IN VITRO STUDIES OF MAMMALIAN SOMATIC CELL VARIATION : II. ISOIMMUNE CYTOTOXICITY WITH A CULTURED MOUSE LYMPHOMA AND SELECTION OF RESISTANT VARIANTS

When long term cultures of mouse lymphoma cells, known to possess the isoantigenic phenotype determined by the H-2^d allele, are incubated with anti H-2d isoantibody and guinea pig complement, slightly more than 99 per cent of cells are killed under optimal conditions. Growth in mass culture and colony formation by single cells after incubation with isoantibody and complement are employed to assess the cytotoxic effect. The cytotoxic action of isoantibody is complement-dependent, for viability of cells exposed to antibody alone is unaltered. When excess isoantibody and optimum concentrations of complement are used, killing begins as soon as these reagents are mixed with the cells, and no further killing occurs after 5 to 15 minutes at 37°C. About 80 per cent of cells are killed with an isoantiserum containing antibody to two isoantigenic components of the H-2d complex. That the cytotoxic action is mediated through the H-2 isoantigen is shown by (a) isoantiserum containing only anti H-2d antibody produces maximal cell killing, and (b) isoantiserum from which anti H-2d antibody has been removed by absorption loses all cytotoxic activity. Variant cells resistant to the cytotoxic action of anti H-2d isoantibody were isolated from lymphoma cell populations surviving multiple exposures to isoantibody and complement. These variants can be distinguished morphologically from the isoantibody-sensitive parent cell line. Although variants are resistant to anti H-2d isoantibody, these cells possess H-2d isoantigen but in a lower concentration than found in cells of the parent line. The basis for resistance to cytotoxic isoantibody is discussed.     

利用流式细胞仪检测人外周血NK细胞毒性方法的建立

目的利用流式细胞仪（FAcs）技术检测人外周血NK细胞的毒性。方法首先将pEGFP-N1质粒转染人NK细胞的天然靶细胞K562，经过G418筛选后进一步单克隆化，得到稳定、均一表达绿色荧光蛋白的K562细胞。取对数期的K562-EGFP细胞与人外周血单个核细胞分别按5：1、10：1、20：1、40：1的比例混合，分别孵育0．5、1、2、4h后用碘化丙啶（PI）标记，用流式细胞仪分析呈红、绿色荧光的细胞数，最后计算NK细胞的杀伤效率。结果0．5、1、2、4h均能得到明显的杀伤效果，而且以2h的杀伤率最高。此时杀伤率与传统乳酸脱氢酶法（LDH）具有显著相关性（7=0．997，P=0．003），而且显示出更强的敏感性。结论利用流式细胞仪检测NK细胞毒活性的方法可作为传统^51Cr释放法、LDH法的一个补充，而且具有经济、快速和敏感的特点。     

利用流式细胞仪检测人外周血NK细胞毒性方法的建立

目的利用流式细胞仪(FACS)技术检测人外周血NK细胞的毒性。方法首先将pEGFP-N1质粒转染人NK细胞的天然靶细胞K562,经过G418筛选后进一步单克隆化,得到稳定、均一表达绿色荧光蛋白的K562细胞。取对数期的K562-EGFP细胞与人外周血单个核细胞分别按5∶1、10∶1、20∶1、40∶1的比例混合,分别孵育0.5、1、2、4 h后用碘化丙啶(PI)标记,用流式细胞仪分析呈红、绿色荧光的细胞数,最后计算NK细胞的杀伤效率。结果0.5、1、2、4 h均能得到明显的杀伤效果,而且以2h的杀伤率最高。此时杀伤率与传统乳酸脱氢酶法(LDH)具有显著相关性(γ=0.997,P=0.003),而且显示出更强的敏感性。结论利用流式细胞仪检测NK细胞毒活性的方法可作为传统51Cr释放法、LDH法的一个补充,而且具有经济、快速和敏感的特点。     

COMMON CELL SURFACE ANTIGEN ASSOCIATED WITH MAMMALIAN C-TYPE RNA VIRUSES : CELL MEMBRANE-BOUND gs ANTIGEN

The indirect membrane immunofluorescence test and the absorption analysis of rabbit anti-FeLV, rabbit anti-FeLVp 30, and rabbit anti-MuLVp 30 antisera yielded the following conclusions. An antigen shared by mammalian (murine and feline) C-type RNA leukemia and sarcoma viruses was detected on the surface of cells infected or transformed by C-type viruses. The antigen was characterized as membrane-bound gs antigen bearing two determinants, membrane-bound gs-1, intraspecies-specific antigenic determinant, and membrane-bound gs-3, interspecies-specific antigenic determinant. Membrane-bound gs antigen was located on the cell surface, frequently near the site of virus budding but not on the envelope of murine C-type RNA virus.     

