IL—2—PE40对小鼠异基因骨髓移植的影响

探讨白细胞介素２（ＩＬ－２）与绿脓杆菌外毒素组成的融合蛋白－ＩＬ－２－ＰＥ４０对小鼠异基因骨髓移植的基因骨髓移植的影响。实验结果表明体外应用ＩＬ－２－ＰＥ４０能够选择性抑制混合淋巴细胞培养（ＭＬＣ）中的异基因反应细胞，清除ＭＬＣ诱导产生的细胞毒效应细胞，保留未活化细胞对ＣｏｎＡ诱导的丝裂原反应；体内应用ＩＬ－２－ＰＥ４０能延长异基因骨髓移植小鼠的存活期，而对ＣＦＵ－ＧＭ产率无明显影响。     

CD_3单抗诱导的T细胞功能检测法

建立了用特异性刺激原白细胞分化抗原３（ＣＤ＿３）单克隆抗体（单抗）诱导外周血淋巴细胞（ＰＢＬ）增殖转化、产生白细胞介素－２（ＩＬ－２）及ＩＬ－２受体（ＩＬ－２Ｒ）表达等检测Ｔ细胞功能的方法，并对其实验影响因素进行探讨。结果表明：ＣＤ＿３单抗诱导ＰＢＬ活化的过程与Ｔ细胞在体内的活化过程相符；ＣＤ＿３单抗的丝裂原作用所需浓度范围广、用量少；由ＣＤ＿３单抗诱导的ＰＢＬ转化能力受ＣＤ＿３单抗的浓度和培养时间的影响；ＣＤ＿３单抗与植物血凝素（ＰＨＡ）活化Ｔ细胞的效应，在Ｔ细胞增殖方面二者呈显著正相关，而在ＩＬ－２活性和ＩＬ－２Ｒ表达方面则无相关性。     

白细胞介素2基因和白细胞介素3基因共转染白血病瘤苗的抗白…

目的：研究白细胞介素２（ＩＬ－２）和白细胞介素３（ＩＬ－３）基因共转染的白血病细胞瘤苗对白血病的小鼠的治疗效果。方法：用ＩＬ－２重组腺病毒（Ａｄ－ＩＬ－２）和（或）ＩＬ－３重组腺病毒转染红白轿病细胞株ＦＢＬ－３，＾６０Ｃｏ射后制备成瘤苗对实验性白血病小鼠进行治疗，观察肿瘤生长，小鼠生存期及治疗后小鼠腹腔巨噬细胞，ＮＫ细胞，诱导杀伤性Ｔ淋巴细胞（ＣＴＬ）杀伤活性，并与低剂量环磷酰胺合用，观察其抗肿瘤     

香菇多糖脂质体联合IL—2体内靶向巨噬细胞抗瘤作用的 …

目的 观察香菇多糖脂质体（Ｌｉｐ－ＬＮＴ）与低剂量ＩＬ－２协同体内靶向扩增、激活腹腔巨噬细胞（ＰＭΦ）的抗瘤作用。方法 Ｂａｌｂ／ｃ纯系小鼠经Ｌｉｐ－ＬＮＴ、ＩＬ－２体内诱导两天，收集、计数腹腔渗出细胞（ＰＥＣ）及ＰＭΦ，监测ＰＭΦ细胞毒活性及ＴＮＦ活性。腹腔接种Ｈ２２肝癌细胞后体内治疗９天，观察生存期。结果 Ｌｉｐ－ＬＮＴ与ＩＬ－２联合可使ＰＥＣ体内大量扩增，ＰＭΦ百分比显著升高，同时ＰＭΦ细胞     

高压氧对小鼠脾细胞IL－2产生及反应能力的影响

Ｃ５７ＢＬ／６小鼠在０．２５ＭＰａ下吸９９．２％氧，其脾淋巴细胞白细胞介素－２（ＩＬ－２）产生能力及对ＩＬ－２反应能力变化如下：高压氧（ＨＢＯ）组均明显低于正常组小鼠，去肾上腺组、去肾上腺髓质组均明显高于ＨＢＯ组、假手术组．实验证明，高压氧可降低脾淋巴细胞ＩＬ－２的产生及对ＩＬ－２的反应能力，在这一影响中肾上腺有一定作用．     

