丝裂霉素或顺铂对离体淋巴因子激活的杀伤细胞增殖和抗膀胱癌细 …

AIM: To study the effect of mitomycin (Mit) or cisplatin (Cis) on the proliferation of lymphokine-activated killer (LAK) cells in patients with transitional cell cancer of bladder and their cytolysis to bladder tumor cells. METHODS: LAK cell proliferation was assayed in the presence of Mit or Cis by cell counting. Bladder cancer cell lines BIU-87 and EJ were cultured as target cells and cytotoxicity of LAK cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: The proliferation of LAK cells induced by recombinant interleukin-2 (IL-2) was inhibited by Cis in a concentration-dependent manner and was decreased to 55.3% at 100 mg.L-1 compared with control at 96 h. The enhanced growth of the LAK cells was observed with Mit 5-10 mg.L-1 from 48 to 96 h. Cis 10 mg.L-1 increased the cytotoxicity against BIU-87 and EJ cells. CONCLUSION: Immunomodulatory effect of chemotherapeutic agents on LAK cell proliferation induced by IL-2 in patients with bladder cancer mainly depends on the drug itself.     

Tretinoin or retinol enhancement of lymphokine-activated killer cell proliferation and cytotoxicity against human bladder cancer cells in vitro.

AIM: To study the effect of tretinoin (Tre) or retinol (Ret) on the proliferation of lymphokine-activated killer (LAK) cells in patients with transitional cell cancer of bladder and their cytolysis to bladder tumor cells. METHODS: LAK cell proliferation was assayed in the presence of either Tre or Ret by cell counting. Human transitional bladder cancer cell lines BIU-87, EJ, or bladder tumor cells (BTC) from patients with bladder cancer were used as target cells and cytotoxicity of LAK cells was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: The proliferation of LAK cells induced by interleukin-2 (IL-2) was stimulated by Tre or Ret (10-100 nmol.L-1). The cytotoxicity of LAK cells against BIU-87, EJ cells, or BTC was enhanced by pretreatment of LAK cells with Tre or Ret 10-100 nmol.L-1. CONCLUSION: Tre or Ret enhances the proliferation and cytotoxicity of LAK cells from patients with bladder cancer. Retinoids are potential in adoptive immunotherapy of bladder cancer.     

The immunostimulating activities of anti-tumor polysaccharide from K1 capsular (polysaccharide) antigen isolated from Klebsiella pneumoniae

We have previously shown that K1 capsular polysaccharide antigen (K1CPS) of Klebsiella exhibits anti-tumor activities. In the present study, we examined the effect of K1CPS on cytotoxic effector cells. We found that K1CPS could activate many cytotoxic effector cells including alloreactive cytotoxic T cells and tumor-infiltrating lymphocytes (TILs). Moreover, K1CPS could increase the anti-tumor activity of lymphokine-activated killer (LAK) cells, both in vitro and in vivo. The i.p. injection of K1CPS in low dose could enhance the LAK cytotoxicity and the effect was further potentiated by coculture of LAK cells with K1CPS and low concentration of murine rIL-2 in vitro. The phenotypic characterization revealed that K1CPS might contribute to the increase in CD3+ LAK cell subpopulation by its in vivo priming effect. In addition, the K1CPS-treated LAK cells were able to inhibit the growth of WEHI-164 tumor cells in vivo in Winn-type inhibition assay. Subcutaneous (s.c.) and intraperitoneal (i.p.) adoptive infusion of LAK cells (splenocytes from K1CPS-treated WEHI-164-bearing mice cultured with K1CPS-plus-rIL-2) into WEHI-164 sarcoma-bearing mice could slightly cause regression in terms of tumor diameter, and more significantly in sarcoma weight.     

