巨噬细胞在新生小鼠移植耐受诱导中的作用

本文采用新生小鼠锈导的移植耐受(NITT)模型。探讨了新生期过继转移成年同系鼠腹腔渗出细胞(PEC)对诱导移植耐受的影响。实验结果表明,新生小鼠(CFW、C57BL/6)单接受杂交一代小鼠(F_1)脾与淋巴结细胞后,诱导了特异性移植耐受(F_1组),耐受鼠百分率分别为57.7％与71.4％。如新生小鼠接受F_1 细胞前先接受 PEC 转移(PEC 组),则耐受的诱导受阻抑,耐受率为 0,MLR 的平均相对反应率(RR ％)增高,P<0.001;而与正常对照组比较,均无显著差异。 实验结果又表明,转移去T细胞的 PEC仍能阻抑CFW与 C57BL/6小鼠耐受的诱导,MLR的RR ％增高,P<0.001及<0.05;而转移非粘附性PEC组的RR ％与 F_1组无显著差异,耐受的诱导未受明显影响(P>0.3)。这提示 PEC中起阻抑耐受诱导作用的主要细胞是 Mφ,新生小鼠Mφ辅助功能缺陷可能是 NITT模型建立的重要原因。     

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。     

抗TCRαβ单抗联合供体骨髓细胞输注诱导小鼠皮肤移植耐受的研究

探讨抗TCRαβ单克隆抗体联合大剂量供体骨髓细胞输注方法对同种异基因小鼠皮肤移植耐受诱导的促进作用。第 0天 ,C5 7BL/ 6 (H 2 b,B6 )小鼠尾静脉注入 2× 10 8BALB/c (H 2 d,B/c )来源的骨髓细胞 ,同时腹腔注射抗TCRαβ单克隆抗体5 0 0 μg ;第 6天进行皮肤移植 ;在不同的时间对B6小鼠进行迟发型超敏反应 (DTH )、混合淋巴细胞反应 (MLR )等耐受状态进行检测 ,并进行MLR的IL 2逆转实验、过继转移实验和嵌合状态分析以初步探讨耐受形成机制。结果显示 ,经抗TCRαβ单克隆抗体联合大剂量供体骨髓细胞输注处理的B6小鼠的供体皮肤移植物平均存活 5 0 4d ,显著长于其他各组 (P <0 0 0 1)。相对于正常对照组小鼠 ,耐受B6小鼠在DTH和MLR中均表现出显著的低反应性 (P <0 0 0 1)。IL 2逆转实验结果表明克隆无能参与了移植耐受的形成。体内、外过继转移实验均显示耐受B6小鼠脾细胞中存在抑制细胞活性。嵌合体检查结果表明在耐受B6小鼠的胸腺和脾脏中均形成了混合嵌合体 ,在皮肤移植后第 15、 30和 70天时脾脏内供体来源嵌合体水平依次为 15 86 % ,10 5 7%和 1 77% ,胸腺中依次为 8 19% ,5 72 %和 1 87% ,嵌合体水平随时间而下降。这些表明 ,抗TCRαβ单抗联合大剂量供体骨髓细胞输注可以在异基因成年小?     

抗TCRαβ单克隆抗体联合大剂量供体骨髓细胞输注诱导小鼠皮肤移植耐受的研究

目的　探讨抗TCRαβ单克隆抗体联合大剂量供体骨髓细胞输注方法在同种异基因小鼠皮肤移植耐受诱导中的作用。方法　第 0天 ,C5 7BL 6 (H 2 b,B6 )小鼠尾静脉注入 2× 10 8BALB c(H 2 d,B c)来源的骨髓细胞 ,同时腹腔注射抗TCRαβ单克隆抗体 5 0 0 μg。第 6天进行皮肤移植。在不同的时间对B6小鼠进行迟发型超敏反应测定 ,混合淋巴细胞反应分析 ,并进行MLR的IL 2逆转实验和过继转移实验 ,初步探讨耐受形成机制。结果　抗TCRαβ单克隆抗体联合大剂量供体骨髓细胞输注处理的B6小鼠中供体皮肤移植物平均存活为 5 0 .4d ,显著长于其它各组 (P <0 .0 0 1)。相对于正常对照组小鼠 ,耐受B6小鼠在迟发型超敏反应和混合淋巴细胞反应中均表现出显著的低反应性 (P <0 .0 0 1)。IL 2逆转实验结果表明 ,克隆不应答 (anergy)可能参与了移植耐受的形成。体内、外过继转移实验均显示耐受B6小鼠脾细胞中存在抑制细胞活性。结论　抗TCRαβ单克隆抗体联合大剂量供体骨髓细胞输注可以在组织相容性抗原完全不相同的成年小鼠间成功地诱导出皮肤移植物的长期耐受。多种耐受机制 ,包括克隆不应答、抑制细胞 ,都参与了耐受的形成。     