IL—6基因转染的骨髓基质细胞系对骨髓移植小鼠造血功能恢复？…

目的：探讨白细胞介素６（ＩＬ－６）基因转染的骨髓基质细胞系ＱＸＭＳＣ１ＩＬ－６对骨髓移植后造血功能的重建作用。方法：将骨髓造血细胞和骨髓基质细胞系一起经尾静脉注射给同系小鼠，建立骨髓移植（ＢＭＴ）模型。小鼠的造血功能用脾结节（ＣＦＵ－Ｓ）、粒－单系祖细胞（ＣＦＵ－ＧＭ）、红系祖细胞（ＣＦＵ－Ｅ、ＢＦＵ－Ｅ）测定及外周血各项血液学指标来确定。结果：ＷＸＭＳＣ１ＩＬ－６转基因骨髓基质细胞可明显增强ＢＭ     

脂质体介导的腹腔内IL-2和IL-6基因疗法对淋巴瘤小鼠巨噬细胞的激活作用

目的：探讨白细胞介素２（ＩＬ－２）和白细胞介素６（ＩＬ－６）基因疗法的抗肿瘤作用机制。方法：将脂质体包裹的ＩＬ－２基因和ＩＬ－６基因直接注射至ＥＬ－４淋巴瘤小鼠腹腔后，研究淋巴瘤小鼠的腹腔巨噬细胞（Ｍφ）在基因治疗后的功能变化。结果：腹腔内单独注射脂质体包裹的ＩＬ－２基因后，可明显提高Ｍφ的ＭＨＣＩａ的表达，增强Ｍφ的杀伤活性，并促进Ｍφ诱导白细胞介素１（ＩＬ－１）及肿瘤坏死因子（ＴＮＦ）的产生；腹腔内单独注射脂质体包裹的ＩＬ－６基因后，对Ｍφ也具有激活作用，但效果不及前者。二者联合后能非常显著地提高荷瘤小鼠的Ｍφ的上述功能。结论：腹腔内脂质体介导的ＩＬ－２基因和ＩＬ－６基因治疗能有效地解除淋巴瘤小鼠体内Ｍφ功能的抑制，激活Ｍφ发挥抗肿瘤作用。     

GATA—2与免疫球蛋白重链胚系基因Cμ在白血病细胞中的表 …

应用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）对不同类型白血病患者骨髓（ＢＭ）或外周血（ＰＢ）细胞ＧＡＴＡ－２和ＩｇＨ胚系基因ＣμｍＲＮＡ的表达进行检测，探讨ＧＡＴＡ－２与ＩｇＨ胚系基因Ｃμ在白血病细胞中的表达与共表达及意义。在正常人ＢＭ和ＰＢ细胞中未检测到ＧＡＴＡ－２和ＩｇＨ胚系基因Ｃμ。在急性髓性白血病（ＡＭＬ）和急性淋巴细胞白血病（ＡＬＬ）以及慢怀粒细胞白血病慢性期（ＣＭＬ－ＣＰ）ＧＡＴＡ－２检出     

IL—12基因转导EL—4细胞对C57BL／6小鼠的肿瘤疫苗作用

应用逆转录病毒构毒建了ＩＬ－１２表达载体,以研究ＩＬ－２基因修饰的肿瘤细胞的肿瘤疫苗作用,将其转染ＥＬ－４胸腺瘤细胞并研究了该基因导入细胞的抗肿瘤免疫效果。当接种了ＥＬ－４／ＩＬ－１２转染细胞后,在Ｃ５７ＢＬ／６同系鼠中其基因导入细胞的致瘤性比ＥＬ－４／Ｗｔ和ＥＬ－４／Ｎｅｏ组明显减少（Ｐ〈０．０１）,在ＥＬ－４／ＩＬ－１２被排斥后,体内试验中实验动物诱发了抗ＥＬ－４／Ｗｔ的系统性、保护性免疫,５     

