Identification of a novel CD56- lymphokine-activated killer cell precursor in cancer patients receiving recombinant interleukin 2.

Circulating lymphokine-activated killer (LAK) cell activity in cancer patients receiving recombinant interleukin 2 (rIL-2) therapy is confined to cells expressing the CD56- surface marker. However, CD56- cells from these patients but not normal individuals have been reported to exhibit LAK cytotoxicity only following in vitro activation with rIL-2. Studies were performed to document the existence of CD56- LAK precursor cells and to phenotypically characterize this population in patients receiving rIL-2 therapy using fluorescence-activated cell sorter-purified CD56- cell subsets. Initial studies confirmed that CD56- cells exhibit NK activity [20 +/- 7 (SE) LU/10(6) cells] but not LAK activity (0 +/- 0 LU/10(6) cells) when evaluated directly from peripheral blood of patients receiving rIL-2. CD56- cells from patients but not normal individuals developed significant LAK cytolytic activity against NK-resistant COLO 205 targets (16 +/- 3 LU/10(6) cells) when cultured for 3 days with 1500 units/ml rIL-2. The CD56- LAK precursor activity was confined to cells expressing a CD56-CD16+ phenotype and a large granular lymphocyte morphology; little or no NK or LAK precursor activity was detectable in CD56-CD5+ T-cells from patients. Phenotypic characterization of CD16+CD56- cells revealed that this population is uniformly CD11a+,CD18+, and CD38+ and is heterogeneous in its expression of CD11b, CD11c, and CD16/Leu 11c. These results indicate that rIL-2 administration induces enhanced LAK precursor activity in a novel population of CD5-CD16+CD56- cells.     

Effects of concurrent cisplatinum administration during radiotherapy vs. radiotherapy alone on the immune function of patients with cancer of the uterine cervix

PURPOSE: To compare the effects of concurrent administration of cisplatinum (40 mg/m(2)/weekly) with radiation therapy (C-RT) to those induced by radiation therapy alone (RT) on the immune function of patients with locally advanced cervical cancer. METHODS AND MATERIALS: In 8 prospectively randomized patients (i.e., 4 receiving RT vs. 4 receiving C-RT), lymphocyte populations including CD3+, CD4+ and CD8+ T-cell subsets, B cells (CD19+) and natural killer cells (CD56+, CD16+, CD3-) were studied before, during, and after therapy. Expression of the activation marker CD25 on CD3+ T cells, intracellular levels of perforin in CD8+ and CD56+ cells, and interferon-gamma (IFN-gamma) and IL-2 in CD4+ and CD8+ T cells was also measured. Finally, lymphoblast transformation and natural killer (NK) cytotoxic activity were assessed. RESULTS: Both RT and C-RT significantly decreased the mean absolute number of all lymphocyte subsets compared to pretreatment levels (p > 0.001). However, no differences were detected in the characteristics or the magnitude of the lymphopenia induced by the two treatments. Both RT and C-RT increased similarly the percentages of CD25-positive lymphocytes (p > 0.001), and significantly decreased PHA-induced T-cell lymphoblast transformation (p > 0.001) and NK cytotoxic activity against K562 cells (p > 0.001). The percentage of perforin-positive and CD8+ T cells was not altered during either treatment, whereas the percentage of perforin-positive and CD56+ cells was significantly reduced during both treatments, and correlated with reduced cytotoxicity against K562 cells. The percentages of CD8+ IFN-gamma+ and CD4+ IFN-gamma+ T cells as well as that of CD8+ IL-2+ and CD4+ IL2+ T cells were not significantly altered by C-RT compared to RT alone. Finally, with both regimens, NK cells and B-cell numbers showed a more rapid recovery than T-cell numbers. CONCLUSION: Administration of concurrent cisplatinum to radiation may synergistically increase cytotoxic effects of radiation on tumor cells but does not alter the magnitude and the characteristics of radiation-induced immunosuppression.     

