双特异性抗体对LAK细胞增殖和细胞毒作用影响的体…

将抗ＣＤ１３与抗ＨＢｓ的单克隆抗体经化学偶联得到的双特异性抗体，观察该双特异性抗体对ＬＡＫ细胞增殖和增强细胞毒性的作用。结果显示双特异性抗体显著提高ＬＡＫ细胞与２．２．１５细胞结合率；促进淋巴细胞增殖．＾１２５Ｉ－ＵｄＲ释放试验检测发现双特异性抗体增强ＬＡＫ细胞对２．２．１５细胞的细胞毒性且与抗体浓度呈正相关 。     

抗CD3—抗B细胞白血病独特型双特异性抗体的制备及细胞毒性

采用化学偶联法制备抗ＣＤ３抗Ｂ细胞性白血病独特型双特异性抗体，研究了该双特异性抗体诱导ＬＡＫ细胞的增殖及其细胞毒作用。结果提示，双特异性抗体可显著提高ＬＡＫ细胞与Ｄａｕｄｉ细胞的结合率；促进淋巴细胞的增殖。乳酸脱氢酶试验检测发现，加入双特异性抗体后，ＬＡＫ细胞对Ｄａｕｄｉ细胞的细胞毒性作用显著高于对照组。     

抗人小细胞型肺癌和抗CD3单克隆抗体的异质交联体的制备及其…

本文报道了用化学交联法制备同时识别人小细胞型肺癌表面相关抗原和人ＣＤ３抗体的双特异性抗体２Ｄ６－ＵＣＨＴ１的实验研究工作。＾５１Ｃｒ释放法测试结果表明：２Ｄ６－ＵＣＨＴ１可显著增强ＬＡＫ细胞对靶瘤细胞的毒性，而且这种增强效应与加入双特异性抗体的浓度相关。     

抗人小细胞型肺癌和抗CD3单克隆抗体的异质交联体的制备及其增强LAK细胞杀伤活性的实验研究

本文报道了用化学交联法制备同时识别人小细胞型肺癌表面相关抗原和人ＣＤ３抗体的双特异性抗体２Ｄ６－ＵＣＨＴ１的实验研究工作。（５１）Ｃｒ释放法测试结果表明：２Ｄ６－ＵＣＨＴ１可显著增强ＬＡＫ细胞对靶瘤细胞的毒性，而且这种增强效应与加入双特异性抗体的浓度相关。     

抗人黑素瘤双特异性抗体增强LAK细胞毒效应的实验研究

抗肿瘤单克隆抗体的高度特异性与杀伤细胞对肿瘤的细胞毒效应相互结合起来，是增强ＬＡＫ细胞抗肿瘤作用的重要途径。本研究用化学连接法制备了既可识别淋巴细胞表面ＣＤ３抗原又可识别ＬｉＢｒ黑素瘤细胞表面肿瘤相关抗原的双特异性抗体ＣＤ３－ＨＢ８７５９。ＦＡＣＳ分析证实，ＣＤ３－ＨＢ８７５９具有结合两种抗原的活性。４ｈ５１Ｃｒ释放细胞毒实验结果表明，ＣＤ３－ＨＢ８７５９在诱导相可显著增强ＬＡＫ对ＬｉＢｒ细胞的细胞毒效应，也能使非ＬＡＫ细胞获得细胞毒性；在杀伤相加入ＣＤ３－ＨＢ８７５９对ＬＡＫ细胞的细胞毒作用也具有促进作用。上述结果表明，ＣＤ３－ＨＢ８７５９是一种抗人黑素瘤的双特异性抗体，为体内应用提供了良好的实验基础。     