白细胞介素2基因和白细胞介素3基因共转染白血病瘤苗的抗白血病作用的实验研究

目的：研究白细胞介素２（ＩＬ－２）和白细胞介素３（ＩＬ－３）基因共转染的白血病细胞瘤苗对白血病小鼠的治疗效果。方法：用ＩＬ－２重组腺病毒（Ａｄ－ＩＬ－２）和（或）ＩＬ－３重组腺病毒（Ａｄ－ＩＬ－３）转染红白血病细胞株ＦＢＬ－３，６０Ｃｏ照射后制备成瘤苗对实验性白血病小鼠进行治疗，观察肿瘤生长，小鼠生存期及治疗后小鼠腹腔巨噬细胞、ＮＫ细胞、诱导杀伤性Ｔ淋巴细胞（ＣＴＬ）杀伤活性，并与低剂量环磷酰胺（ＣＴＸ）合用，观察其抗肿瘤作用。结果：用ＩＬ－２或ＩＬ－３基因转染瘤苗治疗的白血病小鼠肿瘤生长明显减缓，生存期显著延长（Ｐ＜０．０５），用联合转染ＩＬ－２和ＩＬ－３基因的瘤苗治疗较单独转染ＩＬ－２或ＩＬ－３基因的瘤苗治疗效果更佳，与低剂量ＣＴＸ合用后抗肿瘤效果最佳。治疗后小鼠腹腔巨噬细胞、ＮＫ细胞、ＣＴＬ细胞杀伤活性显著增强。结论：ＩＬ－２、ＩＬ－３基因转染的瘤苗能够有效地通过诱导机体抗肿瘤免疫功能而发挥抗肿瘤作用，与低剂量ＣＴＸ合用后，抗肿瘤效果更佳。     

Targeting IL-13Ralpha2-positive cancer with a novel recombinant immunotoxin composed of a single-chain antibody and mutated Pseudomonas exotoxin

We have shown previously that high-affinity receptors for interleukin-13 (IL-13Ralpha2) are overexpressed on a variety of solid cancer cells, diseased fibroblasts, and other cells, and a chimeric fusion protein composed of human IL-13 and mutated Pseudomonas exotoxin (IL-13-PE38) is highly and specifically cytotoxic to these cells in vitro and in vivo. To improve the specificity for the target, we isolated specific antibodies against IL-13Ralpha2 from human single-chain Fv (scFv) antibody phage library and developed immunotoxin by selecting two high-affinity clones of scFv and fused to PE. The fusion chimeric gene was expressed in Escherichia coli, and highly purified IL-13R-specific immunotoxin, termed anti-IL-13Ralpha2(scFv)-PE38, was tested for its cytotoxicity. This molecule was highly cytotoxic to U251 glioma and PM-RCC renal cell carcinoma cell lines in vitro. The cytotoxic activity was neutralized by purified extracellular domain of IL-13Ralpha2 but not by IL-13, indicating that cytotoxic activity is specific. Anti-IL-13Ralpha2(scFv)-PE38 showed significant antitumor activity in immunodeficient mice with s.c. glioma tumors. Both i.p. and i.t. routes of administration showed antitumor activity in a dose-dependent manner. The maximum tolerated dose of anti-IL-13Ralpha2(scFv)-PE38 was 200 microg/kg i.p. twice daily for 5 days. These results indicate that anti-IL-13Ralpha2(scFv)-PE38 is a highly selective therapeutic agent for cancer therapy and should be further tested in animal models of human cancer.     