IN VITRO STUDIES OF MAMMALIAN SOMATIC CELL VARIATION : I. DETECTION OF H-2 PHENOTYPE IN CULTURED MOUSE CELL LINES

The isoantigenic phenotype of the H-2 locus has been detected by isohemagglutinin absorption in a line of mouse lymphoma cells growing continuously in culture for 6 years and in two established lines of fibroblastic mouse cells growing continuously in culture for 1 year. Quantitative absorption studies suggest that the concentration of H-2 isoantigens is higher in the cultured lymphoma cells than in the other two fibroblastic cell lines.     

干扰素-r对胃癌细胞增殖和调变5-FU细胞毒作用的实验研究

目的 :探讨干扰素 -r体外对胃癌细胞增殖抑制作用和调变 5 -氟脲嘧啶对胃癌细胞增殖抑制作用的影响。方法 :采用原代细胞培养和MTT活细胞定量技术 ,观察和比较单独使用不同剂量干扰素 -r和联合 5 -氟脲嘧啶对胃癌原代细胞增殖的抑制效应。结果 :工作浓度为每毫升 2 0 0、10 0 0、2 0 0 0和 30 0 0的干扰素 -r作用于不同胃癌原代细胞株 72h后 ,体外对胃癌原代细胞的抑制率中位值分别是 7 33% ,8 68% ,11 0 0 %和 9 30 % ,组间无统计学显著性差异 (P >0 0 5 )。联合应用工作浓度为 2 0 0的 5 -氟脲嘧啶后 ,上述四种浓度的干扰素 -r对胃癌原代细胞的直接抑制作用中位值分别为 35 45 % ,37 80 % ,38 2 8%和 39 37% ,组间无统计学显著性差异 (P >0 0 5 )。结论 :不同剂量干扰素 -r体外对胃癌原代细胞增殖抑制作用较小 ,无剂量效应相关性 ,不同剂量干扰素 -r与 5 -氟脲嘧啶联合应用未能显示调变 5 -氟脲嘧啶对胃癌原代细胞的增殖抑制效应 ,干扰素 -r体外对胃癌原代细胞的免疫治疗和免疫化疗作用尚不明确     

A PEROXIDASE-MEDIATED, STREPTOCOCCUS MITIS-DEPENDENT ANTIMICROBIAL SYSTEM IN SALIVA

H₂O₂ formation by Streptococcus mitis was measured by the catalase-dependent conversion of [¹⁴C]formate to ¹⁴CO₂ ; it was optimal at pH 6.0–6.5 and required glucose. The H₂O₂ formed by S. mitis could be employed as a component of an antimicrobial system that also included lactoperoxidase (LPO) and either iodide or thiocyanate ions in the concentrations present in saliva. The antimicrobial effect of the LPO-iodide-S. mitis system was measured by the decrease in the viable cell count of the target organisms (Escherichia coli, Staphylococcus aureus, Candida tropicalis). The antimicrobial effect of the LPO-thiocyanate-S. mitis system was measured by the decrease in the rate of growth or the rate of uptake of [¹⁴C]valine by the target organisms (E. coli, S. aureus). Mixed or parotid saliva could replace LPO and thiocyanate ions in the S. mitis-dependent inhibition of bacterial growth and valine uptake. The presence in saliva of a peroxidase-mediated, antimicrobial system dependent on microbial metabolism for H₂O₂ and its role as a natural host defense mechanism are considered.     

A SIMPLIFIED CHEMOSTAT FOR THE GROWTH OF MAMMALIAN CELLS: CHARACTERISTICS OF CELL GROWTH IN CONTINUOUS CULTURE

A simplified technique has been described for the continuous growth of mammalian cells in suspension culture. The cell population density increased as the rate of input of fresh medium was decreased, and the average generation time was concommittantly prolonged. At relatively high input rates, the population remained stabilized for an indefinite period, but at low flow rates, there was sometimes a cyclical variation in population density. The factor limiting growth rate at input rates of approximately 0.2 volumes per day was not the exhaustion of the medium; but in some experiments a non-dialyzable material appeared which inhibited cell growth.     