抗CD3－抗胶质瘤双特异性抗体的制备及其细胞毒活性作用的研究

采用化学偶联法制备抗ＣＤ３－抗胶质瘤双特异性的抗体，其分子结构为杂合的Ｆ（ａｂ′）２片段，结合率试验证明，此双抗能同时识别并结合ＬＡＫ细胞和胶质瘤细胞，在细胞毒性试验中，虽然双抗、ＣＤ３单抗和ＳＺ－３９单抗混和物均能提高ＬＡＫ细胞对胶质瘤细胞的杀伤作用，但双抗的作用特别显著（Ｐ＜０．０１），增强效应为１４８％，而在以非胶质瘤细胞为靶细胞时，双抗虽有增强作用，但低于ＣＤ３单抗的作用（Ｐ＜０．０５）。     

抗CD3—抗胶质瘤双特异性抗体的制备及其细胞毒活性作…

采用化学联法制备抗ＣＤ３－抗胶质瘤双特异性的抗体，其分子结构为杂合的Ｆ（ａｂ‘）２片段、结合率试验证明，此双抗能同时识别并结合ＬＡＫ细胞和胶质瘤细胞，在细胞毒性试验中，虽然双抗，ＣＤ３单抗和ＳＺ－３９单抗混和物均能提高ＬＡＫ细胞对胶质瘤细胞的杀伤作用，但双抗的作用特别显著，增强效应为１４８％，而在以非胶质瘤细胞为靶细胞时，双抗虽有增强作用，但低于ＣＤ３单抗的作用。     

T-AK细胞和IL-2脂质体联合硒酸酯多糖抗白血病效应的研究

目的研究抗ＣＤ３单克隆抗体与ＩＬ－２共同诱导的Ｔ－ＡＫ细胞和ＩＬ－２脂质体（Ｌ－ＩＬ－２）联合硒酸酯多糖（ＫＳＣ）对Ｌ１２１０小鼠白血病的治疗作用。方法用ＤＢＡ／２小鼠建立Ｌ１２１０白血病模型，正常小鼠脾细胞诱生制备Ｔ－ＡＫ细胞，按设计方案转输Ｔ－ＡＫ细胞和ＩＬ－２脂质体，ＫＳＣ灌胃，检测ＮＫ细胞活性、脾淋巴细胞增殖活性和ＩＬ－２诱生水平，观察荷瘤小鼠生存期。结果Ｌ１２１０白血病小鼠的免疫功能急剧降低，生存期为１６．４３±１．９２天；转输Ｔ－ＡＫ细胞（５×１０６）和Ｌ－ＩＬ－２（１０４Ｕ／ｋｇ）能部分逆转白血病小鼠低下的细胞免疫功能，生存期延长（２４．７８±３．９４天），并有１４．３％小鼠长期存活；ＫＳＣ（４０ｍｇ／ｋｇ）对Ｔ－ＡＫ／Ｌ－ＩＬ－２的抗白血病作用有明显的增强效应，荷瘤小鼠细胞免疫功能进一步增强，生命延长率、长期存活率分别提高３６％和９９．９％。结论硒酸酯多糖具有生物反应调节剂（ＢＲＭ）样作用；以应用Ｔ－ＡＫ细胞和ＩＬ－２脂质体为主体，硒酸酯多糖为辅佐的生物治疗方案对白血病小鼠具有显著的免疫抑瘤作用     

