Effect of low dose cyclophosphamide on the immune system of cancer patients: reduction of T-suppressor function without depletion of the CD8+ subset

Low dose cyclophosphamide (CY) can augment the development of delayed-type hypersensitivity to primary antigens in patients with advanced cancer. In this paper, we have considered the hypothesis that the immunopotentiation is related to reduction of T-suppressor activity. Peripheral blood lymphocytes were collected and cryopreserved from 45 patients with metastatic malignancy before and then 3, 7, and 19 days after administration of CY, 300 mg/m2 i.v. The peripheral blood lymphocytes were tested for generation of concanavalin A-inducible suppressor activity, proliferative response to phytohemagglutinin, and phenotype using monoclonal antibodies to CD4 and CD8. Concanavalin A-inducible suppression was significantly reduced by day 3 and declined progressively through day 19. The mean percentage changes in suppression were: day 3, -23.4 +/- 6.8 (SE) (P less than 0.01); day 7, -33.1 +/- 14.3 (P = 0.052); day 19, -43.1 +/- 10.7 (P less than 0.01). In contrast, CY caused no significant changes in phytohemagglutinin proliferation (mean percentage changes: day 3, -4.7 +/- 6.1; day 7, -15.6 +/- 7.5; day 19, -5.5 +/- 8.1), indicating that the reduction in concanavalin A-inducible suppression was not merely a reflection of a general reduction in peripheral blood lymphocyte function. The total number of circulating lymphocytes was not affected by low dose CY. Moreover, flow cytometric analysis showed no significant changes in the percentage of circulating CD8+ or CD4+ T-cells or in the CD4/CD8 ratio at any time point after CY. Thus, administration of low dose CY to these patients caused impairment of nonspecific T-suppressor function without selective depletion of the CD8+ subset that is generally associated with that function. Several immunoregulatory models that are consistent with these observations are discussed.     

The prognostic role of CD4+ and CD8+ lymphocytes during chemoimmunotherapy in metastatic melanoma

We have observed that an early increase in the CD4+/CD8+ ratio of metastatic melanoma patients during chemoimmunotherapy is the most favourable independent prognostic factor. In this study, 87 patients with metastatic melanoma were monitored for peripheral blood lymphocyte subsets (CD4+ and CD8+) before and during chemoimmunotherapy (dacarbazine, vinblastine, lomustine and bleomycin or dacarbazine alone plus interferon-alpha) to confirm our previous observation. Blood samples were systematically obtained from patients who received either of these chemoimmunotherapies. The frequencies of peripheral blood lymphocyte subsets were monitored by flow cytometry using monoclonal antibodies OKT4 (CD4+, T-helper cells) and OKT8 (CD8+, T-suppressor cells). The overall response rate was 46.5%, and the median overall survival was 9.3 months. Patients with pre-treatment CD4+ levels above the mean level, who later responded to therapy, had a median survival of 28.1 months vs. 10.2 months for responders with low pre-treatment CD4+ levels (P=0.053, log-rank test). Responders with decreasing CD8+ levels had a median survival of 27.8 months vs. 10.8 months for responders with increasing CD8+ levels (P=0.04, log-rank test). Similarly, responding patients with an increasing CD4+/CD8+ ratio had a median survival of 28.1 months vs. 10.5 months for those with decreasing ratios (P=0.002, log-rank test). Non-responders showed no difference in median survival irrespective of low or high pre-treatment CD4+ lymphocytes, increasing or decreasing CD8+ levels, or increasing or decreasing CD4+/CD8+ ratios. This follow-up study confirms that the monitoring of CD4+ and CD8+ lymphocytes may provide important predictive and prognostic information in metastatic melanoma patients receiving chemoimmunotherapy.     