Functional and phenotypic characteristics of recombinant interleukin-2 or T-cell growth factor-activated splenic lymphoid cells from patients with gastric or hepatocellular carcinoma

Fourteen days' culture of human spleen cells with recombinant interleukin-2 (rIL-2) or T-cell growth factor (TCGF) results in the generation of lymphokine-activated killer (LAK) effector cells that have the unique property of lysing natural killer (NK)-resistant human tumor cells, Daudi, and NK-sensitive K562 cells. LAK cells were generated from patients with advanced cancer or liver cirrhosis. The splenic LAK-effector cell types were analyzed by two-color flow cytometry. The rIL-2-induced LAK cells showed an increased proportion of CD8+CD11- and CD57+CD16- and a decreased proportion of CD4+Leu-8- cells. In contrast, TCGF-induced LAK cells revealed a significantly increased proportion of CD8+CD11- and CD4+Leu-8- cells and a decreased proportion of CD57+CD16- cells. Thus, splenic LAK cells with different surface phenotypes were induced by the cultivation with rIL-2 or TCGF. Furthermore, TCGF-induced LAK cell activities in patients with cancer were found to be lower than the rIL-2-induced LAK cell activities. It was noted that the TCGF-activated splenic lymphoid cells did not inhibit the effector process of tumor cell lysis by LAK cells that had been activated by rIL-2. Other mechanisms of lower LAK cell activities of TCGF-activated splenic lymphoid cells from patients with cancer were discussed. The findings suggest that spleens of examined patients with gastric or hepatocellular carcinoma do not seem to be responsible for suppression of cell-mediated antitumor immunity.     

Effect of blood transfusion during radiotherapy on the immune function of patients with cancer of the uterine cervix: role of interleukin-10

: To analyze prospectively the effects of blood transfusion administered during radiotherapy (RT) on the immune function of patients with locally advanced cervical cancer.

: In a total of 15 patients, 7 transfused and 8 untransfused, lymphocyte populations, including CD3+, CD4+, and CD8+ T-cell subsets, B cells (CD19+), and natural killer (NK) cells (CD56+, CD16+, CD3−) were studied before (i.e., time 0), during (i.e., times 1 and 2), and after (i.e., time 3) therapy. Expression of the early (CD25) and late (HLA-DR) activation markers on CD3+ T cells, the intracellular levels of perforin in CD8+ and CD56+ cells, and interferon (IFN)-γ, interleukin (IL)-2, and IL-4 in CD4+ and CD8+ T cells were also measured. NK cell cytotoxicity against the NK-sensitive target K-562 cells and CD8+ T-cell-directed cytotoxicity against OKT3 hybridoma cells were also assessed. Finally, the plasma levels of the immunoregulatory cytokine IL-10 were analyzed by enzyme-linked immunosorbent assay.

: The mean absolute number of all lymphocyte subsets compared with pretreatment levels decreased significantly during RT of both transfused and untransfused patients (p >0.001), with no detectable differences between the two groups in terms of total lymphocytes or relative numbers of CD3+ and CD4+ T cells, CD56+ NK cells, or CD19+ B cells. In contrast, concomitant with an inversion of the CD4/CD8 ratio, a significant increase in the number of CD8+ T cells at time 2 and CD3+ T cells, CD8+ T cells, and NK cells at time 3 was found in the transfused patients compared with the untransfused group. The percentages of CD25+/CD3+ T cells and HLA-DR+/CD3+ T cells increased during RT of the untransfused patients, but CD3+ T cells showed decreased CD25 expression and increased HLA-DR expression in the transfused group. An increase of CD8+ IFN-γ+ T cells with a concomitant decrease in CD8+ IL-2+ T cells was found in the transfused vs. untransfused group, and no differences were noted in the percentage of CD4+ IFN-γ+ T cells and CD4+ IL-2+ T cells. The proportion of perforin-positive CD8+ and CD56+ cells was higher in the transfused group than in the untransfused group. However, CD56+ cells and CD8+ T cells from the transfused patients showed markedly diminished cytotoxic function. Finally, IL-10 was detected only in the plasma of the transfused patients.

: Blood transfusion during primary RT for cervical cancer profoundly alters the magnitude and characteristics of radiation-induced immunosuppression. Elevated serum IL-10 in transfused patients may play a role in the disregulation of lymphocyte function, in particular, the depression of NK- and T-cell cytotoxicity. Investigation of alternatives to blood transfusion during RT that do not diminish host immunity is warranted.     

高强度聚焦超声对子宫肌瘤患者免疫功能的影响

目的 比较高强度聚焦超声(HIFU)和子宫肌瘤切除术(Myomectomy)对子宫肌瘤患者的免疫功能的影响.方法 将子宫肌瘤患者80例,随机分为HIFU组或Myo组.于入院时和术后24 h采集静脉血样,流式细胞术检测患者CD4+、CD8+、CD4+/CD8+、NK细胞水平,酶联免疫吸附法(ELISA)检测患者血清IL-2,IL-6和IL-10水平.结果HIFU组患者恢复快,术后并发症较少,住院时间较短(P<0.05).行HIFU术后患者CD4+T细胞、CD8+T细胞、CD4+/CD8+、NK细胞水平无显著变化(P>0.05);与之相反,手术切除后24 h,患者CD4+T细胞、CD8+T细胞、NK细胞水平均显著下降(P<0.05),CD4+/CD8+比例也显著下降(P<0.05).治疗后,两组患者血清IL-6和IL-10水平均显著升高,IL-2水平显著降低;HIFU组患者IL-6和IL-10水平显著低于Myo组,Myo组患者IL-2水平明显高于HIFU组.结论HIFU对患者免疫功能影响较小,更好的免疫功能可能与组织创伤应激减少有关,并有助于减少术后并发症的发生.     