rhIL-15和rhIL-2诱生的LAK细胞特性比较

目的：研究ｒｈＩＬ－１５激活的ＬＡＫ细胞的增殖、细胞毒作用及相关表型的变化。方法：通过用不同浓度的ｒｈＩＬ－１５和ｒｈＩＬ－２分别诱生 ＬＡＫ细胞，研究二者诱生的 ＬＡＫ细胞的增殖状况；当 ｒｈＩＬ－２和 ｒｈＩＬ－１５为 １５００ Ｕ／ｍｌ时，ＭＴＴ法检测 ＬＡＫ细胞的细胞毒作用，利用流式细胞仪检测细胞表面ＣＤ分子表达情况。结果：ＬＡＫ细胞的增殖对ｒｈＩＬ－２和ｒｈＩＬ－１５均具有时间和剂量依赖性，且无明显差异；终浓度为 １５００ Ｕ／ｍｌ时，ｒｈＩＬ－１５诱生的 ＬＡＫ细胞杀伤 Ｋ５６２细胞的能力强于 ｒｈＩＬ－２诱生的 ＬＡＫ细胞，而对Ｒａｊｉ细胞的杀伤活性，ｒｈＩＬ－２诱生的ＬＡＫ细胞却明显高于 ｒｈＩＬ－１５诱生的 ＩＡＫ细胞，且二者杀伤活性均具有时间依赖性；同时ｒｈＩＬ－１５诱生的ＬＡＫ细胞的ＣＤ３－ＣＤ５６＋细胞、Ｃｄ９４＋细胞百分率均高于ｒｈＩＬ－２诱生的ＬＡＫ细胞。结论：ｒｈＩＬ－１５在体内对ＮＫ细胞功能可能具有较强的调节作用。     

黄芪多糖对IL－2／LAK抗肿瘤增强作用的动物实验研究

采用Ｃ５７ＢＬ／６鼠荷其自发产生的黑色素瘤Ｂ＿１６细胞，以生存期为指标，建立了黄茂多糖（ＡＰＳ）增强ＩＬ－２／ＬＡＫ抗肿瘤作用的动物模型，结果表明：ＡＰＳ自身具有一定的抗肿瘤作用，ＡＰＳ（５ｍｇ／ｋｇ）与ＩＬ－２／ＬＡＫ（５×１０￣３Ｕ／ｋｇ、５×１０￣７／ｋｇ）的抗肿瘤作用相似，荷瘤鼠生存期分别为２１．５７±１．８１天和２０．８６±１．８６天（荷瘤对照鼠为１５．７１±０．４９天）；ＡＰＳ对ＩＬ－２／ＬＡＫ的抗肿瘤效应有明显的增强作用，二者在上述剂量下联合应用，荷瘤鼠生存期为２４．８６±２．７３天（Ｐ＜０．０１）。并对荷瘤鼠进行动态细胞免疫功能观察，提示：ＡＰＳ和ＩＬ－２／ＬＡＭ均具有抵抗鼠脾ＮＫ细胞活性，ＩＬ－２产生能力和腹腔Ｍψ吞噬能力下降的作用，ＡＰＳ对ＩＬ－２／ＬＡＫ仍有显著增强效应。     

A novel method for the simultaneous assessment of natural killer cell conjugate formation and cytotoxicity at the single-cell level by multi-parameter flow cytometry

A flow cytometric assay for the combined measurement of cell-mediated cytotoxicity and conjugate formation has been developed. Cytolysis is detected by propidium iodide uptake. Target cells, effector cells and conjugates between targets and effectors are separated by post-culture immunophenotyping and their scatter profiles. Pre-assay staining of cells is thus not required. Each cluster of cells can be further examined at the single-cell level by simultaneously performed additional immunophenotyping. Two applications were established: the assessment of NK cell activity against K562 cells and the evaluation of LAK cell cytotoxicity against both K562 and Daudi cells. A comparison with the standard 51Cr release assay for the detection of NK cytotoxicity showed that the two assays were strongly correlated, but the sensitivity of the flow cytometric assay was significantly higher.     

