新生期移植耐受依赖嵌合体的形成

目的 研究新生期移植耐受机制,探讨免疫系统发育程度、嵌合体在诱导移植耐受中的作用.方法 雄性C57BL/6(或GFP-C57BL/6)小鼠与雌性BALB/c小鼠杂交获得F1(或GFP-F1)小鼠,不同剂量F1或GFP-F1小鼠脾脏细胞(辐照处理的细胞作为对照)静脉注射到新生24hC57BL/6小鼠体内诱导耐受,6周后皮肤移植、混合淋巴细胞反应实验检测小鼠耐受程度,流式细胞分析小鼠外周血细胞嵌合程度.结果 具有增殖活性的F1小鼠脾脏细胞可诱导新生C57BL/6小鼠嵌合体和针对F1小鼠皮肤移植物的特异性耐受;辐照处理的F1小鼠脾脏细胞不能诱导嵌合体,也没有耐受产生;长期耐受小鼠的嵌合程度明显大于慢性排斥小鼠的嵌合程度(分别为6.48％±4.02％和1.57％±0.89％),两组间的差异具有统计学意义;供体细胞剂量高则诱导小鼠的耐受程度高,3×107剂量F1小鼠脾脏细胞可诱导80％的小鼠长期耐受,0.7×107剂量诱导仅使移植物生存时间轻度延长.结论 新生期移植耐受依赖嵌合体的形成,嵌合体使同种异体反应性T细胞特异性克隆清除.     

同种异体反应性细胞与调节性T细胞在新生期诱导的小鼠移植耐受中的作用探讨

目的:初步探讨同种异体反应性T细胞克隆清除与调节性T细胞在小鼠移植耐受中的作用。方法:雌性BALB/c小鼠与雄性C57BL/6小鼠杂交一代获得F1小鼠,不同剂量的F1小鼠脾脏细胞经眶静脉输注给新生24 h C57BL/6小鼠体内诱导耐受,成年后移植F1小鼠来源的皮肤,建立不同耐受程度的小鼠移植耐受模型;耐受小鼠脾脏细胞经CFSE标记后注射到F1小鼠体内,分析耐受小鼠来源的T细胞在体内对F1抗原的增殖能力;流式细胞术、过继转移实验分析CD4+Foxp3+调节性T细胞在移植耐受和移植排斥过程中的表达。结果:C57BL/6小鼠在新生期输注F1小鼠脾脏细胞可诱导移植耐受,耐受程度与输注的脾脏细胞剂量有关,3×107个F1小鼠脾脏细胞可诱导C57BL/76小鼠长期皮肤移植耐受,1×107个细胞诱导可使移植皮肤生存时间显著延长,但在50 d内完全排斥;体内混合淋巴细胞反应实验证明,长期耐受小鼠体内的同种异体反应性T细胞被完全克隆清除,但低剂量组小鼠体内仍存在一定数量的反应性T细胞;流式细胞分析发现,高剂量和低剂量组小鼠体内的CD4+Foxp3+调节性T细胞表达与初始小鼠相比没有显著差异;同种异体反应性T细胞过继转移给耐受小鼠,移植耐受的皮肤发生排斥反应,小鼠体内的调节性T细胞表达升高。结论:小鼠的移植耐受程度与小鼠体内的同种异体反应性T细胞的克隆清除程度有关,与CD4+Foxp3+调节性T细胞的表达没有直接关系;调节细胞在移植排斥过程中表达升高,可能作为一种反馈机制参与耐受的形成。     