Tumor blood supply and tumor localization by adoptively transferred IL-2 activated natural killer cells

The circulatory pattern of IL-2 activated natural killer (A-NK) cells was studied in C57BL/6 mice bearing 10 day-old pulmonary and subcutaneous (s.c.) metastases of the B16 melanoma in order to evaluate the roles of the concentration of A-NK cells in the blood and of tumor blood flow on accumulation of A-NK cells in tumors. Kinetic studies of the presence of A-NK cells in peripheral blood after adoptive transfer revealed that these cells rapidly disappear from the blood. Via intravital microscopy of animals with exposed lung tissue, we have shown that the vast majority of transferred A-NK cells become efficiently arrested within the lung microcirculation at their first encounter with this organ, thereby explaining the fast disappearance of the cells from the bloodstream. Despite the low number of A-NK cells circulating in the blood, systemically injected A-NK cells (20 million per mouse) localized significantly (70-80 million cells/g) into most pulmonary metastases within 8-16 hours. In contrast, very few A-NK cells (< 0.2 million cells/g) were found in the s.c. metastases. Based on measurements of tumor blood flow (showing a classic inverse relationship between tumor size and tumor blood flow) and the blood concentration of A-NK cells, we estimated the highest intratumoral density of A-NK cells that theoretically can be generated by A-NK cells transported to the tumor by way of the blood. In s.c. tumors, the observed density of A-NK cells was at all times lower (10-50 fold) than the estimated density, indicating that only a few percent of the A-NK cells arriving at these tumors become retained in them. In contrast, the observed density of A-NK cells in pulmonary metastases was at all times higher (2-3 fold) than the estimated density. This finding indicates that A-NK cells might not reach the pulmonary metastases solely by way of the blood stream. In conclusion, i.v. injected A-NK cells become immediately entrapped in the lungs and, consequently, circulate poorly. While lung metastases become significantly infiltrated by i.v. injected A-NK cells, metastases in organs down-stream from the lungs become poorly infiltrated. We hypothesize that only a part of the A-NK cells found in lung metastases 8-16 hours following injection reach these metastases by way of the blood-vascular system. They might also migrate into the metastases from the surrounding normal lung tissue.     

碱性成纤维细胞生长因子增强淋巴因子激活的杀伤细胞对人膀胱 …

目的 探讨白细胞介素Ⅱ（ＩＬ－２）和碱性成纤维细胞生长因子（ｂＦＧＦ）对膀胱癌患者淋巴因子激活的杀伤细胞（ＬＡＫ细胞）的作用。方法 用细胞计数观察不同浓度ｂＦＧＦ对ＬＡＫ细胞增殖的影响,以膀胱癌细胞系ＥＪ及新鲜分离患者自体肿瘤细胞（ＢＴＣ）为靶细胞,用ＭＴＴ法测定ＬＡＫ细胞对膀胱癌细胞的细胞毒作用,结果 虽然外周血单核细胞（ＰＢＭＣ）的增殖可被ｂＦＧＦ５μｇ．Ｌ＾－１所抑制,ＩＬ－２所诱导的ＬＡＫ     

PTD-mediated Loading of Tumor-Seeking Lymphocytes with Prodrug-Activating Enzymes

Using the approach of peptide transduction domain (PTD)-mediated loading of interleukin-2(IL-2)-activated natural killer (A-NK) cells, tumor-seeking lymphocytes, with prodrug-activating enzymes, we primarily aim to generate a cytotoxic drug selectively within tumors and minimize damage to normal tissues. A-NK cells are able to accumulate selectively at tumor sites. While these cells by themselves possess significant antitumor effect in vivo, we suggest that they can also serve as Trojan horses, by bringing anticancer agents, such as prodrug-activating enzymes, selectively to tumors. We have successfully demonstrated in a mouse model that A-NK cells can be rapidly loaded with prodrug-activating enzymes, such as alkaline phosphatase (AP) and beta-galactosidase (beta-gal), in vitro using enzyme-conjugated peptide PTD5. Upon adoptive transfer into lung-tumor-bearing animals, the loaded A-NK cells are able to bring their cargo of the prodrug-activating enzymes selectively to pulmonary metastases. The targeting of the AP to the tumor tissues is highly specific, since more than a fivefold higher concentration of AP was found in the tumor tissues compared to the surrounding normal lung tissue at 24 h after injection. The approach of transporting prodrug-activating enzymes selectively into tumors clearly shows potential for future targeted chemotherapy. Ongoing studies in our laboratory are evaluating the antitumor efficacy of cellular-dependent enzyme prodrug therapy.     