Effect of Cytotoxic Cells on Minimal Residual Leukemia

The presence of minimal residual disease is indicated by the high frequency of relapses after twin bone marrow transplants and after allogeneic bone marrow transplants without graft versus host disease (up to 75% and 45% of cases, respectively). The graft versus leukemia effect may be mediated by IL-2 activation of natural killer cells (CD16 +, CD56 +, CD3-, CD8+/-) or cytotoxic T cells (CD3 +, CD56+/-). These activated killer cells can bind to targets and cause their lysis, and then recirculate to kill other targets. Killing can be blocked by anti-perforin antibodies and enhanced by protein kinase C-activation of effectors

There are several studies indicating that a high percentage of leukemic cells can be killed by LAK-cells. However even in the most sensitive cases, lysis of all the cells cannot be achieved. The finding that leukemic clonogenic cells are generally sensitive to both NK and LAK cytotoxicity provides a more hopeful possibility

The lack of tumor specific antigents, leukemic cell-immunoheterogeneity and maturation asynchrony explains why antigen dependent T-cell mediated cytotoxicity is only partly effective in eradicating residual leukemic cells. Future work should therefore include more studies of the mechanism of resistance to LAK cells, possibilities of further enchancing cytotoxicity and the mechanism of graft-versus-leukemia effect     

Down-regulation of IL-18 receptor in cancer patients: its clinical significance

Interleukin-18 (IL-18) is a powerful inducer of interferon-gamma (IFN-gamma), a key immunoregulatory cytokine. Cellular immune responsiveness, as measured by IL-18-induced IFN-gamma production from peripheral blood mononuclear cells (PBMCs) in ELISA assay, was evaluated in 10 patients with advanced cancer and in 10 normal controls. Supernatant levels of IFN-gamma were detected at 2 hours after PBMCs culture and markedly increased thereafter in healthy volunteers. In contrast, IFN-gamma production in cancer patients was not detected during the culture period (0-72 hours). We also measured IL-18-stimulated IL-12 production in healthy volunteers and null response was observed in cancer-bearing patients. Next, we studied mRNA expressions of IL-18 receptor (IL-18R) and IFN-gamma in PBMCs in cancer patients and healthy volunteers by RT-PCR assay. Both mRNA levels of IL-18R and IFN-gamma were significantly decreased in cancer-bearing patients compared with normal controls. These results suggested that IL-18 responsiveness for IFN-gamma production in cancer-bearing patients was impaired. Using flow cytometric analysis, we studied T-cell subsets, CD3- CD56+ (NK cell), CD3+ CD45RO+ (memory T-cell), CD3+ CD95+ (Fas+ T-cell), CD3+ CD4+ (helper T-cell), CD3+ CD8+ (cytotoxic T-cell: CTL) and CD3+ V alpha24+ (NKT-cell), in cancer patients and normal controls. The NK and cytotoxic T-cells significantly decreased and NKT-cells had decreased tendency in cancer patients compared with normal controls. In contrast, memory T cells, Fas+ T-cells and helper T-cells were all significantly increased in cancer patients compared with normal controls. These results suggested that the underlying mechanism of impaired IL-18 responsiveness in PBMCs from cancer-bearing patients was, at least in part, ascribed to a drastic decrease of NK cells and CTL which constitutively and highly express IL-18R and also attributed to null production of IL-12 which up-regulates IL-18R.     

Pilot trial of interleukin-2 with granulocyte colony-stimulating factor for the mobilization of progenitor cells in advanced breast cancer patients undergoing high-dose chemotherapy: expansion of immune effectors within the stem-cell graft and post-stem-cell infusion.