Treatment of metastatic melanoma with an autologous tumor-cell vaccine: clinical and immunologic results in 64 patients

We treated 64 patients with metastatic melanoma using a melanoma vaccine preceded by low-dose cyclophosphamide (CY), and monitored immunologic effects and antitumor activity. On day 0, the patients were given CY 300 mg/m2 intravenously. Three days later, they were injected intradermally with vaccine consisting of 10 to 25 x 10(6) autologous, enzymatically dissociated, cryopreserved, irradiated (25 Gy) tumor cells mixed with bacillus Calmette-Guérin (BCG). This treatment sequence was repeated every 28 days. Of 40 assessable patients with measurable metastases, five had responses, four complete and one partial, with a median duration of 10 months (7 to 84+ months). In six additional patients, we observed an antitumor response that seems to be peculiar to this vaccine therapy: the regression of metastatic lesions that appeared after the immunotherapy was begun. Delayed-type hypersensitivity (DTH) to autologous, mechanically dissociated melanoma cells that had not been exposed to extraneous antigens, such as enzymes or fetal calf serum, increased significantly following immunotherapy (day 0 v day 49, P less than .001; day 0 v day 161, P less than .001; day 0 v day 217, P = .021). Antitumor responses to the vaccine were strongly associated with DTH, as indicated by three observations: (1) eight of 10 patients who exhibited tumor regression had positive DTH, (2) in postsurgical adjuvant patients, there was a highly significant linear relationship (P less than .001) between the intensity of DTH to autologous melanoma cells and the time to recurrence of tumor, and (3) nine patients who developed DTH to the autologous melanoma cells in their original vaccine developed new metastases that failed to elicit DTH or elicited a much smaller response. In three cases, we were able to excise regressing tumors for histologic examination; such tumors were characterized by an intense infiltration of lymphocytes. This demonstration that an immune response to melanoma-associated antigens can be elicited in cancer-bearing patients provides some basis for optimism about the prospects for developing active immunotherapy that has practical therapeutic value.     

Optimization of intracerebral tumour protection by active-specific immunization against murine melanoma B16/G3.12

Development of brain metastases despite extracerebral response to systemic immunotherapy is a common problem in melanoma patients. We have previously described a murine melanoma vaccine of interferon-gamma (IFNgamma)-treated, irradiated syngeneic B16/G3.12 and allogeneic (Cloudman) melanoma cells, plus the adjuvant DETOX, that is protective against subcutaneous (93%) or intracerebral (69%) syngeneic challenge. This study aimed to optimize this vaccine. Groups of nine or 10 mice were immunized five times in 5 weeks with: (i) complete vaccine +/- IFNgamma (VAC+, VAC-); (ii) syngeneic 2 x 106 G3.12 cells plus DETOX (Syn+D), (iii) 2 x 106 allogeneic Cloudman cells plus DETOX (Allo+D); (iv) VAC+ without DETOX (no DETOX); (v) DETOX alone (DETOX); or (vi) phosphate buffered saline (PBS). Mice were challenged subcutaneously with 104 viable G3.12 (or Cloudman cells) and after 35 days intracerebrally with 104 G3.12 cells. Expression of H-2 antigens (measured using fluorescence-activated cell sorting), splenocyte cytotoxicity (measured using 51Cr release) and median overall survival (OAS) were analysed using the log-rank test. VAC+, VAC- and G3.12 mice were equally protected from subcutaneous (s.c.) and intracerebral (i.c.) melanoma challenge (OAS 65 days for s.c., 30 days for i.c.). Protection was less (P < 0.05) in DETOX mice (48 days for s.c.), PBS mice (47 days for s.c., 21 days for i.c.) or no DETOX mice (51 days for s.c.). Allo+D mice showed s.c. (59 days) but not i.c. protection (20 days). IFNgamma incubation did not increase the effect in either the challenge cells or the vaccine cells (P > 0.05). Specific cytotoxicity was seen with G3.12 targets in VAC+ (27%) but not PBS (2%; P < 0.05) mice with equal NK (YAC-1) lysis (10% versus 7%; P< 0.05). Optimal protection against s.c./i.c. experimental murine melanoma was yielded by irradiated syngeneic cells plus DETOX. DETOX alone was not active. Upregulation of H-2 antigens with IFNgamma under these conditions does not augment protection.     