REGULATION OF CELL-MEDIATED CYTOTOXICITY : I. AUGMENTATION OF CELL-MEDIATED CYTOTOXICITY INDUCED BY RADIATION

Mice were treated with sublethal and midlethal doses of irradiation (500–700 rads) and injected intravenously with allogeneic or semiallogeneic F₁ hybrid spleen cells. The cytotoxicity developed by their spleen cells was measured with the lysis of ⁵¹Cr-labeled target cells and was found to be stronger (although delayed in time) than the cytotoxic activity of spleen cells from nonirradiated mice. The injection of syngeneic thymus or spleen cells in the irradiated mice after their treatment with allogeneic spleen cells exerted a suppressor activity, i.e., reduced the level of cell-mediated cytotoxicity (CMC). The majority of effector cells involved in the modified CMC response of irradiated mice was shown to be of host origin and lysed specifically target cells of the same genotype as the donor. A small percentage of the cells obtained from the spleens of irradiated recipients of allogeneic spleen cells was composed of donor cells which lysed specifically target cells of the same genotype as the host. These results demonstrate that the precursors of the cytotoxic cells responsible for target cell lysis are relatively radioresistant and suggest that their response is regulated by radiosensitive thymus-dependent cells.     

REGULATION OF CELL-MEDIATED CYTOTOXICITY : I. AUGMENTATION OF CELL-MEDIATED CYTOTOXICITY INDUCED BY RADIATION

Last line read B16 for SaI     

A NEW SENSITIVE ASSAY FOR ANTIBODY AGAINST CELL SURFACE ANTIGENS BASED ON INHIBITION OF CELL-DEPENDENT ANTIBODY-MEDIATED CYTOTOXICITY : I. Specificity and Sensitivity

Inhibition of cell-dependent antibody-mediated cytotoxicity has been investigated as a new assay for antibody against cell surface antigens. The cytotoxicity system consisted of effector cells (normal mouse spleen cells), target cells (⁶¹Cr-labeled chicken erythrocytes), and antitarget cell antibody. Addition of antibody against cell surface antigens in the effector cell population regularly inhibited the cytotoxicity measured in this system. This cytotoxicity inhibition assay (CIA) detected antibody with a variety of specificities: anti-H-2, anti-Thy 1.2, anti-immunoglobulin, and antimouse bone marrow-derived lymphocyte antigen. When the inhibition by anti-H-2 sera was analyzed using effector cells from congenic mice, the activity was found to be directed against specificities mapping in the H-2K, H-2D, and I regions of the H-2 complex, correlating well with the specificities characterized by complement-dependent assays. A comparison between the sensitivity of the CIA and complement-dependent lysis revealed that the CIA was 2–11 times more sensitive for anti-H-2 antisera and 20–780 times more sensitive for certain antisera against subpopulations of the spleen cells (i.e., T cells or B cells). The CIA proved to be precise, sensitive, and reliable. It may become a very useful antibody assay in various species including man.     

STUDIES ON THE MODE OF ACTION OF DIPHTHERIA TOXIN : VII. TOXIN-STIMULATED HYDROLYSIS OF NICOTINAMIDE ADENINE DINUCLEOTIDE IN MAMMALIAN CELL EXTRACTS

When diphtheria toxin and NAD are added to soluble fractions containing aminoacyl transfer enzymes isolated from rabbit reticulocytes or from HeLa cells, free nicotinamide is released and, simultaneously, an inactive ADP ribose derivative of transferase II is formed. The reaction is reversible, and in the presence of excess nicotinamide, toxin catalyzes the restoration of aminoacyl transfer activity in intoxicated preparations. In living cultures of HeLa cells, the internal NAD concentration is sufficiently high to account for the rapid conversion, catalyzed by a few toxin molecules located in the cell membrane, of the entire cell content of free transferase II to its inactive ADP ribose derivative. Completely inactive ammonium sulfate fractions containing soluble proteins isolated from cells that have been exposed for several hours to excess toxin, can be reactivated to full aminoacyl transfer activity by addition of nicotinamide together with diphtheria toxin. Transferase II appears to be a highly specific substrate for the toxin-stimulated splitting of NAD and thus far no other protein acceptor for the ADP ribose moiety has been found.     

HUMAN ALLERGY TO MAMMALIAN SERA

Four patients allergic to many mammalian sera were found to have circulating, skin-sensitizing antibodies for these sera. A study of these antibodies by in vitro neutralization and subsequent local passive transfer showed that, in the case of each patient, one particular mammalian serum neutralized antibodies for all sera whereas other mammalian sera neutralized antibodies for themselves alone or for themselves plus one or several additional sera but not for all sera. The possibility of multiple common allergenic determinants in mammalian sera is discussed.     