PURPOSE: To evaluate whether administration of interleukin-2 (IL-2) with granulocyte colony-stimulating factor (G-CSF) improves mobilization of immune effector cells into the stem-cell graft of patients undergoing high-dose chemotherapy and autografting. PATIENTS AND METHODS: We performed a trial of stem-cell mobilization with IL-2 and G-CSF in advanced breast cancer patients receiving high-dose chemotherapy with cyclophosphamide, thiotepa, and carboplatin and stem cells followed by IL-2. The trial defined immune, hematologic, and clinical effects of IL-2 in this setting. RESULTS: Of 32 patients enrolled, nine received G-CSF alone for mobilization. Twenty-one of 23 patients mobilized with IL-2 plus G-CSF had stem cells collected with more mononuclear cells than those receiving G-CSF (19.3 v 10.4 x 10(8)/kg; P =.006), but fewer CD34(+) progenitor cells (6.9 v 22.0 x 10(6)/kg; P =.049). The IL-2 plus G-CSF-mobilized patients had greater numbers of activated T (CD3(+)/CD25(+)) cells (P =.009), natural killer (NK; CD56(+)) cells (P =.007), and activated NK (CD56 bright(+)) cells (P: =.039) than those patients mobilized with G-CSF. NK (P =.042) and lymphokine-activated killer (LAK) (P =.016) activity was increased in those mobilized with IL-2 + G-CSF, whereas G-CSF-mobilized patients had a decline in cytolytic activity. In the third week posttransplantation, immune reconstitution was superior in those mobilized with IL-2 plus G-CSF based on greater numbers of activated T cells (P =.003), activated NK cells (P =.04), and greater LAK activity (P =.003). The 16 of 21 IL-2 + G-CSF-mobilized patients with adequate numbers of stem cells (> 1.5 x 10(6) CD34(+) cells/kg) collected engrafted rapidly posttransplantation. CONCLUSION: The results demonstrate that G-CSF + IL-2 can enhance the number and function of antitumor effector cells in a mobilized autograft without impairing the hematologic engraftment, provided that CD34 cell counts are more than 1.5 x 10(6) cells/kg. Mobilization of CD34(+) stem cells does seem to be adversely affected. In those mobilized with IL-2 and G-CSF, post-stem-cell immune reconstitution of antitumor immune effector cells was enhanced.     

DC-CIK联合放化疗对小细胞肺癌患者免疫功能的影响

目的：探讨细胞因子诱导的杀伤细胞（CIK）、树突状细胞（DC）免疫治疗联合放化疗对小细胞肺癌患者外周血淋巴细胞亚群的影响及疗效。方法收集60例小细胞肺癌患者资料,其中32例患者采用DC-CIK联合放化疗（化疗方案为依托泊苷+顺铂/依托泊苷+卡铂/伊立替康+顺铂,放疗方案为适形调强放疗技术）为联合治疗组;28例进行放化疗的患者为对照组。治疗结束2周后采用流式细胞仪检测外周血CD3+、CD3+CD4+、CD3+CD8+、NK细胞、Treg细胞及相关因子IFN-γ、IL-2、IL-10、TGF-β1的变化,并评价疗效。结果联合治疗组IFN-γ治疗后较治疗前升高,IL-10、TGF-β1较治疗前降低（P﹤0.05）;联合治疗组患者外周血CD3+CD8+、CD3+CD4+、NK细胞治疗后水平高于对照组（P﹤0.05）;而Treg细胞、IL-10、TGF-β1治疗后水平低于对照组（P﹤0.05）。对照组患者外周血CD3+、CD3+CD4+、CD3+CD8+、NK细胞治疗后较治疗前下降,差异有统计学意义（P﹤0.05）;细胞因子在治疗前后无变化。虽然联合组疾病控制率较对照组高,但差异无统计学意义（87.50%vs 71.43%,P﹥0.05）。结论自体DC-CIK治疗可以改善小细胞肺癌联合放化疗患者的细胞免疫功能,是否能改善生存需进一步研究。     

Synergistic effect of Nocardia rubra cell wall skeleton and recombinant interleukin 2 for in vivo induction of lymphokine-activated killer cells

C57BL/6 mice inoculated i.p. with 3LL tumor cells were treated by local combination therapy with Nocardia rubra cell wall skeleton (N-CWS) and recombinant interleukin 2 (rIL-2). The combination treatment significantly prolonged their survival and augmented lymphokine-activated killer (LAK) activity of peritoneal cavity lymphocytes (PCL), compared with treatments with rIL-2 alone or N-CWS alone. After in vitro culture of peritoneal exudate mononuclear cells with rIL-2, the nonadherent population derived from N-CWS-injected tumor-bearing mice showed a significantly higher LAK activity than did that population derived from saline solution-injected mice. When N-CWS-induced PCL were cocultured with either N-CWS-induced macrophages or control macrophages in the presence of rIL-2, their LAK activity was higher than that of control PCL. Therefore, it was suggested that N-CWS-induced PCL themselves have a more potent ability as precursors of LAK cells. Phenotypic analysis on PCL populations revealed that N-CWS-induced PCL contained increased proportions of CD3+CD4-CD8- cells and asialo GM-1+ cells compared with control PCL. These results suggest that N-CWS selectively accumulates potent LAK precursors, namely, CD3+CD4-CD8- T-cells and asialo GM-1+ natural killer cells, at the injection site and that LAK cells are efficiently induced by subsequent administration of rIL-2.     