GENERATION OF CYTOTOXIC T LYMPHOCYTES IN VITRO : I. RESPONSE OF NORMAL AND IMMUNE MOUSE SPLEEN CELLS IN MIXED LEUKOCYTE CULTURES

Mouse cytotoxic T lymphocytes (CTL) were generated in mixed leukocyte cultures (MLC) using spleen cells as responding cells and irradiated allogeneic spleen cells as stimulating cells. Cytotoxicity was assessed by a quantitative ⁵¹Cr assay system and the relative frequency of CTL in individual cell populations was estimated from dose-response curves. Inclusion of 2-mercaptoethanol in the MLC medium resulted in a 20–40-fold increase in the relative number of CTL generated at the peak of the response. Under these culture conditions, cell-mediated cytotoxic activity was detectable in MLC populations as early as 48 h after the onset of the cultures. When spleen cells from mice immunized with allogeneic tumor cells 2–4 mo previously were cultured with irradiated spleen cells of the same alloantigenic specificity (MLC-Imm), it was found that the cell-mediated cytotoxic response was detectable earlier and reached higher levels than that observed in a primary MLC. At the peak of the response, MLC-Imm populations were observed to lyse up to 50% of the target cells within 3 h at a lymphocyte: target cell ratio of 0.3:1. Immunological and physical characterization of the effector cells generated in MLC-Imm indicated that they were medium to large-sized T lymphocytes. Altogether, these studies suggested the existence of an anamnestic cell-mediated cytotoxic response in MLC-Imm.     

Increased Production and Expression of Tissue Thromboplastin-Like Procoagulant Activity In Vitro by Allogeneically Stimulated Human Leukocytes

Intravascular coagulation, thrombosis, and fibrin deposition often produce tissue damage in allogeneic inflammatory reactions such as allograft rejection. The mechanisms which initiate blood clotting in these reactions are poorly understood. We find that allogeneic stimulation of human leukocytes in vitro increases production and expression of tissue thromboplastin-like activity. In our experiments mixed leukocyte cultures (MLC) of cells from allogeneic (unrelated) donors produced and expressed more procoagulant activity than control cultures of cells from each donor alone. After 7 days, allogeneic MLC had 5- to 50-fold more total procoagulant activity than controls, as shown by assaying lysed whole cultures. Additionally, allogeneic MLC had 8- to 240-fold more procoagulant activity expressed on leukocyte surfaces and in culture supernates than controls after 7 days, as shown by assaying intact whole cultures and cell-free supernates. These increases were largely accounted for by gains in the amounts of procoagulant activity produced and expressed per cell in MLC as compared to controls. Controls and MLC produced and expressed considerable amounts of procoagulant activity during the 1st day of culture, and there were no differential effects of allogeneic stimulation on day 1. However, after day 1, the total amount of procoagulant activity produced and the amount expressed declined steadily in controls, nearly reaching preculture levels by day 7. In contrast, the total amount of procoagulant activity in allogeneic MLC remained high, and the amount of activity expressed on cell surfaces and in supernates increased severalfold by day 7. MLC of syngeneic (identical twin) cells produced and expressed the same amount of activity as controls over a 7-day period, whereas MLC of cells from each twin and an allogeneic donor produced and expressed more activity than controls (at least 9- and 35-fold more, respectively). Thus, increases of procoagulant activity production and expression were found only in MLC of genetically dissimilar cells. Therefore, these increases must have resulted from allogeneic stimulation.     

Evidence for a B-cell -like helper function in mixed lymphocyte culture between immunocompetent thymus cells

The immunocompetent subpopulation by mouse thymus cell (TH-2) was isolated by buoyant density centrifugation and by hydrocortisone pretreatment. TH-2 cells undergo a proliferative one-way or two-way mixed lymphocyte culture (MLC) response only when cultured with allogeneic or congenic peripheral lymphoid cells. However, mixtures of allogeneic TH-2 cells alone do not proliferate in either one-way or two-way MLC reactions. Such MLC mixtures are proliferative only if mitomycin-blocked peripheral lymphoid cells are also present in the mixture. The peripheral helper cell has been found to be of low net density, non-adherent, insensitive to anti-thy-1 serum cytotoxicity, but sensitive to the cytotoxic effets of anti-immunoglobulin serume plus complement. The helper effect does not depend on proliferation nor does it appear to involve demonstrable soluble mediators. The nature of failure of MLC between TH-2 subpopulations appears to be dependent on the exppression of some product of the K, I regions of the H-2 locus. Possible mechanisms by which a B-cell-like helper cell triggers TH-2 proliferation are discussed terms of the present knowledge of specific alloantigen receptor on T and B cells, and the immunoglobulin Fc region receptors on T cells.     