活性氧自由基对膀胱癌患者淋巴因子激活的杀伤细胞的作用

目的:探讨活性氧自由基对膀胱癌患者淋巴因子激活的杀伤(LAK)细胞的增殖和抗膀胱癌细胞系活性的作用.方法:分别用细胞计数和MTT法测定LAK细胞的增殖和细胞毒作用.结果:羟自由基浓度依赖性地抑制IL-2所诱导的LAK细胞增殖.用抗坏血酸400μmol·L~(-1)和硫酸亚铁40μmol·L~(-1)培养细胞96h,细胞增殖被抑制34.5％.这种抑制可被一定浓度的甘露醇和依地酸(edetic acid)扭转.一定浓度的超氧阴离子或一氧化氮释放剂硝普钠可刺激由IL-2所诱导的LAK细胞的增殖.超氧化物歧化酶(SOD)可抵消超氧阴离子的刺激作用.外源性超氧阴离子可加强LAK细胞对BIU-87和EJ细胞的杀伤.羟自由基和SOD对LAK细胞杀伤作用则影响不明显.结论:超氧阴离子和一氧化氮可增强膀胱癌患者LAK细胞的增殖、激活和抗肿瘤的细胞毒,而羟自由基对此起抑制作用.这两种活性氧自由基对IL-2诱导的LAK细胞增殖的作用是不同的.     

Inhibition of human LAK-cell activity by the anti-depressant trifluoperazine

The anti-depressive drug trifluoperazine (TFP) was studied on in vitro immune responses. TFP proved to be an inhibitor of lymphokine-activated killer (LAK) cells in its generative step, as well as in its effector phase. Natural killer (NK) activity and interleukin-2 (IL-2) or mitogen-induced lymphocyte proliferation were just as sensitive to the drug effects, whereas the division of tumor cells was more resistant. The mechanism through which TFP suppresses these lymphocytic systems remains unclear. It does not, however, affect an early stage of cellular activation as the addition of the drug as late as 24 h after the start of the culture was still inhibitory for lymphocyte mitogenesis. Neither the expression of CD25, nor that of CD56 was affected by TFP, and exogenous IL-2 was unable to overcome the suppression of proliferation. In relation to cell-mediated cytotoxicity, TFP partially interfered with the effector/target binding. However, addition of lectin to the assay did not overcome the inhibition of lysis produced by the drug. Although further work remains to be done, the effect of TFP on immune responses must be taken into consideration when treating immunonosuppressed patients.     

Matrix metalloproteinases of human NK cells

We have previously reported that MMP-2 and MMP-9 are present in rat A-NK cells, and have recently documented that additional MMPs are present in rodent A-NK cells. To our knowledge only proMMP-9 has previously been reported for human NK and A-NK cells. Herein, we report for the first time the presence of MMP-2 and MT1-MMP in human NK cells. The importance of these enzymes for the migration of A-NK cells into tumor metastases is of great potential relevance. MMPs may be rate limiting in A-NK cells, following their adoptive transfer, to traverse basement membrane and accumulate within established cancer metastases, a likely pre-requisite to their cytolytic function. Human NK cells express and produce MMP-2, MMP-9, MT1-MMP and the inhibitor TIMP-1. Moreover, human A-NK cells degrade the extracellular matrix equivalent (Matrigel) in a seemingly IL-2 dependent manner. It is therefore likely that A-NK cell MMPs play crucial roles in contributing to A-NK cell localisation and positioning the cells in vivo to allow for triggering their cytolytic potential.     