Lymphokine-activated killer activity in long-term cultures with anti-CD3 plus interleukin 2: identification and isolation of effector subsets

Peripheral blood lymphocytes cultured in recombinant interleukin 2 during 3 to 5 days (short-term cultures) develop the ability to lyse natural killer-resistant tumor lines and fresh tumor cells, i.e., express lymphokine-activated killer (LAK) function. Phenotypic analysis has shown these cells to be natural killer cells, i.e., CD16+ and/or Leu 19+ cells. CD3+,CD16- T-cells, instead, develop very low LAK function in these cultures. We recently reported the development of long-term (up to 21 days) cultured cells with LAK activity by stimulation with OKT3 + interleukin 2(IL2). These culture conditions repeatedly resulted in a several hundred-fold expansion in cell number. Specific LAK activity on Day 14 of culture was comparable to that of 3-day LAK cultures and could be further enhanced by the addition of interleukin 1 beta, beta-, or gamma-interferon. Total LAK activity was greatly increased in OKT3 + IL2 cultures over that found in short-term cultures. Isolation of effectors mediating LAK function in long-term cultures stimulated with OKT3 + IL2 showed that both CD3+,CD16- cells and CD16+,CD3- cells tested on Day 14 of culture expressed equivalent levels of LAK activity as shown by lysis of natural killer-resistant targets, HL60 and Daudi. Further dissection of the subpopulations developing LAK activity demonstrated that, in addition to CD16+,CD3- cells, CD3+, CD4-,CD8- cells and Leu 19+,CD3-,CD16- cells also developed high LAK activity in long-term cultures with OKT3 + IL2. Further, long-term culture with OKT3 + IL2 induced increases in the numbers not only of CD3+,CD4-,CD8- cells but also of CD16+,CD3- and Leu 19+,CD3-,CD16- cells. Although there is a significant increase in the number of CD3+,CD8+ cells, neither these, nor the CD3+,CD4+ cells, mediate LAK activity to the same extent as the populations mentioned above.     

Lymphokine-activated killer activity induced by in vivo interleukin 2 therapy: predominant role for lymphocytes with increased expression of CD2 and leu19 antigens but negative expression of CD16 antigens

The phenotype and function of lymphocytes from cancer patients treated with repetitive weekly cycles of continuous i.v. infusions of recombinant interleukin 2 (IL-2) were examined. Peripheral blood lymphocytes (PBL) obtained after IL-2 therapy showed an increased percentage of cells bearing the CD16 and leu19 markers which are associated with natural killer cells. These PBL mediated significantly increased levels of IL-2-dependent lymphokine-activated killer (LAK) activity against the Daudi cell line. Depletion of CD16+ cells from PBL obtained after in vivo IL-2 caused only slight inhibition of their LAK activity or their proliferative response to IL-2 in vitro. This indicates that CD16+ cells are involved but play only a minor role in these responses. In contrast, depletion of leu19+ cells, from PBL activated in vivo with IL-2, virtually abrogated their LAK activity and their proliferative response to IL-2. Two-color flow cytometry studies showed that a leu19+/CD16- population was expanded by in vivo IL-2 therapy and was responsible for the majority of LAK activity by in vivo-activated PBL. Moreover, this CD16- population showed an increased density of leu19 and CD2 (E rosette receptor) antigens when compared to the resting PBL obtained prior to IL-2 treatment. These data show that the predominant population mediating in vitro LAK activity, induced by in vivo IL-2 therapy, consists of activated natural killer cells with a high density of leu19 and CD2 antigens but negative for the CD16 antigen.     

Human interleukin 4 regulates the phenotype of lymphocytes generated during mixed lymphocyte culture and inhibits the IL-2-induced development of LAK function in normal and leukaemic cells

This study examined the immunoregulatory role of recombinant interleukin 4 (IL-4), also known as B-cell stimulating factor 1, on the generation of cytotoxic effector cells from normal and leukaemic human blood mononuclear cells. When tested on cells from normal individuals, the addition of IL-4 to mixed lymphocyte cultures led to a dose-dependent proliferation of T-helper cells (CD3, 4 positive) with a concomitant decrease in phenotypic and functional cytotoxic T cells and natural killer (NK) cells. IL-4 also inhibited the interleukin-2 (IL-2)-induced generation of lymphokine-activated killer (LAK) activity when added at the beginning of mixed lymphocyte culture. When tested on mature leukaemic NK cells, IL-4 also inhibited the ability of IL-2 to induce LAK function using a short-term culture system. These results show that IL-4 acts on both normal and leukaemic cells and suggests that it acts at more than one level during the development of LAK function.     