Active immunotherapy of human melanoma exploiting the immunopotentiating effects of cyclophosphamide

Malignant tumors may escape rejection by the immune system because they induce a state of immunological tolerance mediated by tumor antigen-specific suppressor T cells. In animal systems, cyclophosphamide can reverse the tolerance and thereby facilitate immunologically mediated tumor destruction. We have applied these concepts to the immunotherapy of human malignant melanoma. Forty-three patients with metastatic disease were treated with a whole cell vaccine 3 days after intravenous administration of cyclophosphamide, 300 mg/m2. The vaccine consisted of cryopreserved, irradiated autologous melanoma cells, obtained from metastatic masses by dissociation with collagenase and DNAse, mixed with bacillus Calmette-Guerin (BCG) and injected intradermally. The cyclophosphamide (CY) + vaccine combination was repeated every 28 days. Delayed-type hypersensitivity (DTH) was tested by injecting 1 x 10(6) melanoma cells intradermally and measuring the diameter of induration at 48 h. Most patients had minimal pretreatment DTH responses to melanoma cells (mean +/- SE, mm = 2.4 +/- 0.5). After two vaccine treatments, the responses increased significantly (mean increase +/- SE = 12.1 +/- 1.6 p less than .001) and that level of response was maintained after 4, 6, and 8 treatments. The patients were also skin-tested with a mixture of the enzymes used to dissociate the tumors. No patients exhibited DTH to collagenase + DNAse prior to vaccine injection, but every patient developed DTH to the mixture following two treatments (mean, mm = 26.4 +/- 3.9). Although extracting viable cells from tumor tissue without the use of enzymes proved difficult, we were able to test DTH to mechanically dissociated tumor cells in 23 patients. After two vaccine treatments, there was a significant increase in DTH to enzyme-free autologous melanoma cells (mean DTH +/- SE, mm: 5.4 +/- 1.0, p less than .01). Whereas 5 of 23 patients had positive DTH responses (5 mm induration or greater) before treatment, 11 of 23 were positive after two treatments. Further significant increases in DTH enzyme-free cells were observed after 6 and 8 treatments. Thus, it appears that patients receiving CY + vaccine developed DTH to tumor-associated antigens as well as to residual collagenase and DNAse on the cell surface. Thirty-three patients could be evaluated for antitumor effects of cyclophosphamide + vaccine. There were 3 complete remissions, 1 partial remission, and 2 minor responses. Two complete responders remain alive and free of disease after 57 and 12 months, respectively, and the third died after 39 months. The partial remission consisted of 75% regression of a pulmonary metastasis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dominance of CD4+ alpha/beta T-cells and inferior role of innate immune reaction in liver metastases

BACKGROUND: Tumor infiltrating lymphocytes (TIL) are frequently present in human tumors with CD8+(-)T-cells as effector and CD4+ T-cells as helper cells. Despite the well established knowledge about primary tumors, only little is known about metastatic disease, especially for liver metastases. The role of the innate immune system in the tumor defence is still enigmatic. MATERIALS AND METHODS: We performed a subtyping of TIL in 20 liver metastases. Using immunohistochemistry, CD20+, CD3+, CD56+, CD4+ and CD8+ lymphocytes, gamma/delta-T-cells and alpha/beta-T-cells in the tumor, the peritumoral region, portal tracts and lobules were investigated. RESULTS: The immune response was highly accentuated in the surroundings of the metastases with only few lymphocytes in the tumor itself. There was a dominance of CD3+(-)CD4+(-)alpha/beta-T-cells with a lower number of CD8+(-)T-cells. The CD4+/CD8+ ratio was 6:1. CD56+(-)NK/NKT-cells and gamma/delta-T-cells were rare. No differences were found between metastases from different primaries or according to the number or diameter of the metastases. CONCLUSION: TIL are part of an interaction between the metastatic tumor and the liver. Among them CD4+ T-cells seem to have a unique independent function in tumor response. The localization of the immune response in the tumor periphery might be a reason for insufficient tumor defense. A defect in the innate immune system could be a reason for the escape of the metastatic tumor cells from tumor surveillance.     