阿霉素对人胚胸腺细胞IL-2产生及LAK活性的影响

某些抗癌药物的免疫调节效应在其抗肿瘤机制中可能起着重要作用.本研究表明,低浓度(0.04mg·L~-1)阿霉素对人胚胸腺细胞IL-2产生和LAK活性的诱导具有促进作用;高浓度(4mg·L~-1)阿霉素虽对LAK活性没有影响,但却明显抑制IL-2的产生.提示低浓度阿霉素对人胚胸腺细胞具有一定的免疫增强效应.     

A novel drug delivery system using IL-2 activated NK cells and Zyn-linked doxorubicin

Adoptively transferred IL-2 activated NK (A-NK) cells selectively accumulate within tumor metastases which recommends them as vehicles for locoregional drug delivery. Zyn-Linkers are membrane-binding lipophilic dyes which can be coupled by a variety of conjugation chemistries to therapeutic agents. We have previously demonstrated that A-NK cells labeled with PKH26 are able to accumulate within established B16 melanoma pulmonary metastases by 16 h at a concentration of over 600 cells/mm2 of tumor tissue (Basse et al. J. Exp. Med. 174: 479 1991). Zyn-205 is a prodrug in which doxorubicin is attached to a similar Zyn-Linker through an acid-sensitive bond. We have optimized the ex vivo labeling conditions and found that a 10 min incubation with 25 microM Zyn-205 results in the uptake of over 10(8) drug molecules per cell with no effect on either cell viability or cytolytic activity up to 24 h after labeling. Given these parameters, the amount of drug which may be carried to and concentrated in metastatic lesions represents a local concentration of approximately 15 microM. In addition, A-NK cells carrying Zyn-Linked doxorubicin at an equivalent dose of 25 micrograms/kg was therapeutically comparable to a systemic dose of 8 mg/kg (320x more) in the 3LL model of experimental metastasis. These data indicate that A-NK cells bearing Zyn-Linked chemotherapeutic agents represent a unique and feasible method to target chemotherapeutic agents to cancer metastases and that therapeutic doses can be attained without unwanted systemic exposure.     

Anti-tumor activity of ex vivo expanded cytokine-induced killer cells against human hepatocellular carcinoma

Cytokine-induced killer (CIK) cells are ex vivo expanded T cells with natural killer cell phenotypes and functions. In this study, the anti-tumor activity of CIK cells against hepatocellular carcinoma was evaluated in vitro and in vivo. In the presence of anti-CD3 antibody and IL-2 for 14 days, human peripheral blood mononuclear cell population changed to heterogeneous CIK cell population, which comprised 96% CD3(+), 3% CD3( inverted exclamation mark(c))CD56(+), 32% CD3(+)CD56(+), 11% CD4(+), 75% CD8(+), and 30% CD8(+)CD56(+). CIK cells produced significant amounts of IFN-gamma and TNF-alpha; however, produced only slight amounts of IL-2, IL-4, and IL-5. At an effector-target cell ratio of 30:1, CIK cells destroyed 33% of SNU-354 human hepatocellular carcinoma cells, which was determined by the (51)Cr-release assay. In addition, a dose of 1x10(6) CIK cells per mouse inhibited 60% of SNU-354 tumor growth in irradiated nude mice. This study suggests that CIK cells may be used as an adoptive immunotherapy for patients with hepatocellular carcinoma.     

Interaction between lymphokine activated killer cells generated in the presence of cisplatin/FK-565 and tumor cells: transmission electron microscopy analysis.

Lymphokine activated killer (LAK) cells generated in the presence of cisplatin/FK-565 along with interleukin-2 (IL-2) showed enhanced binding to tumor cells as compared with LAK cells generated in the presence of IL-2 alone. The LAK cells showed a reorientation of cellular organelles in the presence of tumor cells. LAK-tumor conjugate pairs showed vesicle-like structures in the extracellular pockets. The number of vesicles in the extracellular pockets was found to be increased in the case of LAK cells generated in the presence of cisplatin/FK-565 along with IL-2. The closer interaction between the LAK cell-target cell and the large number of vesicles in the extracellular space of conjugate pairs may explain the increased lysis of target cells by LAK cells generated in the presence of cisplatin/FK-565 along with IL-2 as compared with IL-2 alone.     