Natural killer and lymphokine-activated killer activities in stomach cancer patients with special emphasis on the effect of 5-fluorouracil, adriamycin and mitomycin-C chemotherapy

The cytotoxicities of peripheral blood lymphocytes (PBL) and lymphokine-activated killer (LAK) cells were studied to evaluate the effect of chemotherapy on cellular immunity, in 18 patients with unresectable stomach cancer before and after chemotherapy with 5-fluorouracil, adriamycin and mitomycin-C (FAM), and in 21 healthy volunteers. LAK cells were generated in vitro by culturing PBL with 100 U recombinant human interleukin-2 (rH-IL-2)/ml for 72 h. K562 (human myelogenous leukemia), MKN-45 (human stomach adenocarcinoma) and PC-14 (human pulmonary adenocarcinoma) were used as target cells. The cytotoxicity of PBL to K562 and MKN-45 was suppressed in patients with stomach cancer before chemotherapy, compared with that in healthy volunteers (P less than 0.05). The cytotoxicity of LAK cells was significantly higher to all three cell lines tested than that of PBL in both the healthy volunteers and stomach cancer patients (P less than 0.01); however, a lower level of LAK activity was generated in patients with cancer compared to that in the healthy volunteers. FAM therapy did not suppress the cytotoxicities of PBL and LAK cells. The surface markers of PBL and LAK cells were measured, demonstrating that there was no significant change in the percentage of lymphocytes with CD3+, CD4+, CD8+, CD16+ or CD19+ after chemotherapy. The ratios of CD4+ to CD8+ cells in PBL and LAK cells were also not significantly changed after chemotherapy. In the present study, we have demonstrated that the PBL of stomach cancer were defective in generating LAK activity compared to those of controls, but the LAK activity generated from PBL receiving chemotherapy was similar to that from PBL without chemotherapy in stomach cancer patients.     

Interleukin-2 Therapy for Myelodysplastic Syndrome: Does It Work?

Recent clinical studies suggested that interleukin-2 (IL-2) has therapeutic potential for some hematologic malignancies, but the therapeutic role of IL-2 for myelodysplastic syndrome (MDS) is still unclear. MDS is a clonal malignant disorder which often involves a variety of immunologic abnormalities. Examination of the effects of IL-2 on MDS in vitro yielded the following results: (1) IL-2 did not induce the proliferation of blasts in most MDS cases. (2) The cytotoxicity of IL-2-induced lymphokine-activated killer (LAK) cells for cell lines and MDS blasts was reduced in the high-risk MDS group (refractory anemia with excess blasts (RAEB), RAEB in transformation and MDS transformed to acute leukemia), but it was still preserved in the low-risk MDS group (refractory anemia (RA) and RA with ringed sideroblasts). However, considerable variation in LAK cell cytotoxicity was noted in each group. (3) The reduced LAK cell cytotoxicity observed in MDS was explained, at least in part, by the presence of a reduced of number of natural killer (NK) cells amongst the LAK cells. (4) MDS patients who have a high blood soluble IL-2 receptor (sIL-2R) level often had defects in NK and CD8+ T cells. These in vitro findings suggest that the response to IL-2 is heterogeneous in MDS patients, and those who have a low-risk MDS subtype and/or a low blood sIL-2R level, may be prone to respond to IL-2 therapy. Clinical trials are mandatory in order to elucidate the efficacy of IL-2 therapy in the treatment of MDS.     

Effect of anti-CD3 antibody on the generation of interleukin-2-activated lymphocytes from tumor tissues of gastrointestinal cancer.