IL-6受体α亚单位mRNA和蛋白在人白血病细胞中的表达

为了探讨IL－6R的α亚单位基因与蛋白在人白血病细胞中的表达，为临床利用IL－6／IL－6R系统介导重组IL－6－PE40外毒素融合蛋白靶向杀伤白血病细胞提供可靠依据，采用RT－PCR半定量技术、直接免疫荧光标记及流式细胞术检测了IL－6R的α亚单位基因及蛋白在多种人白血病细胞细胞系中的表达。结果表明，粒系、单核系、红白血病细胞系HL－60，KG－1，U937和TF－1均高表达IL－6R的α亚单位基因和蛋白；淋巴系白血病细胞系Raji亦有一定量的基因表达；而淋巴系细胞等CEM和HuT28以及慢性粒细胞白血病细胞系K562无论是IL－6Rα亚单位的基因还是蛋白均为阴性。相对表达丰度依次为KG－1＞TF－1＞U266＞U937＞HL－60＞Raji。鉴于粒系、单核系、红白血病细胞高表达IL－6Rα亚单位的基因和蛋白，而IL－6Rα亚单位蛋白在正常人外周血细胞均为阴性表达这一事实，提示可以利用IL－6／IL－6R系统介导重组IL－6－PE40外毒素融合蛋白进行靶向杀伤和治疗这些白血病，而且不会对正常血细胞产生毒副作用。     

RESPONSIVENESS OF GERMFREE MICE IN MIXED LEUKOCYTE CULTURE : REEXAMINING THE NATURE OF THE RESPONDING UNIT

Conventional (cv) and germfree (gf) mice are able to give a good proliferative response to allogeneic cells in the mixed leukocyte culture (MLC) test, while the response to xenogeneic stimulating cells has been in question. Previous studies by others have suggested only a low MLC response in cv animals and none in gf ones. We have found that both cv and gf animals can give a good MLC response to xenogeneic as well as allogeneic cells. These findings are of importance for our understanding of both MLC stimulation and response.     

Lysis of tumor biopsy cells by autologous T lymphocytes activated in mixed cultures and propagated with T cell growth factor

Blood lymphocytes from tumor patients were cocultivated with allogeneic lymphocytes (MLC) or autologous tumor cells (ATS), and their cytotoxicity was characterized. The main objective of the study was the lysis of autologous tumor biopsy cells by such effectors. Lymphocytes of patients activated in MLC lysed allogeneic third-party cells and in some cases also lysed autologous tumor cells. Allogeneic but not autologous PHA blasts were also damaged by these effectors. The cytotoxic potential of MLC-activated lymphocytes from healthy donors was similar; allogeneic tumors and phytohemagglutinin (PHA) blasts but not autologous PHA blasts were lysed. The cytotoxicity of lymphocytes activated in ATS were specific for the stimulator because they acted only on the autologous tumor cells. Allogeneic tumors and autologous and allogeneic PHA blasts were not lysed. The pattern of cytotoxicity with regard to this target panel was maintained when the MLC or ATS cultures were further propagated with TCGF. Results obtained in cold target competition assays suggested (a) activated lymphocyte lyse the third party tumor targets because of alloantigen recognition; (b) in MLC several different sets of alloreactive cytotoxic lymphocytes are present simultaneously; and (c) the alloreactive cells are different than those that act on the autologous tumor cells. Thus, the lysis of allogeneic tumor cells by lymphocytes of the patient is not due to recognition of cross-reacting tumor-related antigens, and the autotumor cytotoxicity of the patients' MLC-activated lymphocytes if performed by specifically reacting cells.     