Binding of Leukaemic Blasts to Various Subpopulations of Lymphokine-Activated Killer Cells

Conjugate formation by natural killer (NK)-resistant and N K-sensitive leukaemic cell lines with fresh and IL-2-stimulated (lymphokine-activated killer, LAK) peripheral blood lymphocytes (PBL) was studied by a flow cytofluorometry method with double staining, A significant difference in binding of NK-resistant T-cell lymphoma (HuT 78) and NK-sensitive myeloid (K562) blasts to fresh PBL was observed ( P <0.01). Activation of lymphocytes with IL-2 resulted in a significant increase of binding and killing of both K562 and HuT 78. However, in the case of blasts from NK-resistant cell line Daudi a similar conjugate formation with fresh PBL and LAK effectors was observed, despite a significant increase in killing. Various subpopulalions of LAK effectors (CD3, CD16 and CD56 positive) displayed similar binding activity towards myeloid (K562) and lymphoid (Raji) blasts, which shows that conjugate formation occurs not only with NK-cell-derived. but also with T-cell-derived LAK cells. The blocking of CD71 antigen (transferrin receptor) on K562 blasts inhibited binding of cytotoxic lymphocytes, which was mostly due to the blocking of binding of CD56⁺ subpopulation.
Our results indicate that the resistance of leukaemic blasts to cell-mediated cytotoxicity may depend on different (and probably several) steps of this process.     

Lymphokine-activated killer-cell-mediated killing of WiDr colon carcinoma cells is inhibited by K562 erythroleukemia cells.

Lymphokine activated killer (LAK) activity was induced in human peripheral mononuclear blood cells by human recombinant interleukin-2. Monocytes were required for optimal rapid proliferation of cells with LAK activity. They had no influence on the expression of tumoricidal activity by the LAK cells. The effector cells killed K562 erythroleukemia cells and WiDr colon cells differently, i.e. contact areas with WiDr cells were limited, whereas the contact areas between effector cells and K562 cells were much longer. Using mixtures of hot and cold target cells it was shown that effector cells preferably bind with K562 cells, impeding the binding of WiDr cells. Differences in expression of cytotoxicity of LAK cells against WiDr and K562 cells respectively was also observed after culturing the LAK cells for a relatively longer period. Cytotoxicity against WiDr was maximal at 3-16 days after starting LAK cell generation, whereas cytotoxicity against K562 was kept constantly high for at least 21 days. The addition of biological response modifiers [PHA and anti-CD3 antibody (OKT3)] during the LAK cell induction also had different effects on the expression of LAK activity against WiDr and K562 cells. Whereas PHA, in combination with rIL-2 had no significant influence on the cytotoxicity against WiDr cells, the cytotoxicity against K562 was significantly inhibited. Addition of anti-CD3 antibody diminished the cytotoxicity against WiDr target cells and had no influence on the cytotoxicity against K562 cells.     

Peripheral blood lymphocytes resistant to Epstein-Barr virus immortalization manifest high natural killer (NK) type activity against NK-resistant target cells

Epstein-Barr virus (EBV) readily immortalizes human peripheral blood lymphocytes (PBL) in vitro. We found recently that PBL from two EBV-seropositive healthy adults were exceptionally resistant to immortalization by EBV. In contrast to PBL from other EBV-seropositive donors sensitive to immortalization by EBV (S-PBL), the "resistant" PBL (R-PBL) respond to EBV infection with an early interleukin-2 (IL-2) synthesis and high interferon gamma (IFN gamma) production. In order to determine whether these differences in cytokine responses between R-PBL and S-PBL could be associated with a detectable difference in lymphocyte cytotoxicity, we compared the natural killer (NK) activity of R-PBL and S-PBL effectors by using both NK-sensitive (i.e. K562) and NK-resistant (i.e. Raji) targets. We found that, while effectors from EBV-infected R-PBL and S-PBL cultures exhibited comparable NK activity against the K562 targets, they differed remarkably in their cytolytic activity against Raji cells. At days 3 and 5 of culture, effectors from EBV-infected R-PBL showed a significantly higher lytic activity against Raji targets, whereas S-PBL did not. Culture of EBV-infected R-PBL and S-PBL effectors in the presence of recombinant IL-2 (rIL-2) for 5 days resulted in increases of their lytic activity against Raji cells, whereas pretreatment of these effectors with recombinant IFN gamma (rIFN gamma) was found to increase only R-PBL cytotoxicity. These results suggest that the resistance of R-PBL to EBV immortalization could be associated with a lymphokine-mediated early cellular cytotoxic response of the NK/LAK (lymphokine-activated killer cell) type against EBV-infected cells.     