CD_3单克隆抗体加白细胞介素2活化的杀伤T细胞系治肿瘤的安全性

目的 :探讨CD3单克隆抗体 (CD3McAb)与小剂量白细胞介素 2 (IL 2 )活化的杀伤T细胞系治疗肿瘤病人的安全性。方法 :从健康人外周血、脐血、人胎胸腺或病人自体血分离淋巴细胞 ,经诱导、激活传代后获淋巴因子活化的杀伤细胞 (LAK)和CD3AK细胞系 ,先分别试用 10例病人 ,(1～ 5 )×10 8,iv ,每个疗程 5次 ,显示安全有效后增加病例 ,LAK细胞组 5 0例 ,CD3AK细胞组 4 0例。结果 :效应细胞生长形态良好 ,多数能长出细胞集落。生长曲线表明CD3AK细胞比LAK细胞增殖快 ,生长时间长 ;CB CD3AK和PBL CD3AK细胞对数生长期为d 9～ 18,能从 10 6 扩增到 10 8,而其他细胞对数生长期为d 6～ 15 ,扩增到 10 7,110例病人经iv 5 48次治疗 ,不良反应主要为发热和寒颤 ,偶有恶心、呕吐 ,眩晕 ,皮疹 ;HFT LAK发生率为 11% ,其他约 5 % ,CD3AK平均不良反应发生率为 3.6 %。结论 :CD3McAb与小剂量IL 2活化的杀伤T细胞系可以安全地用于治疗肿瘤。     

地佐辛静脉自控镇痛对肺叶切除术后免疫功能的影响

目的:观察地佐辛静脉自控镇痛对肺叶切除术后免疫功能的影响.方法:选取60例拟行开胸肺叶切除术的患者随机分为对照组和DEZ组,每组30例.DEZ组术后PCA泵注0.5 mg·ml^-1地佐辛,对照组术后PCA泵注0.9%氯化钠.采集患者麻醉诱导前(T0)、术后4 h(T1)、术后24 h(T2)和术后48 h(T3)血标本,测定血清CRP、TNF-α、IL-2、IL-6和IL-10水平,以及外周血淋巴细胞(L)计数及淋巴细胞中CD4⁺T淋巴细胞、CD8⁺T淋巴细胞、自然杀伤细胞(NK)比例的变化.于T1、T2和T3时点随访患者,记录患者视觉模拟评分(VAS)和数字镇静评分(NSS).结果:肺叶切除术后患者血清CRP、TNF-α、IL-6和IL-10的水平较术前明显升高(P <0.05),IL-2较术前降低(P <0.05);术后外周血淋巴细胞计数及CD4⁺T淋巴细胞比例和NK细胞比例较术前降低(P <0.05);地佐辛静脉自控镇痛能明显降低术后血清CRP、IL-6,升高IL-2及CD4⁺T淋巴细胞比例(P <0.05);地佐辛组患者术后24 h的VAS评分低于对照组(P <0.05).结论:地佐辛静脉镇痛具有免疫调节作用,可改善肺叶切除术后的免疫抑制,并减轻炎症反应.     

Suppressor function of lymphocytes activated in in vitro co-culture against autologous tumor cells and expanded in interleukin-2.

The phenotypic and functional nature of the lymphocytes that have been activated against autologous tumor cells and expanded in Interleukin-2 (IL-2) was studied in the context of their suitability for use in adoptive immunotherapy for cancer. While long-term co-cultures between autologous lymphocytes and tumor cells in the presence of exogenous IL-2 occasionally induced CD8+ cytolytic T cells, a substantial majority of such co-cultures generated predominantly CD4+ non-cytolytic or poorly cytolytic effector cells. In addition, these CD4+ non-cytolytic effector populations, and a number of CD4+ T cell clones derived from them, behaved like functional T suppressor (Ts) cells by elaborating a factor(s) that had profound negative effect(s) on activation of fresh T cells (inhibition of IL-2 synthesis, inhibition of IL-2 receptor [IL-2]-alpha expression, and inhibition of proliferation). Accordingly, infusion of these non-cytolytic populations capable of exhibiting such regulatory properties may have a substantial negative effect on host response toward cancer.     