新生小鼠胸腺细胞免疫抑制功能探讨——Ⅱ.对同种半心移植排异反应的抑制作用

经~(60)Co轻度照射(200Rad)之成年CFW小鼠,在接受2×10~8同品系新生早期胸腺细胞及供体新生心肌细胞混悬液腹腔注射后,其对同种半心移植(C57BL CFW)的排异反应明显延缓。新生鼠胸腺细胞组之移植物平均存活期(20.0±1.8天)比对照组(12.9±1.5天)和成年鼠胸腺细胞组(16.2±3.0天)显著延长。提示新生小鼠胸腺细胞对排异反应有抑制作用。 就各种术前处理对移植物存活期影响所作的分析表明,照射对移植物存活期无显著影响,在照射后的5—7天,受鼠免疫系统功能已回复正常水平。而照射联合新生供体心肌细胞注射,则在一定程度上延长移植物的存活期。进一步的分析显示,成年鼠胸腺细胞组移植物的存活期之所以较长,可能是由于受照射联合新生供体心肌细胞注射的影响。     

巨噬细胞在新生小鼠移植耐受维持和崩溃中的作用

本文采用体外混合淋巴细胞反应(Mixed Lymphocyte Reaction,MLR)与体内细胞过继转移试验进一步对巨噬细胞(Mφ)在新生小鼠移植耐受维持和崩溃中的作用进行了研究。体外MLR中,耐受小鼠的脾脏单层粘附细胞不能重建去粘附细胞的同系正常鼠的MLR,相反呈现出抑制作用。另一方面,正常小鼠的脾脏与腹腔单层粘附细胞却能恢复同系耐受鼠脾细胞的MLR至正常水平。体内实验中,过继转移正常小鼠腹腔Mφ 能恢复同系耐受鼠(切除或未切除脾脏、淋巴结)对同种心移植物的排斥反应。这种作用明显地见于新移植心,而对已耐受的移植心则作用不明显。本文结果提示,耐受鼠Mφ 递呈耐受原功能的缺陷以及Mφ 抑制作用可能是免疫耐受得以维持的重要原因,一旦功能恢复(体内外重建)则耐受崩溃。     

移植器官体积对小鼠皮肤移植物生存时间影响的实验研究

目的 探讨移植器官体积对移植器官生存时间的影响.方法 C57BL/6小鼠与BALB/c小鼠杂交获得F1小鼠,1.5×107个F1小鼠脾脏细胞输注给新生C57BL/6小鼠;混合淋巴细胞反应检测体内同种异体反应性T细胞的克隆清除;Na(i)ve(初始)C57BL/6小鼠和新生期处理的C57BL/6小鼠移植F1小鼠皮肤,皮片大小分别为0.5 cm2和1 cm2,观察皮片的生存时间.结果 新生期C57BL/6小鼠输注F1小鼠脾脏细胞后体内同种异体反应性T细胞数量显著减少,对移植抗原的反应性减弱;Na(i)ve小鼠迅速排斥F1小鼠皮肤移植物,0.5 cm2和1 cm2组皮肤移植物的生存时间分别为(8.9±1.0)d和(9.3±1.0)d,两组间差异没有统计学意义(P＞0.05);新生期处理的C57BL/6小鼠移植F1小鼠皮肤,1 cm2组皮肤移植物生存时间为(63±28)d,大于0.5 cm2组皮肤移植物(28±16)d,两组间差异具有统计学意义(P＜0.05).0.5 cm2组皮肤移植物在60d内完全排斥,1 cm2组皮肤移植物在100 d时仍有部分存活,形成长期耐受.结论 降低移植受体排斥强度的同时增加移植器官的体积可延长移植器官的生存时间.     

髓腔内骨髓移植诱导异基因小鼠皮肤移植耐受的研究

探讨髓腔内骨髓移植(IBM-BMT)对异基因小鼠皮肤移植耐受的诱导效果。受鼠为雌性C57BL/6(H-2b,B6)小鼠,于第0天接受60Coγ射线全身照射(TBI),4 h内输注雄性BALB/c(H-2d)小鼠来源的骨髓细胞(BMC),2 d后腹腔注射环磷酰胺(CTX)。通过皮肤移植、混合淋巴细胞反应(MLR)检测耐受状态,并通过骨髓染色体分析了解BMC的植入程度。结果显示,接受IBM-BMT的B6小鼠对BALB/c小鼠的皮肤移植物平均存活时间(MST)超过300 d,显著长于其余两组(P<0.001);MLR结果证明,实验组B6小鼠获得供体特异性耐受,在耐受小鼠骨髓中可检测到一定比例的Y染色体存在。以上表明髓腔内骨髓移植可以保证异基因骨髓细胞的植入并形成混合嵌合状态,从而有效地诱导免疫耐受。     