Ts细胞在自身免疫耐受中的作用

用同系小鼠红细胞在体外刺激BALB/C小鼠脾脏细胞,然后检查受刺激的脾脏细胞产生抗体形成细胞的数量,确定其是否对同系小鼠红细胞发生免疫反应。作者发现2～3个月范围内的幼龄BALB/C鼠脾细胞对同系鼠红细胞不发生反应,而13～14个月范围内的老龄BALB/C鼠脾细胞却对同系鼠红细胞发生反应。这说明幼龄BALB/C鼠对同系     

自身反应性T细胞系免疫生物学活性研究

目的：探讨自身反应性T细胞系过继转移对受体小鼠免疫应答的影响，为在BXSB小鼠探索T细胞疫苗的可行性奠定基础。方法：应用体外扩增的自身反应性T细胞过继转移给同系小鼠体内：^3H－TdR掺入法检测细胞增殖，ELISA检测抗体细胞因子水平。结果：将自身反应性T细胞系过继转移到年轻未发病的同系小鼠体内可以诱导血清中自身抗体和外来抗原反应性抗体以及细胞因子IFN－γ水平显著升高，可以降低受体小鼠对外来抗原（OVA）体液免疫应答能力。结论：自身反应性T细胞的过继转移可以影响机体自免疫应答和对外来抗原的体液免疫应答能力。     

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.     

肿瘤特异性T杀伤细胞系的建立及其生物学特性

<正> 用小鼠T淋巴细胞性克隆瘤细胞LAC-1免疫同系SW_1小鼠,诱导对该瘤株特异性T杀伤细胞,并采用TCGF条件培液在体外扩大培养,建立体外长期培养肿瘤特异性T杀伤细胞系。实验结果表明:经体内3～4次免疫的小鼠脾脏、腹腔淋巴结细胞均对LAC-1癌细胞有明显的特异性杀伤效应,并随效/靶比例增高,杀     

树突状细胞在榄香烯复合瘤苗主动免疫效应中的作用

本实验以HCa F或L6 1 5榄香烯复合瘤苗 (H TCV或L TCV )、H TCV溶解物 (TH )、短小棒状菌 (CP )免疫小鼠 ,分离制备其脾脏DC ,在体外分别用相应瘤细胞溶解物 (H或L )和瘤苗溶解物 (TH或TL )冲激后 ,以MTT法检测其诱导同系小鼠脾不粘附细胞增殖的能力。结果表明用榄香烯复合瘤苗或其溶解物免疫DC供鼠和体外冲激DC ,可增强其诱导同系小鼠脾不粘附细胞增殖的作用 ,给DC供鼠注射CP可进一步增强DC的这一作用     

雷帕霉素对同种移植耐受模型CD4+CD25+ T细胞活性的影响

目的：研究雷帕霉素（Rapa）对同种移植耐受个体CD4^＋CD25^＋ T细胞体内负免疫调节作用的影响.方法：建立同种皮肤移植模型, 受体鼠术前预输注供体鼠脾细胞, 术后给予环孢素A（CsA）进行耐受诱导.移植后第14天提取耐受诱导模型鼠的T细胞, 经不同浓度Rapa和/或IL-2体外处理后, 混合淋巴细胞反应（MLR）确定T细胞特异增殖水平;流式细胞术（FCM）检测CD4^＋CD25^＋ T细胞比例变化;RT-PCR检测Foxp3 mRNA表达情况;ELISA检测细胞培养不同时间后上清中IL-10的变化.然后将Rapa和/或IL-2处理的T细胞过继转移给同种移植后的BALB/c-SCID鼠, 观察移植物存活状态.结果：CsA加供体脾细胞预先注射可明显延长小鼠移植皮片的存活期（P＜0.05）;移植耐受状态的T细胞经Rapa和/或IL-2体外处理后CD4^＋CD25^＋ T细胞比例升高、增殖水平明显降低、 Foxp3表达量明显增加;过继转输给同种移植SCID鼠后, 其移植皮片存活时间显著延长（P＜0.05）.结论：Rapa可体外扩增耐受诱导模型中CD4^＋CD25^＋ T细胞, 使CD4^＋CD25^＋ T细胞相关的Foxp3和IL-10明显升高, 过继免疫后, 小鼠同种移植物存活时间明显延长, 而低浓度IL-2可以协同Rapa的这一作用.     

Surface Lyt phenotype of suppressor cells in C57BL/6 mice infected with Mycobacterium lepraemurium.