Changes in natural immunity during the course of HIV-1 infection.

The role of natural killer (NK) and lymphokine-activated killer (LAK) cell-mediated cytotoxicity in AIDS has yet to be established. The objective of this study was to determine inducible LAK cell responses at different stages of HIV-1 infection, and specifically to establish the participation of CD8 lymphocytes in these responses. Peripheral blood lymphocytes (PBL) were isolated from healthy seronegative (CDC-0) subjects and HIV-1+ individuals who were clinically asymptomatic (Centre for Disease Control group 2, CDC-2) or symptomatic (CDC-4) with regard to secondary opportunistic infection (OI). LAK cells were generated upon incubation of PBL with IL-2 and their cytolysis of K562 and U-937 targets was determined using chromium release assays. The role of CD8+ lymphocytes as progenitors and effectors of these LAK cell responses was determined by immunomagnetic depletion of CD8+ cells from precursor PBL and LAK cells, respectively. LAK cell-mediated cytotoxicities in HIV-1-infected individuals were reduced compared with seronegative controls without any corresponding changes in the relative proportions of CD56+ (NK) cells among groups. Depletions of CD8+ subsets from either PBL or LAK cells dramatically reduced total LAK cytotoxic responses and LAK activities per unit CD56+ cell in the OI-/CDC-2 seropositive population. No corresponding changes in LAK activities in seronegative control or HIV+/OI+/CDC-4 groups were observed. Levels of LAK activity against K562 targets in CDC-0/HIV- and CDC-4/HIV+ groups correlated with the percentage of CD56+ LAK cells; corresponding LAK activity in the CDC-2/HIV+ group correlated with the percentage of both CD56+ and CD8+ subsets. These findings suggest that adaptive changes in non-MHC restricted cytotoxic responses occur in HIV-1 individuals at early stages post-HIV infection, before the onset of opportunistic infection.     

丝裂霉素C对人胎脾LAK细胞活性的影响

本文对人胎脾LAK细胞活性及丝裂霉素C(MMC)对LAK活性的影响作了初步研究。结果显示,人胎脾淋巴细胞可诱导产生明显的LAK活性,对K562和Raji瘤细胞的杀伤活性分别为74.4％和69.4％(E:T=50:1)。诱生的LAK细胞经不同浓度MMC处理后,DNA合成受到显著抑制(p<0.O1),但仍可保持较高水平的LAK活性。提示细胞增殖对MMC的抑制作用更为敏感,LAK细胞在效应阶段并非绝对有赖于细胞增殖。     

Limiting dilution analysis (LDA) of cells responding to recombinant interleukin-2 without previous stimulation: evidence that all responding cells are lymphokine-activated potent effectors

The relationship between peripheral blood mononucleated cells spontaneously bearing the IL-2 receptor (IL-2R) and cell cytotoxicity for the natural killer (NK)-sensitive K562 target cell line was investigated. For this purpose, three types of experiments were performed. (i) Positive selection of cells spontaneously bearing the IL-2R was carried out by culturing peripheral blood lymphocytes (PBL) in the sole presence of recombinant IL-2 (rIL-2). Cytotoxicity was assessed at Day 6 of the culture in a 4 hr cytotoxic assay. (ii) Negative selection was performed by complement mediated lysis using the B1.49.9 monoclonal antibody which is specific for the IL-2R. (iii) Limiting dilution analysis of non-adherent PBL was carried out in the presence of rIL-2 alone. The colonies obtained were divided and daughter colonies assayed for anti-K562 cytotoxicity in a 6 hr cytotoxic assay and for proliferation. The results show that: (i) a 6-day culture of human non-adherent PBL in the presence of rIL-2 alone leads to a sharp increase in anti-K562 cytotoxicity; (ii) depletion of B1.49.9 positive PBL strongly decreases cytotoxicity against K562 targets; (iii) limiting dilution analysis indicates that all colonies grown without activation in the presence of autologous serum and rIL-2 can mediate cytotoxicity against K562 targets, which is not the case when the starting population is activated. Thus, our data taken together strongly suggest that lymphocytes spontaneously bearing the IL-2R are directly involved in K562 lysis by fresh PBL (classical NK activity). Moreover, we demonstrate that all colonies able to proliferate without any activation, in the sole presence of rIL-2, are potent K562 killers (in this case, these cells correspond to the so-called lymphokine activated killers, LAK).     