抗MHC-Ⅰ类分子单抗促进异基因小鼠皮肤移植耐受

目的通过静脉注射异基因脾细胞和抗H-2b单抗加腹腔注射环磷酰胺诱导成年B6小鼠对异基因皮肤移植物产生免疫耐受.方法经C57BL/6(H-2b,B6)小鼠尾静脉注射BALB/c小鼠(H-2d)脾细胞,2 d后腹腔注射环磷酰胺(CP),随后2次尾静脉注射抗小鼠的H-2b单克隆抗体,然后进行皮肤移植,并于耐受30d对受体B6小鼠作混合淋巴细胞反应(mixed lymphocyte reaction,MLR)、迟发型超敏反应(delayed type hypersensitivity,DTH)等耐受状态检测.结果 BALB/c小鼠的皮肤移植物在耐受B6小鼠中存活期特异延长.MLR和DTH检验证明:B6小鼠对BALB/c小鼠的脾细胞产生特异性耐受,对无关第3者KM小鼠的脾细胞仍表现出强烈的免疫反应.结论抗MHC-Ⅰ类分子单克隆抗体可明显促进"细胞+CP"诱导的异基因皮肤移植耐受;克隆排除是诱导耐受的主要因素.     

腺病毒载体介导的CTLA4-Ig基因转移促进异基因小鼠皮肤移植耐受

目的　探讨CTLA4 Ig重组腺病毒 (AdCTLA4 Ig)促进“脾细胞 (spleencells ,SP) 环磷酰胺 (cyclophosphamide ,CP)”系统诱导移植耐受的作用及其机制。方法　BALB c(H 2 d)小鼠经尾静脉注射C57BL 6(H 2 b,B6)小鼠脾细胞和AdCTLA4 Ig颗粒 ,48h后腹腔注射环磷酰胺 ,并同天进行皮肤移植 ,于耐受 2 5d时对受体小鼠进行混合淋巴细胞反应 (mixedlymphocytereaction ,MLR)、迟发型超敏反应 (delayedtypehypersensitivity ,DTH)等耐受状态的检查。结果　B6小鼠的皮肤移植物在耐受的BALB c小鼠中存活期特异延长。MLR和DTH检验证明BALB c小鼠对B6小鼠的脾细胞产生特异性耐受 ,对无关第三者ICR小鼠的脾细胞仍表现出强烈免疫应答。结论　AdCTLA4 Ig颗粒可以明显促进“细胞 环磷酰胺”诱导的异基因皮肤移植耐受 ;克隆排除和克隆无能是耐受诱导的主要因素     

测定小鼠脾混合淋巴细胞反应的新方法——SCM测定法

本文利用近年来新发展的胞浆基质结构性(SCM)方法检测了小鼠脾混合淋巴细胞反应。 实验结果指出,组织相容(近交系动物)个体之间 MLR不表现 SCM反应;组织不相容(不同品系之间)个体则表现 SCM 反应。从 CFW,C_(57)BL,C_(57)BL_6,ICR,615,BALB/c,F_1ICR/615小鼠得到的结果表明,当组织不相容时,RR_(SCM)((P_(MLR)/P_C)×100％)=75±5％,CV<20％,与传统的同位素掺入检测MLR方法结果相符。 实验提示,脾淋巴细胞密度、分离条件及试剂的准备对成功地导出SCM反应有很大关系。本文对SCM反应细胞群等也作了探讨。 该方法与传统的 MLR(5～7天)方法相比,具有快速(全过程 4～5 小时)这一明显优点。本文亦讨论了在临床移植配型工作中应用该方法的可能性。     

