Angelo Nicolin

Clonal analysis of T lymphocytes isolated from ovarian carcinoma ascitic fluid. Phenotypic and functional characterization of T-cell clones capable of lysing autologous carcinoma cells

T lymphocytes isolated from ascitic fluid of patients with stage III–IV ovarian carcinoma were cloned by means of a microculture system that allows cloning of virtually all peripheral blood human T lymphocytes. Under these experimental conditions, 15% to 42% of ascitic T cells gave rise to clonal progenies that were analyzed for different functional capabilities. Of the clones obtained, 36%–70% had cytolytic activity in a PHA‐dependent assay (using P815 as target cells) that allowed detection of cytolytic cells of any specificity. About one‐half of the cytolytic clones lysed the NK‐sensitive K562 target cells as well. In addition, 30%–50% of the total clones released IL‐2 upon stimulation with PHA for 24 hr. In all patients analyzed a variable proportion (11%–56% of all cytolytic clones) had cytolytic activity against autologous tumor cells. Some of these clones have been analyzed in more detail: 18/21 expressed the T4−T8+phenotype, whereas the remaining 3 were T4+T8−. Only one out of 6 clones tested lysed allogeneic ovarian carcinoma cells as well, while 5/8 had a definite NK‐like activity. Finally, all 8 clones tested were inhibited by anti‐T11 and 7/8 by anti‐T8 monoclonal antibody.

Thymidine labelling index as prognostic factor in resected non-small cell lung cancer

To assess the prognostic value of tumor proliferative activity, 89 patients with operable non-small cell lung cancer were studied. Tumor samples were obtained during surgery and cell kinetics were analyzed by the in vitro thymidine labelling index (TLI). The overall median TLI (2.9) was used to identify two subsets of patients with high and low proliferating tumors. In univariate analysis survival was significantly longer in patients with lower TLI (P = 0.047) and with stage I-II (P = 0.003) and T1-T2 tumors (P = 0.043). In multivariate analysis, stage was the most important prognostic parameter (P = 0.004). The risk of death for patients with TLI higher than 2.9 was increased (hazard ratio = 2.01, CI = 0.96-4.27).

Generation and Characterization of a Low-Degree Drug-Resistant Human Tumor Cell Line

A 2780 human ovarian cancer cells, obtained from an untreated patient, have been exposed to a relatively low, clinically maintainable dose (10 nmol/l) of the anthracycline doxorubicin (DX) to derive a low-degree (5-fold) drug-resistant subline (A2780-DX1). Compared to parental cells, these DX-resistant cells have increased size (+60% of cell volume) and contain a greater number of cytoplasmic vacuoles as determined by electron microscopy. When exposed to several other antiproliferative drugs, A2780-DX1 cells were highly cross-resistant (greater than 10-fold) to epirubicin, mafosfamide and cisplatin and slightly cross-resistant (2- to 3-fold) to navelbine and bleomycin, while they retained the original sensitivity to vinblastine, Ara-C and fluorouracil. Gel electrophoresis of cytoplasmic membrane proteins showed differences between the pattern of parental A2780 sensitive and A2780-DX1 cells as far as low-molecular-weight proteins (less than 45 kD) are concerned, while no clear overexpression of P-glycoprotein (P-170) could be detected. Membrane modifications yielding a decrease of both DX uptake and retention, increased content of intracellular glutathione (+32%) and reduced DNA double-strand breaks seem to be involved in the resulting multidrug-resistant phenotype of A2780-DX1 cells.

Peripheral blood and tumor infiltrating lymphocytes in non-small cell lung cancer: analysis at the population and clonal level.

Tumor infiltrating (TIL) and peripheral blood lymphocytes (PBL) were isolated from 18 patients with non-small cell lung cancer undergoing radical surgery. Surface marker analysis revealed that TILs and PBLs mainly consisted of CD3+T cells and that TILs generally displayed a lower CD4/CD8 ratio. Differences were found in the expression of CD25 (IL-2 receptor) and DR (MHC class II) antigens, which were increased in TILs, and in the percentage of CD16+ natural killer (NK) cells, which was reduced in TILs as compared to PBLs. Accordingly, the NK activity of TILs was lower than that of PBLs, whereas neither TILs nor PBLs expressed spontaneous cytolytic activity against fresh autologous tumor cells, melanoma cells and the « NK-resistant » A549 lung carcinoma cell line. After 4 days of culture in medium with recombinant-inter-leukin-2 (rIL-2), TILs and PBLs acquired cytolytic activity against all cell targets, but TILs expressed higher levels of cytotoxicity than autologous PBLs only in 3 patients out of 16 tested. More importantly, both TILs and PBLs displayed similar levels of cytotoxic activity against autologous tumor cells. TILs and PBLs from 8 patients were also analyzed by a limiting dilution microculture system. Cloning efficiency was remarkably lower in TILs, and surface marker analysis of T cell clones confirmed that an accumulation of CD8+ lymphocytes, which displayed cytolytic activity in a lectin-dependent assay, occurred at the tumor site. The non-MHC-restricted cytolytic activity of TIL- and PBL-derived T cell clones against K562, A549, and allogeneic melanoma cells and the cytolytic activity against autologous tumor cells showed no significant differences. Only 53% of TIL clones released IL-2 in response to PHA+TPA stimulation, whereas 68% of PBL-derived clones were IL-2 producers. Moreover, most PBL- and TIL-derived clones released tumor necrosis factor alpha in response to mitogen stimulation.