Human recombinant IL-4 suppresses the induction of human IL-2 induced lymphokine activated killer (LAK) activity.

The effect of recombinant human interleukin 4 (rhIL-4) on the induction in vitro of human lymphokine activated killer cell (LAK) activity was investigated. Peripheral blood mononuclear cells (PBMC) from normal healthy donors were incubated for 4 days with or without recombinant human interleukin-2 (rhIL-2) in the presence or absence of rhIL-4. LAK activity was measured against the NK-resistant colon adenocarcinoma cell line SW742, and NK mediated cytotoxicity was determined using NK sensitive K562 cells. Unlike previous reports using mouse effector cells, rhIL-4 neither induced LAK activity nor augmented the cytotoxic response induced by rhIL-2. In four out of six experiments there was a significant reduction of rhIL-2 induced LAK in the presence of rhIL-4, accompanied by a reduction of Tac antigen expression by rhIL-2 activated cells. Recombinant hIL-4 failed to influence the effector phase of the activated PBMC against SW742 or K562 targets.     

Adhesion molecule-mediated signals regulate major histocompatibility complex-unrestricted and CD3/T cell receptor-triggered cytotoxicity.

Appropriate experimental conditions were devised to demonstrate that CD58 (LFA-3), CD54 (ICAM-1) and CD11a/CD18 (LFA-1) adhesion molecules are the source of signals that regulate nonspecific major histocompatibility complex-unrestricted and CD3/T cell receptor (TcR)-triggered cytotoxicity. Using anti-LFA-3 monoclonal antibody (mAb)-treated, interleukin-2 (IL-2)-cultured peripheral blood lymphocytes (PBL) or cloned CD3+/CD8+ cells as lymphocyte-activated killer (LAK) effectors, and ligand (CD2)-negative tumor cell lines as targets, a down-regulation of CD3- and CD3+ cell-mediated LAK activity was consistently observed. Anti-LFA-3 mAb also down-regulated tumor cell lysis when T cell clones were triggered to kill P815 cells through stimulation of the CD3/TcR complex by an anti-CD3 mAb. The inhibitory effect of anti-LFA-3 mAb was not prevented by stimulatory anti-CD2 mAb. Anti-ICAM-1 mAb treatment of IL-2-cultured PBL consistently up-regulated LAK cytotoxicity against tumor target cells. However, this effect was only exerted on CD3- LAK effectors. Anti-LFA-1 mAb blocked conjugate formation between effector cells and tumor target cells, thus rendering this model unsuitable to evaluate the regulatory role of LFA-1. Therefore, a cytotoxicity model system was applied in which a hybrid anti-CD3/anti-human red blood cell (HuRBC) mAb triggers cytolytic T cells to lyse HuRBC. In these experiments, anti-LFA-1 mAb markedly up-regulated the lytic ability of IL-2-cultured PBL. We conclude that mAb against LFA-3, ICAM-1 and LFA-1 molecules deliver regulatory signals for LAK cells and cytotoxic T lymphocytes. As these stimuli may be delivered by ligands expressed on tumor targets as well as on other immune competent and inflammatory cells, the present observations are relevant in the context of both the host's immune response against tumors and the general functioning of the immune system.