BACKGROUND. The efficiency of anti-CD3 antibody (OKT3) for adoptive immunotherapy using lymphokine-activated killer (LAK) cells generated from tumor-infiltrating lymphocytes (TIL), regional lymph node lymphocytes (RLNL), and peripheral blood lymphocytes (PBL) was investigated. METHODS. TIL, RLNL, and PBL derived from 39 patients with gastrointestinal cancers (16 gastric cancers, 17 colorectal cancers, and 6 esophageal cancers) were cultured for 4 weeks with 200 U/ml of recombinant interleukin-2. To one group, solid-phase 10 micrograms/ml OKT3 was added during the initial culture period (day 2 or 4). Cytotoxicity against K562 cells (NK-like activity) and Daudi cells (LAK activity) and the phenotypes of effector cells generated after culturing for 2-3 weeks were studied. RESULTS. Proliferative responses were significantly increased by OKT3 in each type of effector cell (P less than 0.01); in particular, TIL expanded more by OKT3 than PBL and RLNL (P less than 0.01). The population of CD8+ CD11b- cytotoxic T-cells in OKT3-stimulated groups was significantly larger than that in unstimulated groups (P less than 0.01), whereas no differences were observed with CD4+ cells (helper/inducer T-cells) and CD8+ CD11b+ cells (suppressor T-cells). OKT3 enhanced the NK-like activity of TIL and PBL but did not affect their LAK activity. OKT3 suppressed the NK and LAK activity of RLNL. CONCLUSIONS. OKT3 stimulation did not significantly enhance the LAK activity, but the authors propose that OKT3 could be an effective addition to adoptive immunotherapy using TIL due to an increased proliferation and generation of a large cytotoxic T-cell population.     

康赛迪胶囊对食管癌放疗患者细胞免疫功能的影响

目的　观察中药复方制剂康赛迪胶囊对食管癌患者放疗前后细胞免疫功能的影响。方法　采用FACSCalibur流式细胞仪 ,SimulSETv3 1分析软件 ,分别检测 2 0例食管癌患者在放疗 6 0Gy前后 (对照组 )和口服康赛迪同时放疗 6 0Gy前后 (实验组 )患者外周血NK细胞活性、T淋巴细胞亚群 (CD3+、CD4 +、CD8+)、CD4 +/CD8+、B淋巴细胞 (CD19+)、NK淋巴细胞 (CD16 +,CD5 6 +)水平。结果　二组患者放疗前外周血NK细胞活性、CD3+、CD4 +、B淋巴细胞百分数、CD4 +/CD8+的平均值均低于健康人参考值 ,其中CD4 +细胞和B淋巴细胞明显下降 ,CD8+细胞明显升高。对照组患者放疗 6 0Gy后 ,除CD8+细胞和NK淋巴细胞百分数稍有升高外 ,其他细胞均略有下降 (P >0 0 5 )。实验组患者在治疗后CD3+、CD4 +、NK淋巴细胞百分数、CD4 +/CD8+明显升高 ,并明显高于对照组 (P <0 0 5或P <0 0 1)。结论　食管癌患者在放疗前后细胞免疫功能持续低下。放疗 6 0Gy后患者细胞免疫功能受到轻度抑制 ,康赛迪可以明显改善食管癌患者放疗后的细胞免疫功能状态。提示食管癌患者在放疗的同时应加强免疫治疗 ,以改善患者的免疫抑制状态。     

B-NHL患者NK细胞中CD16ζ表达及利妥昔单抗与LAK细胞的联合抗瘤作用

背景与目的:自然杀伤细胞(nature killer cell,NK)是抗体依赖细胞介导的细胞毒作用的主要效应细胞,肿瘤患者普遍存在NK细胞活化功能的缺陷可能会影响单克隆抗体的治疗效果.因此如能逆转NK细胞的CD16ζ链信号转导的功能缺陷,并与单克隆抗体联合免疫治疗,可能会产生协同抗肿瘤作用.本研究的目的是了解B细胞非霍奇金淋巴瘤(B-cell non-Hodgkin's lymphoma,B-NHL)患者是否存在NK细胞的活化障碍,体外白细胞介素-2(interleukin-2,IL-2)是否能完全逆转其活化障碍,并观察利妥昔单抗与LAK细胞联合对肿瘤细胞的杀伤作用.方法:使用密度梯度离心方法分别分离69例B-NHL患者和30例健康志愿者外周血单个核细胞(peripheral blood mononuclear cell,PBMC),将两种PBMC在体外与1 000 U/ml IL-12共同培养制备LAK细胞,流式细胞仪检测PBMC和LAK细胞中CD16ζ链的阳性率和平均荧光强度.流式细胞仪检测Raji细胞表面CD20的表达;Annexin V/PI方法检测利妥昔单抗单药对Raji细胞的促凋亡作用,乳酸脱氢酶(lactate dehydrogenase,LDH)释放实验进行杀伤活性的检测.结果:在B-NHL组和健康对照组,CD56 细胞表达CD16ζ链的阳性率为(63.3±16.4)%、(97.8±3.1)%(P＜0.001),CD16ζ链MFI值分别为1.3±1.3和3.6±1.7(P＜0.001).在体外1 000 U/ml的IL-2共培养的LAK细胞中,两组CD16ζ链的阳性率分别为(99.3±4.1)%和(99.7 3.9)%,其MFI值分别为29.2±12.5和31.4±13.8,均无显著性差异(P=0.15和P=0.44).40 μg/ml利妥昔单抗可以完全结合细胞表面CD20抗原,在24 h时才开始出现对Raji细胞的明显的凋亡作用.利妥昔单抗与LAK细胞联合对Raji细胞的杀伤率在不同的浓度组均明显高于不加利妥昔单抗组(P＜0.05).LAK细胞与Herceptin(40 μg/ml)联合的杀伤率与不加Herceptin组相比,在各效靶比浓度梯度均无明显提高(P＞0.05).LAK细胞与利妥昔单抗联合对Jurket细胞的杀伤率在各效靶比浓度梯度均与不加利妥昔单抗组无显著性差异(P＞0.05).结论:B-NHL患者普遍存在NK细胞CD16ζ链的表达下调,高剂量的IL-2可以显著增强CD16ζ链的表达,利妥昔单抗与LAK细胞联合可增强对Raji细胞的抗肿瘤作用.     