C57BL/6 were infected intravenously with 10(7) Mycobacterium lepraemurium (MLM). At increasing time intervals after infection different isolated splenic cell subpopulations were tested for their ability to suppress the mixed lymphocyte reaction (MLR) of normal syngeneic mouse splenocytes. During the first 6 months after infection neither T depleted nor plastic adherent spleen cells from infected mice exerted a suppressive activity on the normal mouse allogeneic proliferative response. Conversely, splenic T cells from MLM infected mice exhibited suppressive activity as early as 2 months after infection. Attempts to characterize the Lyt phenotype of splenic suppressor T cells from 6 months infected mice showed that both Lyt 1+ 2- and Lyt 2+ enriched cell subsets possessed the ability to suppress the MLR of the normal mouse spleen cells and Lyt 1+ 2- T cells were shown to be more efficient suppressors than Lyt 2+ cells.     

Immunization against abolition of transplantation tolerance*

Attempts were made to break the tolerance of murine transplantation antigens of strain A in CBA mice by adoptive transfer of: 1) normal, syngeneic (CBA) lymphoid cells, or 2) moderate doses of sensitized, syngeneic (CBA anti-A) lymphoid cells followed later by high doses of CBA anti-A cells. Both kinds of attempt failed. Tolerance could be broken when a large enough dose of sensitized cells was given. Prior “immunization” of CBA mice tolerant to A with small doses of CBA spleen cells protected against doses of sensitized cells otherwise successful in breaking tolerance. Determinations of anti-recognition structure (anti-RS) antibody and of alloantibody activities in sera of tolerant mice, of mice which were successful in defending tolerance, and of mice in which tolerance could be abolished revealed an important role for anti-RS antibodies. No clear role could be assigned to alloantibodies present in the same sera. The function of anti-RS antibodies was shown by the failure of spleen cells from immunized tolerant mice to recognize tolerated antigen. This suggests that anti-receptor antibodies are active in neutralizing receptors of responding lymphoid cells emerging from a stem cell reservoir.     

Variations in macrophage antigen phenotype: a correlation between Ia antigen reduction and immune dysfunction during tumor growth

Variable Ia antigen expression by macrophages (M phi) was examined during tumor growth by measuring: Ia antigen masking and immunofluorescence by anti-Ia antibody, accessory cell function in concanavalin A (Con A) and mixed lymphocyte reaction (MLR)-induced T cell proliferation, and M phi stimulatory function in the MLR. Tumor-induced progressive loss of Ia antigen expression was shown by immunofluorescence and corroborated by anti-Ia blockade of MLR stimulatory activity of normal but not tumor-bearing hosts (TBH) splenic M phi. The TBH splenic M phi supported Con A-induced proliferation of syngeneic T cells (Ia antigen-independent) but did not support syngeneic T cell proliferation in the MLR (Ia antigen-dependent). Irrespective of tissue source, normal and TBH M phi differed in their MLR stimulatory capabilities. In general, splenic M phi preparations were better stimulators of allogeneic T cell blastogenesis in the MLR than thioglycollate-elicited peritoneal M phi. Kinetic studies with TBH M phi showed a significant progressive loss in MLR stimulatory activity, which was especially pronounced with peritoneal M phi. Expression of Ia antigens by normal but not TBH M phi were diminished by 24-h in vivo plating of the peritoneal M phi. Indomethacin treatment showed Prostaglandin E2 was not a direct in vitro factor in Ia antigen-mediated reduction of splenic M phi MLR stimulatory activity. Taken together, these data delineate a loss of M phi Ia antigen expression, resulting in a decrease in Ia antigen-mediated functional activities during tumor growth.     

Inside the thymus, Mls antigen is exclusively presented by B lymphocytes.

The ability to stimulate an Mls-1 mixed lymphocyte reaction (MLR) is predominantly expressed by low density B lymphocytes in the spleen and peritoneal cavity of normal adult mice, and is absent in splenic B cells 1 month after lethal irradiation and reconstitution from autologous bone marrow. Coreconstitution of these mice with normal syngeneic peritoneal cells restores the stimulatory potential of splenic B cells, but sorted CD5+ or CD5- IgM+ lymphocytes from peritoneum are equally good stimulators, suggesting that functional Mls-1 expression may require long life spans and selection. Bone-marrow-reconstituted DBA/2 mice that fail to express Mls-1 antigens in the periphery nevertheless maintain T-cell receptor V beta 6 and 8.1 deletions among the newly formed T cells. These findings led us to directly investigate the Mls stimulatory ability of purified antigen-presenting cell populations inside the thymus. We report here that thymic B lymphocytes seem to represent the only intrathymic cell population able to stimulate Mls-1 MLR.     