裸鼠与正常鼠杂交子一代的免疫功能研究

将BALB/cAnN-nu裸鼠与BALB/cAnN正常鼠杂交的子一代(F_1)与其亲代进行免疫功能比较,单向与双向混合淋巴细胞反应(MLR)。结果表明,F_1与亲代裸鼠在遗传关系上更接近。淋巴细胞转化试验表明F_1由裸鼠遗传获得对ConA诱导T细胞亚群转化活性低、B细胞功能活性高、以及对异种C57BL/6J小鼠MLR反应性低等性状。F_1具有部分细胞免疫功能较亲代裸鼠高而低于亲代BALB/c小鼠的特性,可考虑供细胞免疫功能低下的试验小鼠模型之用。     

抗独特型抗体对小鼠心肌移植耐受的诱导作用

目的探讨抗独特型抗体对小鼠移植耐受的诱导作用.方法以C57BL/6小鼠脾细胞免疫Balb/c小鼠制备抗同种异品系抗体(Ab1),Ab1交联KLH后,免疫Balb/c小鼠诱导产生抗独特型多克隆抗体(Ab2),以之为移植受体,观察Ab2对小鼠心肌移植耐受的诱导作用.结果Ab1交联KLH和弗氏佐剂免疫可以有效诱导抗独特型抗体(Ab2),和对照组相比,Ab2诱导组的小鼠移植物的存活时间明显延长.结论移植前在受体体内诱导产生以移植物抗原为模拟抗原的抗独特型的抗体,可以对小鼠特异性低免疫反应状态起到有效的诱导作用.     

Effect of anti-carrier antibody on carrier-determined tolerance

These experiments were originally designed to determine whether an anti-carrier antibody, e.g., anti-allotype could break hapten-specific tolerance in vivo. Tolerance to 2,4-dinitrophenyl (DNP) was induced in C57BL/6J mice using DNP-BALB/c IgG_2a conjugate. When anti-allotype serum was injected in C57BL/6J mice one day after a single injection of DNP-IgG_2a the mice were not tolerant. In contrast, when tolerance was induced by four weekly injections of tolerogen, the anti-allotype serum had no effect on the tolerant state. This effect was specific for tolerance-inducing carrier. Anti-carrier antibody injected in C57BL/6J mice one day after DNP-IgG_2a produced a small but significant anti-DNP response without administration of the immunogen, whereas the tolerogen (DNP-IgG_2a) by itself was not immunogenic. Similarly, despite multiple injections of DNP-IgG_2a, bearing the foreign allotype, only one out of 7 C57BL/6J mice showed a weak anticarrier response. In contrast, a marked anti-carrier (IgG_2a) response was obtained when the anti-allotype antibody was passively administered in C57BL/6J mice. In conclusion, these experiments suggest that tolerance to an antigenic determinant may be broken by an antibody directed not to this determinant, but to another on the same molecule. The significance of this finding in relationship to the mechanism of the carrier-determined tolerance and the breakdown of self-tolerance is discussed.     

同基因造血干细胞移植延长同种异体小鼠皮肤移植物存活时间的实验研究

为了研究同基因造血干细胞移植诱导器官移植免疫耐受的可行性。建立小鼠异基因皮肤移植模型,术后2周给予FK506腹腔注射,3周起行全身照射及同基因骨髓移植,观察记录小鼠和移植物存活情况,以流式细胞检测受体GFP嵌合表达,混合淋巴细胞反应、迟发型超敏反应检测诱导耐受的特异性和效能。实验组小鼠移植物存活时间达(29.14±4.92)d,显著长于对照组(P<0.05);GFP在BMT后4周、6周嵌合程度达到82%、91%;实验组MLR、DTH结果与对照组差异显著,提示诱导耐受具有高度特异性和高效性。同基因造血干细胞移植联合免疫抑制剂治疗可以有效诱导小鼠皮肤移植的免疫耐受。     

Successful pancreatic allografts in combination with bone marrow transplantation in mice.