Proliferative and/or cytotoxic activity of lymphocyte clones to autologous human melanoma

Peripheral blood lymphocytes (PBL) of a patient with metastatic melanoma were cultured with autologous melanoma cells (Auto-Me) and recombinant interleukin 2 (IL-2) (MLTC-PBL). Thirty-five days later, when no cytotoxicity against Auto-Me or K562 was detectable, MLTC-PBL were cloned in the presence of Auto-Me, IL-2 (25 U/ml) and Daudi cells as feeder. Eighty-one growing clones were simultaneously screened for proliferative and cytotoxic activity to Auto-Me. Twenty-two clones proliferated in the presence of Auto-Me only, 29 in the presence of IL-2 only and 41 in the presence of Auto-Me plus IL-2; 12 clones showed cytotoxic activity against Auto-Me. Six clones expressed both cytotoxic and proliferative activity to Auto-Me. The phenotype of 6 proliferative clones tested was CD3+, CD4+, WT31+, CD8-, CD16-, Leu19-, whereas that of 2 cytotoxic-proliferative clones tested was CD3+, CD8+, Leu19+, WT31+, CD4-, CD16-. Specificity analysis of proliferative response of 6 clones and of cytotoxicity of 7 clones, tested on a panel of 14 different target cells, revealed a complex pattern of reactivity, each clone expressing a peculiar specificity. Our results suggest the possibility of isolating, from melanoma patients' PBL, T-cell clones with proliferative activity to Auto-Me and Auto-Me plus IL-2, and T-cell clones which apparently express both proliferative and cytotoxic activity to Auto-Me.     

转移性肾癌自体DC-CIK细胞免疫治疗疗效观察

目的:观察自体树突状细胞（dendritic cell,DC）激活的细胞因子诱导的杀伤（cytokine-induced killer,CIK）细胞治疗转移性肾癌的临床效果及安全性。方法:32例转移性肾癌患者,采集外周血单个核细胞（peripheral blood mononuclear cell,PBMC）,体外制备成DC-CIK细胞,12天后连续三天回输,输注结束后次日开始下一个疗程,共计3个疗程。观察治疗前、治疗后6个月外周血中T细胞亚群及细胞因子IL-2、IFN-γ、IL-4及IL-10的变化,并统计治疗3年后生存率以及生活质量评分（KPS）,观察不良反应。结果:治疗前后比较CD4+无显著差异,CD8+显著降低,CD3+、CD56+以及CD4+/CD8+有显著差异（P<0.05）。两组治疗后6个月 IL-2、IFN-γ与治疗前比较均升高（P<0.05）。IL-4、IL-10与治疗前比较均降低（P<0.05）。治疗前、治疗后2周患者WBC、ALT、AST以及BUN、Cr比较无显著性差别（P>0.05）。3年生存率90.63%,治疗后1年生活质量评分平均升高24.7,显著高于治疗前的11.8(P<0.05)。不良反应轻微。结论:自体DC-CIK细胞输注后可显著提升转移性肾癌（RCC）患者免疫水平,安全性良好,可作为晚期RCC患者可选择的治疗方法。     

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.     