Analysis of immunological tolerance to major histocompatibility complex antigens. I. High frequencies of tolerogen-specific cytotoxic T lymphocyte precursors in mice neonatally tolerized to class I major histocompatibility complex antigens

Injection of (CBA X A)F1 cells into neonatal CBA mice rendered them tolerant to skin grafts of (CBA X A)F1 origin. Limiting dilution analysis revealed a very low frequency of tolerogen-inducible cytotoxic T lymphocyte precursors (CTL-P) in spleens of tolerant mice. Two in vitro procedures allowed, however, the induction of tolerogen-specific CTL-P of high frequencies in tolerant mice: (a) the "by-pass" activation of spleen cells from tolerant mice by concanavalin A under short-term bulk culture conditions followed by culture of limiting numbers of activated responder cells, and (b) absorption of spleen cells from tolerant mice on monolayers of tolerogen-activated T cells from normal syngeneic mice. Furthermore, spleen cells from tolerant mice, recently challenged with a tolerogen-bearing skin graft, specifically suppressed the activation of tolerogen-reactive splenic CTL-P from normal CBA mice under limiting dilution conditions. These data confirm the presence of tolerogen-specific CTL-P of high frequency in tolerant mice and suggest their functional inactivation through a suppressive mechanism.     

Use of mice tolerant to lipopolysaccharide to demonstrate requirement of cooperation between macrophages and lymphocytes to generate lipopolysaccharide-induced colony-stimulating factor in vivo

Injection of lipopolysaccharide (LPS) into mice was followed by a rapid elevation of colony-stimulating factor (CSF) in the serum. A second, challenging injection of LPS given 3 to 4 days later failed to induce elevated levels of CSF in the serum. Such mice tolerant to LPS were used as an experimental tool to identify the CSF-producing cells which respond to LPS. We observed that generation of LPS-induced CSF in mice tolerant to LPS could be restored by an intraperitoneal injection of spleen cells 24 h before the challenging injection of LPS. Depletion of the adherent cells from the spleen cells reduced the ability of the splenic lymphocytes to restore the capacity of the mice tolerant to LPS to generate serum CSF. Reconstitution of the splenic lymphocytes with 5% thioglycolate-elicited peritoneal macrophages, however, reestablished the restorative capacity of these cells, whereas almost no restoration was observed after direct injection of elicited peritoneal macrophages. These data suggest that the spleen cells are active in generating CSF, provided that macrophages are present and can interact with the splenic lymphocytes to generate LPS-induced CSF in the serum.     

Hapten-specific tolerance in mice. II. Adoptive transfer studies and evidence for unresponsiveness in the B cells

The suppression fo the anti-NIP ((4-hydroxy-5-iodo-3-nitrophenyl)acetyl) plaque-forming cell (PFC) response elicited in mice by treatment with NIP-coated syngeneic erythrocytes could be transferred by spleen cells into irradiated recipients. This was evidenced by the lack of an indirect anti-NIP PFC response 7 days after cell transfer and challenge with NIP. FGG (fowl IgG). This state of specific unresponsiveness could be serially transferred by spleen cells into a second irradiated recipient. The hapten-specific suppression in the first recipient could be reversed by addition of normal spleen cells, but not by cortisone-resistant thymus cells. The lesion appears to be in the T cell-depleted population, since the suppression could be reversed by supplementing with T cell-depleted normal spleen cells. In vitro incubation of spleen cells from tolerant animals did not restore the capacity of these cells to produce an anti-NIP PFC response in irradiated recipients. In vitro incubation of normal spleen cells with sera from tolerant animals did not prevent these spleen cells from producing a normal anti-NIP PFC response in irradiated recipients. The adoptive secondary response response to NIP. FGG was inhibited by injection of NIP-coated syngeneic erythrocytes on the same day as the adoptive transfer of the NIP. FGG primed spleen cells and the challenging antigen. It would seem that NIP-coupled syngeneic erythrocytes, which are presumably poor stimulators of T cells, can suppress NIP-specific B cells, perhaps by gaining direct access to the surface of these cells.     

In vitro proliferative response of BALB/c mouse spleen cells stimulated with trinitrophenylated syngeneic spleen cells.

Spleen cells from normal BALB/c mice showed in vitro proliferative response against hapten-conjugated syngeneic spleen cells. Trinitrophenylated (TNP) spleen cells were prepared by treating normal spleen cells with sodium 2,4,6-trinitrobenzenesulphonate (TNBS). Four-day cultures of TNP-labelled spleen cells incorporated 2.5-7.4 times more [3H]thymidine than similar cultures of untreated spleen cells. An obviously positive mixed lymphocyte reaction (MLR) by normal spleen cells against mitomycin C (MC) treated TNP-labeled syngeneic spleen cells was observed after 4 days of culture. The MLR to TNP-labelled syngeneic cells was inhibited in the presence of epsilon-TNP-L-lysine by 23-37%. The spleen cells from the mice injected intraperitoneally with TNP-labelled syngeneic spleen cells showed a higher MLR against TNP-labelled spleen cells than normal spleen cells. The sensitized spleen cells also showed an increased response to MC-treated spleen cells. These results suggest that normal spleen cells include cells which can recognize the hapten and new antigenic determinants introduced into syngeneic spleen by chemical modification.