intratracheal delivery of C57BL/10 donor splenocytes prolonged graft survival significantly (MST 84 days). Mice given intratracheal delivery of alloantigen plus anti-CD70, anti-CD134L, or anti-CD153 mAb, but not those given intratracheal delivery of alloantigen plus anti-CD137L mAb, rejected their graft acutely (MST 16, 14, 10, and 65 days, respectively). Adoptive transfer of splenocytes from mice pretreated with intratracheal delivery of alloantigen plus anti-CD70, CD134L, or CD153 mAb did not prolong survival of C57BL/10 cardiac grafts in naive secondary CBA recipients (MST 14, 11, and 11 days, respectively), whereas adoptive transfer of splenocytes from mice given intratracheal delivery of alloantigen plus anti-CD137L mAb did (MST 75 days). CONCLUSION: The CD27/CD70, CD134/CD134L, and CD30/CD153 pathways are independently required for generation of regulatory cells in our model.     

Cross-species costimulation: relative contributions of CD80, CD86, and CD40

BACKGROUND: The response of human CD4+ T cells against porcine cells is of comparable magnitude to that induced by human leukocyte antigen-mismatched allogeneic cells. This reflects productive interactions between key costimulatory molecules across the species barrier. Inhibition of these molecular interactions will be crucial in overcoming CD4+ T-cell-mediated rejection of xenografts. We have performed a detailed investigation to determine the expression profiles and relative contributions of the three key costimulatory molecules in the porcine-human xenogeneic response. Whereas only porcine CD86 is constitutively expressed on resting endothelial cells, both CD40 and CD80 are rapidly expressed after activation. All three costimulatory molecules are expressed by professional antigen-presenting cells. METHODS: We have isolated full-length cDNA clones for human and porcine CD80, CD86, and CD40. Human fibroblast cell lines (M1) coexpressing DR1 were transfected with these cDNAs and used in mixed lymphocyte reactions and flow cytometric studies in vitro. RESULTS: These data provide the first characterization of the expression profile and functional role of porcine CD80. Functional assays demonstrate that pCD40, pCD80, and pCD86 are independently capable of costimulating human CD4+ T cells, albeit with differing kinetics. Proliferative responses were of comparable magnitude to those obtained when costimulation was provided by human CD40, CD80, and CD86. CONCLUSIONS: These data have implications for therapy targeting the direct pathway of xenorecognition; costimulatory molecule blockade must be directed against both the B7/CD28 and CD40/CD40L pathways.     

CD52 ligation induces CD4 and CD8 down modulation in vivo and in vitro

To successfully induce donor-specific tolerance after immune depletion, it is essential to understand the residual and recovering immune system in the context of the depleting agent because the properties of such a recovering immune system differ based on the depleting agent used. In this study, we investigate the phenotypic and functional characteristics of T cells exposed to Campath-1H in vivo and in vitro. Recovering T cells demonstrated down modulated surface CD4 and CD8 (by flow cytometry) for up to 45 days after Campath-1H administration. Additionally, these T cells had an activated phenotype. To determine whether this CD4/8 down modulation was due to T-cell activation only or in part due to Campath-1H, whole blood from healthy volunteers was exposed to Campath-1H and the surviving lymphocytes isolated. Flow cytometry revealed a dose-dependent down modulation of CD4/8 without T-cell activation. Additionally, these Campath-1H-treated T cells were immunocompetent as indicated by increased surface CD69 and interleukin-2 (IL-2) production following stimulation by soluble anti-CD3 mAb. In conclusion, Campath-1H by itself down modulates surface CD4 and CD8 without activating T cells.     

Peripheral T-cell lymphomas expressing CD30 and CD15

Coexpression of CD30 and CD15 is typically associated with classic Hodgkin's lymphoma (HL). Peripheral T-cell lymphomas (PTCLs) can often display histologic features that simulate classic HL. However, reports of PTCLs coexpressing both CD30 and CD15 have been infrequently described. We report 11 cases of PTCL in which at least a subset of the neoplastic cells coexpressed CD30 and CD15. The patients included 4 women and 7 men and age ranged from 43 to 83 years (median, 62 years). Nine of 10 patients had advanced stage III or IV disease at presentation. Nodal involvement predominated in 8 of 11 patients, whereas 2 patients presented primarily with skin involvement. Two distinct groups were identified based on morphologic and immunophenotypic features. The first group of 5 cases had histologic features mimicking classic HL with CD30+, CD15+ Reed-Sternberg (RS)-like cells in an inflammatory background of varied extent and composition. The background lymphoid cells showed minimal cytologic atypia. The RS-like cells were negative for CD20 and CD79a in all cases, and CD45 expression was absent in 4 of 5 cases. The RS-like cells expressed CD25 and at least one T-cell-associated marker in all cases. The background T-cell population showed convincing subset predominance in 4 of 5 cases and loss of T-cell-associated antigens in 3 of 5 cases and coexpression of CD30 and CD15 in one case. The second group of 6 cases had morphologic features more in keeping with PTCL than classic HL. The proportion of neoplastic cells coexpressing CD30 and CD15 varied. Loss of T-cell antigens was noted in all cases and CD4 predominated in 4 of 5 cases. Three of the 6 cases expressed CD45. PCR analysis revealed clonal T-cell receptor gamma (TCR-gamma) chain gene rearrangements in 9 of 11 cases, but no immunoglobulin heavy (IgH) chain gene rearrangements. In situ hybridization studies for Epstein-Barr virus were negative in all cases. In some PTCL cases, the overlap with classic HL can be striking, and combined immunophenotypic and molecular studies are often necessary to confirm the diagnosis.     

CD4+CD25+ cells regulate CD8 cell anergy in neonatal tolerant mice

BACKGROUND: Injection of neonatal BALB/c mice with semi-allogeneic splenocytes leads to antigen-specific tolerance lasting into adulthood. Tolerant mice accept A/J skin grafts and fail to generate CD8 cytotoxic T lymphocyte (CTL) activity against A/J targets. Anergic CD8 T cells are present in tolerant mice, and CD4 regulatory cells function to maintain CD8 cell anergy. METHODS: Neonatal BALB/c mice were injected with 108 live CAF, splenocytes, and mice were deemed tolerant by accepting A/J grafts over 40 days. CD8 cell proliferation was measured by in vitro incorporation of bromodeoxyuridine coupled with fluorescence-activated cell sorter analysis. Alloantigen-specific cytotoxicity was tested using 51Cr release assays of A/J or third-party targets. RESULTS: We demonstrate that A/J-specific anergic CD8 cells are present in neonatal primed mice that develop tolerance but not in neonatal primed mice that reject A/J skin grafts. Anergic CD8 cells show decreased proliferation and no CTL activity against A/J targets. Addition of interleukin-2 (IL-2) to unfractionated cultures fails to restore CTL activity against A/J targets. However, addition of IL-2 to CD4-depleted cultures restores A/J-specific CD8 CTL activity. Removal of CD4+/CD25+ cells, but not CD4+/CD25- cells, also restores CD8 CTL activity against A/J in the presence, but not the absence, of IL-2. Moreover, when added back into cultures, purified CD4+/CD25+ cells from tolerant mice inhibit the generation of CD8 CTL against A/J targets. CONCLUSION: These data indicate that CD8 anergy is associated with the state of tolerance, and that CD4+CD25+ cells from tolerant mice function to maintain A/J-specific CD8 cell anergy in vitro.