Induction of cell-mediated immunity to autologous melanoma cells and regression of metastases after treatment with a melanoma cell vaccine preceded by cyclophosphamide

There is considerable evidence in animal tumor systems that antitumor immunity is modulated by suppressor T-lymphocytes, and that the cytotoxic drug cyclophosphamide (CY) can abrogate that suppression. We measured the acquisition of delayed-type hypersensitivity (DTH) to autologous melanoma cells in 19 patients with metastatic malignant melanoma. The patients were treated with an autologous melanoma cell vaccine, either given alone, or given 3 days after the administration of CY, 300 mg/m2 i.v. The DTH responses of CY-pretreated patients were significantly greater than those of control (vaccine only) patients. Thus, after two vaccine treatments, the median DTH responses (mm induration) were as follows: controls, 4 mm; CY pretreated, 11 mm; P = 0.034, Mann-Whitney U test, 2-tailed. Whereas seven of eight CY-pretreated patients developed DTH to autologous melanoma cells of at least 5 mm, only two of seven controls did so (P = 0.034, Fisher's exact test). Two patients had significant antitumor responses to treatment with CY plus vaccine, consisting of complete disappearance of skin metastases and a pulmonary nodule in one, and regression of s.c. and liver metastases in the other. Both patients remain free of melanoma after 42 and 33 mo, respectively.     

联合检测外周血T淋巴细胞AgNOR活性及T淋巴细胞亚群评估食管癌围手术期细胞免疫变化规律

背景与目的食管癌患者存在着细胞免疫功能抑制,测定T淋巴细胞核仁区的AgNOR活性及T淋巴细胞亚群,可以了解T淋巴细胞的免疫活性状态,对研究肿瘤的生物学行为具有重要意义。本研究旨在通过动态观察食管癌患者围手术期外周血T淋巴细胞AgNOR的活性及T淋巴细胞亚群的变化规律,研究细胞免疫机能状态及手术对食管癌患者细胞免疫功能的影响。方法分别抽取75例健康人外周血和70例食管癌患者术前、术后第3天、术后第9天的外周血,利用KL型肿瘤免疫图像分析系统检测AgNOR活性,流式细胞术分析T淋巴细胞亚群。结果食管癌患者术前AgNOR活性[(5.5±0.8)%]明显低于健康人[(7.1±0.8)%](P<0.00),CD3 、CD3 CD4 无明显变化(P>0.05),CD4 CD25 、CD4 /CD8 明显上升(P<0.05),CD3 CD8 、CD8 CD25 亚群明显下降(P<0.00)。术后第3天外周血T淋巴细胞AgNOR、CD3 、CD3 CD4 与CD4 /CD8 最低,术后第9天最高;CD3 CD8 亚群、CD4 CD25 亚群、CD8 CD25 亚群则从第3天开始逐渐恢复,术后第9天最高。食管癌患者术后第9天CD3 、CD3 CD4 与健康人相比无显著性差异(P>0.05);外周血T淋巴细胞AgNOR、CD3 CD8 与CD8 CD25 仍未恢复到健康人水平(P<0.05);CD4 CD25 仍明显高于健康人(P<0.00)。结论手术创伤引起食管癌患者暂时性细胞免疫抑制,但从第3天开始可观察到细胞免疫功能恢复,术后第9天继续恢复,但仍未恢复到正常水平。     

超声引导下胸椎旁神经阻滞复合全身麻醉对乳腺癌患者术后镇痛及免疫细胞功能的影响

目的:探讨超声引导下胸椎旁神经阻滞复合全身麻醉对乳腺癌患者术后镇痛及免疫细胞功能的影响。方法:将78例行乳腺癌根治术的患者随机分成全身麻醉组及复合麻醉组。复合麻醉组患者在麻醉诱导前经超声引导下胸椎旁神经阻滞,麻醉诱导后吸入七氟醚;全身麻醉组患者在麻醉诱导后吸入七氟醚,术后均采用瑞芬太尼静脉自控镇痛(PCIA)。比较两组患者在术后1天、术后2天及术后3天视觉模拟评分(VAS)及两组患者术后镇痛持续时间和24 h内舒芬太尼用量;分别于术前、手术结束时、术后1天及术后3天时采用流式细胞仪分析法测定两组患者T淋巴细胞亚群及NK细胞值。结果:复合麻醉组患者在术后1天及术后2天时VAS评分均低于全身麻醉组(P＜0.01),两组患者在术后3天时VAS评分无统计学差异(P＞0.05);复合麻醉组患者镇痛持续时间多于全身麻醉组患者(P＜0.01);复合麻醉组患者24 h内舒芬太尼用量少于全身麻醉组患者(P＜0.01);两组患者在手术结束时、术后1天及术后3天时CD3+、CD4+、CD4+/CD8+及NK细胞值均低于术前(P＜0.05);复合麻醉组患者在手术结束时、术后1天及术后3天时CD3+、CD4+、CD4+/CD8+及NK细胞值均高于全身麻醉组患者(P＜0.05)。结论:乳腺癌根治术麻醉诱导前行超声引导下胸椎旁神经阻滞复合全身麻醉能够提供更加显著的镇痛效果,对T淋巴细胞的抑制更少,同时能够保护NK细胞活性,对患者免疫功能的影响更小。     