3种同种异基因混合骨髓移植小鼠〔A＋B＋C→A）的模型

本研究目的为建立 3种异基因小鼠 (BALB/c ,H 2 d;C5 7BL/ 6 ,H 2 b 和CBA/N ,H 2 k)混合骨髓移植模型(A +B +C→A) ,观察此模型能否延长骨髓移植后受体鼠的存活时间。采用受致死量照射的小鼠接受同基因与 2种异基因 3种混合 (比例为 1∶4∶4 )的骨髓移植。同时还观察了 3种混合淋巴细胞培养及 2种不同抗原混合刺激的迟发型超敏反应。结果显示 ,混合骨髓移植的小鼠存活时间明显长于单纯异基因移植的小鼠 ,混合移植组平均存活时间为 5 6 .6± 2 7天 ,最长者为 10 3天 ,而单纯异基因移植的对照组存活时间为 15± 5天 ,两组比较有显著性差异(P <0 .0 0 1) ;3种混合的淋巴细胞培养 ,不管是 2种反应细胞与 1种刺激细胞混合培养 ,还是 1种反应细胞与 2种刺激细胞混合培养 ,均表现明显低于一对一的混合淋巴细胞反应 ,2种混合细胞刺激的迟发型超敏反应亦明显低于1种抗原刺激的反应。结论 :同基因与 2种异基因 3种混合的骨髓移植可使受髓小鼠存活时间明显延长。     

Antigen recognition by human T cell receptor gamma-positive lymphocytes. Specific lysis of allogeneic cells after activation in mixed lymphocyte culture

These experiments were designed to define the ability of human TCR-gamma+ cells to recognize allogeneic cells. TCR-gamma+-enriched populations were obtained by treating peripheral blood E-rosetting cells with anti-CD4 and anti-CD8 mAbs. The resulting populations were CD2+4-8- expressed variable proportions of CD3+ cells (40-90%), and did not react with the WT31 mAb, which is specific for a framework determinant of the alpha/beta heterodimer that serves as receptor for antigen on most human T lymphocytes. After mixed lymphocyte culture with irradiated allogeneic cells for 7 d and 3 additional days in rIL-2 (100 U/ml), cells underwent proliferation in three of five individuals tested. In addition, MLC-derived cells lysed 51Cr-labeled PHA-induced blasts derived from the allogeneic cells used as stimulator, but not allogeneic unrelated or autologous blast cells. No cytotoxicity against autologous or allogeneic target cells could be induced by culturing CD3+4-8-WT31- lymphocytes in MLC with irradiated autologous cells. Surface iodination of allogeneic MLC-activated CD3+4-8-WT31- cells followed by lysis in 1% digitonin and immunoprecipitation with anti-CD3 mAb indicated that the CD3-associated molecules consisted of a major 45-kD band and a minor band of 43 kD. Northern blot analysis showed that mRNA for the gamma chain was expressed at high levels, whereas mRNAs for alpha and beta chains were missing. These data support the notion that TCR-gamma rather than TCR-alpha/beta is expressed in allospecific CD3-4-8-WT31- cell populations. Clones were further derived from MLC-stimulated CD3+4-8-WT31- populations. All the seven clones studied in detail maintained the surface phenotype as well as the cytolytic pattern of the original MLC populations, thus only specific allogeneic PHA-induced blasts were lysed. NK-sensitive as well as NK-resistant tumor targets were variably susceptible to lysis; therefore, specific cytolytic activity against allogeneic cells was not necessarily linked to the expression of MHC-nonrestricted cytotoxicity against tumor cells.     

PROLIFERATION OF B AND T CELLS IN MIXED LYMPHOCYTE CULTURES

Electrophoretically fractionated CBA/Ca spleen T cells alone respond to allogeneic cells in one-way MLC and to PHA. They do not respond to E. coli LPS. B cells alone do not respond to allogeneic cells nor to PHA, but do respond to LPS. When karyotypically distinguishable syngeneic mixtures of T and B lymphocytes are stimulated with allogeneic cells, at the most 5% of mitoses on 5–9th culture day are of B cell origin. This indicates that B cells are not substantially recruited to proliferate in the MLC.