We have established a new method for pancreatic allografts in mice by combining pancreatic transplantation with allogeneic bone marrow transplantation. In this approach, we first transplanted bone marrow to induce tolerance to both donor-type and host-type major histocompatibility complex (MHC) determinants. Pancreatic tissue from the same mouse strain as bone marrow donor was then grafted under the renal capsule. Acceptance of the grafts was confirmed by histopathological and immunohistochemical techniques. BALB/c mice reconstituted with C57BL/6J bone marrow cells accepted pancreatic tissue from both bone marrow donor (C57BL/6J)-type and host (BALB/c)-type mice. An immunohistochemical study revealed the presence of functional islets under the renal capsules. Assays for both mixed lymphocyte reaction (MLR) and induction of cytotoxic T lymphocytes indicated that the newly developed T cells are tolerant of both donor (stem cell)-type and host-type MHC determinants. By contrast, the T cells of these chimeras showed a significant responsiveness to third party MHC determinants. These findings suggest that pancreatic allografts combined with bone marrow transplantation may become a viable strategy for the treatment of patients with diabetes or patients who have undergone pancreatectomy.     

体外联合应用IL-10和甲基强的松龙处理树突状细胞诱导移植的免疫耐受

目的探讨体外联合应用白细胞介素10(IL-10)和甲基强的松龙(Medron)处理供体树突状细胞(DC)对小鼠皮肤移植术后免疫耐受的诱导效果,为抗移植术后免疫排斥反应治疗提供依据。方法以健康成年C57BL/6小鼠为供体。BALB/c小鼠为受体,随机分为11组。除A组外,其余各组均于皮肤移植前3d自尾静脉输入对应的供体DC。具体对应关系如下A组为空白对照,尾静脉输入生理盐水;B组为输入未修饰的DC;C1组为10μg/LIL-10处理组;C2组为30μg/LIL-10处理组;D1组为10mg/LMedron处理组;D2组为20mg/LMedron处理组;E1~E4组分别为10μg/LIL-10 10mg/LMedron处理组、10μg/LIL-10 20mg/LMedron处理组、30μg/LIL-10 10mg/LMedron处理组、30μg/LIL-10 20mg/LMedron处理组。同时设立F组,为BALB/c对BALB/c的同种同基因皮片移植。各组行皮肤移植术,观察受体移植皮片存活情况。结果相对于生理盐水组,E3组(30μg/LIL-10 10mg/LMedron处理组)的移植皮片存活时间最长,经统计学分析两种药物之间具有交互作用(P<0.05)。结论用IL-10和修饰的供体树突状细胞对受体进行预处理,可明显延长移植皮片的存活时间。     

Deletion of alloantigen-reactive thymocytes as a mechanism of adult tolerance induction following intrathymic antigen administration

Direct injection of foreign antigen into the adult thymus is a potent route of antigen delivery for the induction of tolerance in vivo. In this report, we demonstrate that tolerance to C57BL/10 (H2^b/BL10) alloantigens can be induced in CBA/Ca (H2^k/CBA) mice by intrathymic (IT) administration of BL10 spleen leukocytes coincident with transient peripheral immunomodulation of CD4⁺ T cells using a depleting anti-CD4 monoclonal antibody. T cell receptor (TCR) transgenic mice (BM3.6; H2^k) expressing a CD8-independent TCR specific for H2K^b were used as recipients to facilitate investigation of the mechanisms responsible for tolerance induction by allowing visualization of events in the thymus following IT injection. IT administration of 5 × 10⁷ BL10 spleen leukocytes and concomitant transient peripheral T cell depletion in BM3.6 mice resulted in a substantial H2K^b-specific deletion of transgenic-TCR⁺ (tg-TCR) thymocytes which was dependent on the level of tg-TCR expression. IT deletion and the failure to export CD8⁺ T cells to the peripheral lymphoid organs correlated with the induction of tolerance to H2K^b; TCR transgenic mice that had received IT injection of BL10 splenocytes and peripheral T cell depletion accepted a H2K^b+ cardiac allograft indefinitely. Analysis of tolerant BM3.6 mice revealed that there were low numbers of CD8⁺ T cells in the periphery giving rise to a substantially reduced reactivity in vitro despite the fact that no donor cells or IT deletion were observed in the thymi of the majority of tolerant mice. These results demonstrate for the first time that IT injection of foreign alloantigen into an adult thymus results in the deletion of thymocytes expressing a TCR specific for the injected alloantigen and suggest that this is an important mechanism of tolerance induction following IT injection of alloantigen in vivo. Furthermore, analysis of tolerant TCR-transgenic mice suggests that IT deletion is not required for the maintenance of tolerance, and that peripheral mechanisms enforce continued hyporesponsiveness to H2K^b following transplantation.     