DNA vaccination of HSP105 leads to tumor rejection of colorectal cancer and melanoma in mice through activation of both CD4 T cells and CD8 T cells

We report that HSP105, identified by serological identification of antigens by recombinant expression cloning (SEREX), is overexpressed in a variety of human cancers, including colorectal, pancreatic, thyroid, esophageal, and breast carcinoma, but is not expressed in normal tissues except for the testis. The amino acid sequences and expression patterns of HSP105 are very similar in humans and mice. In this study, we set up a preclinical study to investigate the usefulness of a DNA vaccine producing mouse HSP105 whole protein for cancer immunotherapy in vivo using BALB/c and C57BL/6 mice, Colon26, a syngeneic endogenously HSP105-expressing colorectal cancer cell line, and B16.F10, a melanoma cell line. The DNA vaccine was used to stimulate HSP105-specific T-cell responses. Fifty percent of mice immunized with the HSP105 DNA vaccine completely suppressed the growth of subcutaneous Colon26 or B16.F10 cells accompanied by massive infiltration of both CD4+ T cells and CD8+ T cells into tumors. In cell transfer or depletion experiments we proved that both CD4+ T cells and CD8+ T cells induced by these vaccines play critical roles in the activation of antitumor immunity. Evidence of autoimmune reactions was not present in surviving mice that had rejected tumor cell challenges. We found that HSP105 was highly immunogenic in mice and that the HSP105 DNA vaccination induced antitumor immunity without causing autoimmunity. Therefore, HSP105 is an ideal tumor antigen that could be useful for immunotherapy or the prevention of various human tumors that overexpress HSP105, including colorectal cancer and melanoma.     

Phenotype analyses and cellular mechanisms of the pre-effector T-lymphocyte response to a progressive syngeneic murine sarcoma

Lymph nodes draining the progressively growing, weakly immunogenic, MCA 105 sarcoma contained tumor-sensitized but not fully functional pre-effector T-cells. These cells could further differentiate to acquire full antitumor effector function for adoptive therapy in an established in vitro sensitization (IVS) procedure. In this study, we utilized selective depletion with antibodies of lymphocyte subsets bearing the L3T4 (CD4) or Lyt-2 (CD8) antigen and of cells bearing the asialo-GM1 (ASGM-1) glycosphingolipid to identify the phenotype of pre-effector cells elicited during progressive tumor growth. Cells from lymph nodes draining a progressive MCA 105 tumor in the footpad were treated with antibodies plus complement prior to IVS. The antitumor efficacy of resulting IVS cells was assessed in adoptive therapy of 3-day established pulmonary MCA 105 metastases. Depletion of Lyt-2+ cells eliminated in vivo antitumor reactivity with concurrent elimination of in vitro cytotoxic activity against the MCA 105 tumor, whereas depletion of L3T4+ cells did not have an impact on either in vivo or in vitro antitumor reactivities. Treatment with ASGM-1 antiserum plus complement was also found to abrogate therapeutic efficacy. However, the in vitro cytotoxic activity was not affected. These results indicate that the pre-effector cells were Lyt-2+, L3T4-, and ASGM-1+. We next examined whether the sensitization of pre-effector cells in vivo required the participation of L3T4+ helper cells. To approach this, mice were depleted of L3T4+, Lyt-2+, or ASGM-1+ cells by antibody injections before tumor inoculation. Treatment with Lyt-2 monoclonal antibody abrogated the pre-effector cell response in the draining lymph nodes, as evidenced by failure to generate therapeutically effective cells following IVS. On the other hand, neither L3T4 nor ASGM-1 antibody treatment affected the generation of pre-effector cells. Thus, sensitization of Lyt-2+ pre-effector cells in response to progressive tumor occurred in the absence of L3T4+ helper cells.     