ESCAPE FROM ISOANTISERUM INHIBITION OF LYMPHOCYTE-MEDIATED CYTOTOXICITY

Isoantiserum (IS) inhibition of lymphocyte-mediated cytotoxicity (LMC) was studied using an in vitro ⁵¹Cr release assay system. In the early phase of incubation, LMC was competitively inhibited by IS. However, as the incubation continued, LMC irreversibly overcame IS inhibition (the "escape" phenomenon). Addition of fresh antiserum did not alter the course of the escape. Low-temperature incubation of isoantibody-coated target cells delayed the onset of the escape. We have excluded the possibility that the escape phenomenon is induced by complement or by LMC mediated by antigen-antibody complex. It is hypothesized that antibody directed toward an actively metabolizing target cell induces an alteration in the cell membrane that alters further interaction with the antibody. However, sensitivity to lymphocyte cytotoxicity is maintained.     

LYMPHOCYTE-MEDIATED CYTOTOXICITY IN VITRO : EFFECT OF ENHANCING ANTISERA

The ability of antisera to suppress immune responses either in vivo or in vitro is well known. A variety of lymphocyte-target cell systems have been employed to demonstrate inhibition of cell-mediated immunity by antisera in vitro, and skin, tumor, and kidney graft survival have been prolonged by passively administered antiserum in vivo. An in vitro lymphocyte-tumor cell assay system was developed for the purpose of studying the effects of enhancing antisera (in vivo) on lymphocyte-mediated cytotoxicity in vitro. The characteristics of this system with respect to route of immunization, time of harvest of immune cells, lymphocyte:tumor cell ratio, and effect of nonimmune or nonspecifically immune lymphoid cells are presented. Sera capable of enhancement in vivo were tested in this system and shown to inhibit cell-mediated immunity in vitro. Further, in both instances the immunosuppressive effect is mediated by antigen-antibody complexes and not by free antibody alone. Experiments were also carried out to determine the site of action of these suppressive antigen-antibody complexes. Presensitized lymphocytes were exposed to antigen-antibody complexes, washed, and then allowed to interact with fresh tumor cells (not antibody treated). Lymphocytes treated in this manner are incapable of exhibiting cell-mediated immunity in vitro. This evidence supports the concept that the antigen-antibody complexes have a direct immunosuppressive effect on the lymphocyte.     

THE MODULATING INFLUENCE OF CYCLIC NUCLEOTIDES UPON LYMPHOCYTE-MEDIATED CYTOTOXICITY

The capacity of allosensitized thymus-derived lymphocytes to destroy target cells bearing donor alloantigens is modulated by the cellular levels of cyclic AMP and cyclic GMP. Increases in the cyclic AMP levels of attacking lymphocytes by stimulation with prostaglandin E₁, isoproterenol, and cholera toxin inhibit lymphocyte-mediated cytotoxicity; whereas, depletion of cyclic AMP with imidazole enhances cytotoxicity. The augmentation of cytotoxicity produced by cholinergic stimulation with carbamylcholine is not associated with alterations in cyclic AMP levels and is duplicated by 8-bromo-cyclic GMP. The effects of activators of adenylate cyclase, cholinomimetic agents, and 8-bromocyclic GMP are upon the attacking and not the target cells and occur at the time of initial interaction of attacking and target cells. Indeed, the level of cyclic nucleotide (cyclic AMP and cyclic GMP) at the time of initial cell-to-cell interaction determines the extent of cytotoxicity.     

INHIBITION OF SPECIFIC CELL-MEDIATED CYTOTOXICITY BY ANTI-T-CELL RECEPTOR ANTIBODY

The present study describes a method for the production of a specific anti-T-cell receptor antiserum, and characteristics of its ability to block specific cell-mediated cytotoxicity in vitro. Immunization and antiserum adsorption procedures were designed to select for idiotypic differences in the recognition units of C3H lymphocytes immune to two different strains of mouse cells, such that the reactivity of only one population of effector cells is inhibited by this antiserum. Both in vivo and in vitro sensitized effector T cells are subject to this inhibition. That the site of the antiserum blockade is clearly on the effector cell and not on the target cell is demonstrated.     

IMMUNOLOGIC CROSS-REACTIONS AMONG MAMMALIAN ACUTE PHASE PROTEINS

Crystalline rabbit Cx-reactive protein has been compared immunologically with the analogous crystalline C-reactive protein of man. Immunologic cross-reactivity has been demonstrated between the acute phase proteins of man, rabbit, and monkey. Double-diffusion reactions in agar and passive cutaneous anaphylaxis reactions in vivo both indicate that these acute phase proteins are antigenically closely similar but not identical. Guinea pigs with delayed hypersensitivity to C-reactive protein exhibit delayed skin reactions when tested with Cx-reactive protein and vice versa.