Effect of Salmonella-infected human monocytes on natural killer cell cytotoxicity. In vitro studies.

Various chemicals, including some bacteria-derived components, modulate natural killer cell (NKC) activity. We have analyzed the effect of wild-type Ty2 and of mutant strain TYT1231 Salmonella typhi-infected monocytes (U937 cells and human autologous monocytes) on NKC cytotoxicity of peripheral blood mononuclear cell (PBMC) and highly purified NKC (HPNKC; CD16+/56+ > 95%; the rest corresponding to CD3+ T-cells). PBMC's co-culture with either S. typhi strain infected U937 cells (medium or non-infected U937 cells as controls) resulted in the induction of lymphocyte activated killer (LAK) cell activity showing cytotoxicity against target human NKC-resistant lymphoblastoid Daudi cell line. Comparable experiments using autologous monocytes gave similar results. Co-culture of HPNKC preparations with either S. typhi strain infected U937 cells resulted in increased LAK cell activity against target Daudi cells in each and everyone of the five samples tested; paired Student's t-test p < 0.01 for both times (20 and 40 h) tested. Similar to the results observed in the experiments using PBMC, we did not find significant differences in the ability between medium and non-infected cells, or between wild-type S. typhi Ty2 and mutant strain TYT1231 infected U937 cells, to induce LAK activity in HPNKC preparations. PBMC co-incubation with either S. typhi strain infected U937 cells or autologous monocytes resulted in significant increases in IL-12, TNF-alpha, and IFN-gamma secretion. In similar experiments using HPNKC samples instead, infected U937 cells significantly increased IL-12 and IFN-gamma, but not TNF-alpha secretion. PBMC co-incubation with non-infected U937 cells, but not with non-infected monocytes, significantly increased supernatant IL-12 and TNF-alpha levels (no significant changes in IFN-gamma were recorded). Secreted cytokines remained essentially unchanged after co-incubating HPNKC preparation with non-infected U-937 cells. Incubation of PBMC or HPNKC preparations with either S. typhi strain infected U937 cells failed to produce significant changes in the expression of NKC lineage (CD16+/56+) or activation (CD28+, CD69+ and CD95+) markers. The ability of infected monocytes to induce LAK activity, release NKC cytokines and upmodulate NKC's CD95+ marker expression was essentially the same for both infecting Salmonella strains used. These results suggest a role for NKC in the physiological defensive response against intracellularly infected monocytes representing, perhaps one of the earliest antimicrobial mechanisms of the innate immune system.     

Swainsonine,a glycosylation inhibitor,enhances both lymphocyte efficacy and tumour susceptibility in LAK and NK cytotoxicity

Swainsonine (SW) inhibits the formation of N-linked complex oligosaccharides and has previously been shown to inhibit experimental metastasis in nude mice models. The present studies with human effector cells have shown that SW enhanced both lymphokine activated killer cell (LAK) and natural killer (NK) cytotoxicity in standard ⁵¹Cr-release assays. SW also increased the susceptibility of human K562 and Colo 320 target cells to NK and LAK cytotoxicity. The peak response of both LAK effectors and targets to SW occurred at 1–2 μg/ml SW. A novel finding was that SW enhanced the interleukin 2 (IL-2) β chain receptor subunit expression on both LAK and NK cells to a greater extent than its enhancement of the IL-2Rα (CD25 or TAC) receptor expression on LAK effectors. In addition, increases in both these receptors occurred at the doses of SW which augmented LAK cytotoxicity. We conclude that the anti-metastatic effects of SW have an immunological component which is maximal at 1–2 μg/ml SW. This suggests that dosage may be an important consideration to obtain optimal potential of SW in any future human cancer therapy.