Lineage-specific chimaerism after stem cell transplantation in children following reduced intensity conditioning: potential predictive value of NK cell chimaerism for late graft rejection.

Chimaerism of FACS-sorted leucocyte subsets (CD14+, CD15+, CD3-/56+, CD3+/4+, CD3+/8+, CD19+) was monitored prospectively between days +14 and +100 in 39 children undergoing allogeneic stem cell transplantation with reduced intensity-conditioning regimens. Cell subsets exceeding 1% of nucleated cells were subject to cell sorting. Chimaerism was analysed by dual-colour FISH and/or by short tandem repeat-polymerase chain reaction. The chimaerism pattern on day +28 was evaluated with regard to its correlation with graft rejection. Of 39 patients, nine patients had donor chimaerism (DC) in all subsets. Mixed/recipient chimaerism (MC/RC) was detectable within T cells in 62%, within NK cells in 39% and within monocytes and granulocytes in 38% of the patients. The correlation of secondary graft rejection with the chimaerism pattern on day +28 revealed the strongest association between RC in NK-cells (P<0.0001), followed by T cells (P=0.001), and granulocytes and monocytes (P=0.034). Notably, patients with RC in T cells rejected their graft only if MC or RC was also present in the NK-cell subset. By contrast, none of the children with DC in NK cells experienced a graft rejection. These observations suggest that, in the presence of recipient T-cell chimaerism, the chimaerism status in NK-cells on day +28 might be able to identify patients at high risk for late graft rejection.     

非小细胞肺癌患者引流区淋巴结中CD4+CD25+调节性T细胞的检测及临床意义

目的分析非小细胞肺癌(NSCLC)患者引流区淋巴结中CD4 CD25 调节性T细胞在淋巴结局部免疫抑制状态的形成以及在肺癌发生发展中的作用。方法手术切除53例NSCLC患者引流区淋巴结,采用双标记的间接免疫荧光法检测CD4 CD25 调节性T细胞数量,实时荧光定量逆转录-聚合酶链反应(RT-PCR)法检测细胞因子TGF-β1、IL-10的表达水平,常规免疫组化方法检测CD8 T细胞的数量。结果NSCLC患者引流区转移淋巴结中,CD4 CD25 调节性T细胞(28.80%±8.06%)明显高于未转移淋巴结(15.48%±4.66%,P<0.01)。随肺癌的进展,引流区淋巴结中CD4 CD25 调节性T细胞数量增多。在转移的纵隔淋巴结(N2)和肺内淋巴结(N1)中,CD4 CD25 调节性T细胞数量分别为32.58%±7.52%和22.76%±4.67%(P<0.01)。在进展期(Ⅲ)和早期(Ⅰ Ⅱ)NSCLC患者引流区淋巴结中,CD4 CD25 调节性T细胞数量分别为30.42%±7.47%和16.22%±4.88%(P< 0.01)。NSCLC患者引流区淋巴结中的CD4 CD25 调节性T细胞数量与其自身的CD8 T细胞的数量呈负相关(r=-0.756,P<0.001)。在NSCLC患者引流区淋巴结中,CD4 CD25 调节性T细胞数量与抑制性细胞因子TGF-β1和IL-10的表达水平呈正相关(TGF-β1:r=0.645,P<0.001;IL-10:r=0.769,P<0.001)。结论NSGLC患者引流区淋巴结的CD4 CD25 调节性T细胞数量与肺癌的发展密切相关。一方面,检测肺癌患者引流区淋巴结的免疫状况为评价NSCLC患者疾病的进展程度和预后提供了一个新的免疫学指标;另一方面,在NSCLC的生物治疗中,控制CD4 CD25 调节性T细胞数量,阻断其发挥免疫抑制作用,具有广阔的临床应用前景。     

