"Anomalous" Thy-1+ killer cells in allogeneic and F1-anti-parental mixed leukocyte culture. Relation to natural killer cells and allospecific cytotoxic T lymphocytes.
"Anomalous" Thy-1+ killer cells in allogeneic and F1-anti-parental mixed leukocyte culture. Relation to natural killer cells and allospecific cytotoxic T lymphocytes.
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DOI：
10.1084/jem.157.2.385
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发表时间：
1983-02-01
期刊：
影响因子：
--
通讯作者：
Kiessling R
中科院分区：
文献类型：
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作者：
Kärre K;Seeley JK;Eriksson E;Burton RC;Kiessling R
材料和方法动物。在我们自己的动物群中饲养了以下近交系和F1杂交种的小鼠：CBA、A/Sn（A）、C57 B 1/6（C57 B 1）、C57 B 1+/bg d、C57 B 1 bg J/bg d（C57 B 1 bg/bg）、DBA/2（DBA）、（CBA XA）F1和（AX C57 B1）F1。BALB/c、BALB/c nu/nu、BALB/c+/nu和C57 B1 nu/nu小鼠购自GI。Bomhotg~. rd有限公司，8860 Ry，丹麦。随机繁殖的Swiss+/nu和nu/nu同窝仔是来自德克萨斯州休斯顿的Stehlin癌症研究基金会的Beppino Giovanella博士的友好礼物。成人胸腺切除术。对3-4周龄的麻醉动物进行手术胸腺切除术。3 wk后，切除胸腺的小鼠和对照同窝小鼠接受650 rad全身照射，并通过静脉注射5-15× 106个同基因胎肝细胞进行重建。根据该方案，经成年胸腺切除、辐射和胎肝重建（AT X FL）处理的小鼠在用胸腺依赖性抗原马红细胞攻击后没有产生抗体应答（滴度< 1/10），而对照小鼠（C× FL）在重建后3-4周进行溶血试验时，滴度> 1/80（未发表的观察结果）。YAC-1是来源于A/Sn株的莫洛尼病毒诱导的淋巴瘤。YACACG、YAC-ACK和YAC-8是YAC-1的变体亚系，其已通过体外和体内重复暴露于来自CBA XA小鼠的高效NK细胞而被选择为低NK敏感性。该程序和变异亚系已在最近的报告中详细描述（17）。RBL-5是Rauscher病毒诱导的C57 B1淋巴瘤。MPC-11（矿物油诱导的浆细胞瘤）、RL-d（辐射诱导的淋巴瘤）和WEHI-164（甲基胆蒽诱导的纤维肉瘤）均来源于BALB/c品系。将细胞系作为悬浮培养物维持在补充有青霉素、链霉素和10%胎牛血清（FCS）或新生牛血清（NCS）的F-13培养基中。该培养基也用于细胞制备以及细胞毒性和竞争试验。细胞和培养物的制备。在F-13 NCS中制备淋巴结或脾细胞的单细胞悬液。洗涤和计数后，将细胞直接用于细胞毒性试验（第0天，新鲜脾细胞）或用于培养：将20-30× 106（通常为25× 106）个细胞单独或与6-15× 106（通常为10× 106）个经辐照（2，000 rad）的刺激细胞一起接种于50 ml Falcon组织培养瓶中（Falcon Labware，奥克斯纳德，CA）。因此，MLC中的最终应答者/刺激物比率在2：1和3：1之间变化，这产生了类似的结果。加入补充有10%FCS的培养基（含青霉素-链霉素、3× 10-5 M2-巯基乙醇和10 mMHepes缓冲液的RPMI）至终体积为10 ml，并将培养物在37 ℃、5%CO2下孵育。
Materials and Methods Animals. Mice of the following inbred strains and F1 hybrids were bred in our own animal colony: CBA, A/Sn (A), C57B 1/6 (C57B 1), C57B 1+/bg d, C57B 1 bgJ/bg d (C57B 1 bg/bg), DBA/2 (DBA),(CBA XA) F1, and (AX C57B1) F1. BALB/c, BALB/c nu/nu, BALB/c+/nu, and C57B1 nu/nu mice were purchased from GI. Bomhotg~. rd Ltd., 8860 Ry, Denmark. Randombred Swiss+/nu and nu/nu littermates were a kind gift from Dr. Beppino Giovanella, The Stehlin Foundation for Cancer Research, Houston, TX. Adult thymectomy. Surgical thymectomy was performed on 3-4-wk-old, anesthesized animals. 3 wk later, the thymectomized mice and control litter mates received 650 rad whole-body irradiation and were reconstituted by intravenous injection of 5-15× 106 syngeneic fetal liver cells. Mice treated with adult thymectomy, irradiation, and fetal liver reconstitution (AT X FL) according to this protocol did not give antibody responses (titers< 1/10) after challenge with the thymus-dependent antigen horse erythrocytes, whereas control mice (C× FL) gave titers> 1/80 when tested in a hemolysis assay 3-4 wk after reconstitution (unpublished observation).Tumors. YAC-1 is a Moloney virus induced lymphoma derived from the A/Sn strain. YACACG, YAC-ACK, and YAC-8 are variant sublines of YAC-1, which have been selected for low NK sensitivity by repeated exposures to highly efficient NK cells from CBA XA mice in vitro and in vivo. The procedure and the variant sublines have been described in detail in a recent report (17). RBL-5 is a Rauscher virus-induced C57B1 lymphoma. MPC-11 (mineral oilinduced plasmocytoma), RL-d (radiation-induced lymphoma), and WEHI-164 (methylcholanthrene-induced fibrosarcoma) are all derived from the BALB/c strain. The cell lines were maintained as suspension cultures in F-13 medium supplemented with penicillin, streptomycin, and 10% fetal (FCS) or newborn calf serum (NCS). This medium was also used for preparations of cells as well as in cytotoxicity and competition assays. Preparation of Cells and Cultures. Single-cell suspensions of lymph node or spleen cells were prepared in F-13 NCS. After washing and counting, cells were used either directly in a cytotoxic assay (day 0, fresh spleen cells) or for cultures: 20-30× 106 (usually 25× 106) cells were plated alone or with 6-15× 106 (usually 10× 106) irradiated (2,000 rad) stimulator cells in 50-ml Falcon tissue culture flasks (Falcon Labware, Oxnard, CA). Thus, the final responder/stimulator ratio in MLC varied between 2: 1 and 3: 1, which yielded similar results. Culture medium (RPMI with penicillin-streptomycin, 3× 10-5 M 2-mercaptoethanol, and 10 mM Hepes buffer) supplemented with 10% FCS was added to a final volume of 10 ml, and the cultures were incubated at 37 C, 5% CO2.