Differential activation of lymphokine-activated killer cells with different surface phenotypes by cultivation with recombinant interleukin 2 or T-cell growth factor in gastric cancer patients

Fourteen days' culture of human peripheral blood lymphocytes (PBL) with recombinant interleukin 2 (rIL 2) or T cell growth factor (TCGF) results in the generation of lymphokine-activated killer (LAK) effector cells which have the unique property of lysing natural killer (NK)-resistant human tumor cells, Daudi, as well as NK-sensitive, K562 cells. LAK cells were generated from both normal and gastric cancer patients' PBL. However, LAK cell activities induced by rIL 2 or TCGF decreased with the progress of the tumor growth. In addition, TCGF-induced LAK cell activities were found to be lower than the rIL 2-induced LAK cell activities. Different mechanisms may be involved in the decreases of the rIL 2-induced and TCGF-induced LAK cell activities. This study further demonstrates that the cell types involved are also heterogeneous, as determined by phenotypic characteristics. The LAK-effector cell type was analyzed by two-color flow cytometry. RIL 2-induced LAK cells showed increased proportions of CD4+Leu 8- and Leu 7+CD16-, and a decreased proportion of CD8+CD11- cells, which are believed to be associated with killer T cell functions. In contrast, TCGF-induced LAK cells revealed significantly increased proportions of CD8+CD11- and CD4+Leu8- cells, and a decreased proportion of Leu 7+CD16- cells. Thus, LAK cells with different surface phenotypes were induced by the cultivations with rIL 2 and with TCGF.     

Heterogeneity of human lymphokine (IL-2)-activated killer (LAK) precursors and regulation of their LAK induction by blood monocytes

Highly purified lymphocytes (greater than 99%) and monocytes (greater than 90%) were isolated by CCE from peripheral blood of healthy donors. Blood lymphocytes were separated by this CCE into 9 subpopulations. The NK activities of these lymphocyte fractions against NK-sensitive K-562 cells and their LAK activities against NK cell-resistant target (Daudi) cells were assayed promptly or after incubation of the fractions for 4 days with or without an optimal concentration of IL-2. NK and LAK activities were measured by 4-hr 51Cr-release assay. On the basis of their NK and LAK activities, these lymphocyte fractions were classified into 3 subpopulations of LAK precursors: one lacking both NK and LAK activities (Fr.2), one with moderate NK activity but low LAK activity (Fr.5), and one possessing both NK and LAK activities (Fr.8). Addition of autologous fresh monocytes to the lymphocyte cultures resulted in a significant increase in induction of LAK activity in Fr.2 and Fr.5. This up-regulation of lymphocytes in Fr. 2 and Fr.5 by monocytes was confirmed in parallel experiments by measuring the blastogenic response of the lymphocytes to IL-2. Deletion of lymphocytes in Fr. 8 of CD16+ (Leu-11+) NK cells resulted in 74% reduction in LAK induction, whereas depletion of mixtures of monocytes and lymphocytes in Fr. 2 of cells reacting with CD3+ (OKT3+) antibody resulted in a 66% reduction in LAK induction. This up-regulation of LAK cell induction from LAK precursors by monocytes was confirmed using 4 lines of human lung cancer cells as targets for LAK activity. These results clearly indicate that human monocytes may cause up-regulation of the expression of IL-2-induced LAK activity in T cells and in a subpopulation of NK cells.