培美曲塞联合含铂方案化疗对非小细胞肺癌患者外周血淋巴细胞表型的影响

目的探讨培美曲塞（pemetrexed）联合顺铂（cisplatin）或卡铂（carboplatin）治疗方案对非小细胞肺癌患者外周血淋巴细胞表型的影响。方法45例经病理学或细胞学确诊的非小细胞肺癌患者,采用培美曲塞500 mg/m2,第1天静脉滴注;顺铂25 mg/m2,第1～3天或卡铂按AUC=5计算剂量,第2天静脉滴注,21天为一周期,毎例患者至少治疗2周期,有可测量病灶者进行疗效评价。应用流式细胞仪检测化疗前和化疗后第7天外周血CD3+、CD4+、CD8+、CD16+56+、CD19+细胞的百分率和CD4+/CD8+比值。结果非小细胞肺癌患者经培美曲塞联合含铂方案化疗后CD4+、CD4+/CD8+、CD16+56+较化疗前升高（P=0.032）,CD3+、CD8+、CD19+化疗前后差异无统计学意义（P>0.05）;临床分期为ⅢB～Ⅳ期的患者化疗前CD3+、CD4+、CD4+/CD8+和CD16+56+较ⅠB～ⅢA期患者低（P=0.029）,化疗后两者比较差异无统计学意义（P>0.05）;化疗有效者CD3+、CD4+、CD4+/CD8+比值均显著增高（P=0.008）。结论非小细胞肺癌患者,尤其是晚期患者机体免疫功能低下。培美曲塞联合含铂方案化疗打破了原有的抗肿瘤免疫抑制状态,机体有可能通过免疫重建增强抗肿瘤免疫应答。     

Activation and growth of murine tumor-specific T-cells which have in vivo activity with bryostatin 1.

We examined the ability of bryostatin 1 (Bryo), a novel protein kinase C activator, plus ionomycin (Io), a calcium ionophore, to activate T-cells with specific antitumor activity. Lymphocytes from the draining lymph nodes (DLN) of MCA-105 tumor-bearing host mice were stimulated with Bryo/Io, either fresh or after in vitro stimulation with autologous tumor, and then were incubated in interleukin-2 at 20 units/ml. Lymphocytes sensitized with tumor cells in vitro and then stimulated with Bryo/Io exhibited significant expansion (12-fold) after a total of 3 weeks in culture and moderate cytolytic activity (40% at an effector:tumor cell ratio of (80:1) and were exclusively CD8+ T-cells. DLN cells activated immediately with Bryo/Io, without tumor antigen sensitization in vitro, displayed marked growth (130-fold expansion) over 3 weeks in culture, had weak cytolytic activity (8% at an effector:tumor ratio of 80:1), and were a mixed population of CD8+ and CD4+ cells. Despite the differences in phenotypes and in cytotoxicity, both groups of DLN cells were highly effective in vivo against MCA-105 pulmonary metastases. Bryo/Io-activated DLN cells from MCA-105 tumor-bearing hosts had no therapeutic efficacy against B16 melanoma or MCA-203 sarcoma metastases. Lymph node cells from normal mice and non-draining lymph node cells from tumor-bearing hosts could be expanded with Bryo/Io to a degree similar to that of DLN cells but had no antitumor activity. Phenotypic analyses and in vitro and in vivo depletion studies demonstrate that CD8+ cells mediated tumor regression.