Genetic background affects the expansion of macrophage subsets in the lungs of Mycobacterium tuberculosis‐infected hosts

M1 macrophages are more effective in the induction of the inflammatory response and clearance of Mycobacterium tuberculosis than M2 macrophages. Infected C57BL/6 mice generate a stronger cellular immune response compared with BALB/c mice. We hypothesized that infected C57BL/6 mice would exhibit a higher frequency and function of M1 macrophages than infected BALB/c mice. Our findings show a higher ratio of macrophages to M2 macrophages in the lungs of chronically infected C57BL/6 mice compared with BALB/c mice. However, there was no difference in the functional ability of M1 and M2 macrophages for the two strains in vitro. In vivo, a deleterious role for M2 macrophages was confirmed by M2 cell transfer, which rendered the infected C57BL/6, but not the BALB/c mice, more susceptible and resulted in mild lung inflammation compared with C57BL/6 mice that did not undergo cell transfer. M1 cell transfer induced a higher inflammatory response, although not protective, in infected BALB/c mice compared with their counterparts that did not undergo cell transfer. These findings demonstrate that an inflammation mediated by M1 macrophages may not induce bacterial tolerance because protection depends on the host genetic background, which drives the magnitude of the inflammatory response against M. tuberculosis in the pulmonary microenvironment. The contribution of our findings is that although M1 macrophage is an effector leucocyte with microbicidal machinery, its dominant role depends on the balance of M1 and M2 subsets, which is driven by the host genetic background.     

Chimerism but not neonatal antigen exposure induces transplant tolerance

Acquired transplant tolerance could be readily induced during foetal or neonatal period through donor cell infusion, but it is not the case in adults. This phenomenon has been attributed to the variation of immune system development in neonatal and adult periods. To investigate the role of immature immune system and chimerism in neonatal transplant tolerance, irradiated spleen cells or cell fraction from F1 (BALB/c × C57BL/6) or GFP-F1 mice were injected intravenously into neonatal C57BL/6 mice to induce tolerance. Irradiated cells or cell fraction could not induce chimerism and transplant tolerance in neonatal mice, even increasing the dose of donor cells to 5 × 10(7). Living donor cells induced tolerance in neonatal mice, and the quantity of living cells was correlated with the degree of chimerism and tolerance. At the amount of 3 × 10(7) F1 spleen cells, skin grafts were survived permanently in more than 80% of treated mice. However, the amount of 0.7 × 10(7) F1 spleen cells could only slightly prolong allografts' survival. The more donor cells were infused, the higher level of chimerism was achieved, and the higher frequency of alloreactive T cells was deleted. Chimerism is prerequisite for the induction and maintenance of tolerance. Chimerism in long-term tolerant mice was significantly higher than that in chronic graft rejected mice, with 6.48 ± 4.02% versus 1.41 ± 0.77%. It implies that transplant tolerance depends on the establishment of chimerism, but not on antigen exposure to immature immune system in foetus or neonates.