Cyclosporin-A inhibits IL-2 production by all human T-cell clones having this function, independent of the T4/T8 phenotype or the coexpression of cytolytic activity

Cyclosporin A (CsA) is an immunosuppressive drug that acts, at least in part, by blocking IL-2 release. Since IL-2-producing human T cells are heterogeneous with respect to their functional capabilities and surface phenotype, we investigated whether differences in sensitivity to CsA existed among different IL-2-producing T-cell clones. Preliminary dose/response experiments showed that 100 ng/ml CsA completely inhibited the PHA- or OKT3-induced IL-2 production by four representative T4+/T8- clones. On the other hand, this drug concentration had virtually no inhibitory effect on the proliferation of CTL-L murine indicator cells to exogeneous IL-2. Clones were derived directly from peripheral blood by applying a microculture system that allows clonal expansion of essentially all T cells: under these experimental conditions growing clones are therefore highly representative of the starting T-cell populations. Among clones so derived, 28 were selected according to their capability to release IL-2 upon PHA stimulation. Six of such clones displayed cytolytic activity in a PHA-dependent assay against P815 murine target cells. CsA (100 ng/ml) abrogated IL-2 production of all clones, including those displaying cytolytic activity and expressing the T4-/T8+ phenotype.

Inhibition of cellular DNA synthesis and lack of antileukemic activity by non-photoactivated hematoporphyrin derivative.

It has been reported that cytocidal activity of light-activated hematoporphyrin (HPD) within the cells might be exploited in the therapy of experimental and human cancer. As part of a project from this laboratory aimed to study some major biologic features of HPD, it was found that [3H]thymidine incorporation in tumor cells was highly inhibited as a consequence of HPD treatment. HPD-mediated inhibition, obtained by a treatment either in vitro or in vivo, was long lasting and independent of light activation. Cellular DNA synthesis was inhibited by non toxic doses of HPD which were not influential either cell viability or cell oncogenicity. In preliminary studies, HPD-treated cells accumulated in the G1 phase of the cell cycle as detected by cytofluorometric analysis. This finding is in keeping with a likely inhibition exerted in late G, or at the beginning of the S phase of cell the cycle and might exclude a direct damage of the DNA synthetic machinery. Definitive loss of cell viability and cellular DNA inhibition was obtained immediately after the exposure of HPD-treated cells to He-Ne laser light. HPD-mediated cell lysis was dose dependent and in the order of magnitude of cytocidal doses in different cell systems. HPD antileukemic activity or HPD interactions with chemotherapeutic drugs was ruled out in L1210 leukemic mice.

Pharmacokinetics of zidovudine in HIV-positive patients with liver disease

The pharmacokinetics of zidovudine (ZDV) have been studied in eight AIDS patients with normal liver function, and in four AIDS patients with liver disease. Patients who were previously untreated with ZDV were given 250 mg ZDV, and plasma levels of ZDV and its glucuronic metabolite, GZDV, were determined at 0.5, 1, 1.5, 2, 3, and 4 hours after the dose. In patients with liver disease, Cmax and AUC of ZDV were higher, the oral clearance was only one‐eighth that of patients without liver disease, and the elimination half‐life was longer. There was a trend for concentrations of the principal metabolite, GZDV, to be lower in patients, and, therefore, the ratio of the AUC for GZDV to that for ZDV was much lower in patients with liver disease. Therefore, HIV‐seropositive patients with liver disease had the same markedly altered disposition of ZDV as seronegative patients with liver disease. Although this therapy was not clearly associated with a higher incidence of toxicity, some patients with liver disease had to discontinue therapy because of intolerance; therefore, plasma levels of these patients should be monitored when such therapy is undertaken.

Phenotypic and functional characterization of T cell clones following allogeneic bone marrow transplantation.

T cell clones (n = 456) were derived from 9 patients following allogeneic bone marrow transplantation (BMT) with or without acute graft versus host disease (aGVHD) and from 4 healthy donors. The cloning efficiency was 63.2% in controls, 13.2% and 12.1% in patients with or without aGVHD. Once established, T cell clones were typed for surface markers (CD3, CD4, CD8) and tested for production of IL-2 and expression of cytolytic activities in a lectin-dependent cellular cytotoxicity assay (LDCC) and against the K562 target cell line to detect natural killer activity. We found the expected imbalance of CD4/CD8 clones in BMT patients, as compared to controls. A higher proportion of IL-2-producing clones was observed in patients with aGVHD (83.5%; P less than 0.02) as compared to patients without aGVHD (64.8%) and controls (68.5%). No major differences were found in terms of LDCC, whereas an increased percentage of clones with NK-like activity was found in patients with aGVHD (34.7%, P less than 0.05) as compared to patients without aGVHD (29.5%) and controls (21.3%). The clones were also tested for inhibition of IL-2 production mediated by cyclosporine. Such inhibition could be obtained in virtually all clones both in patients with or without aGVHD, suggesting that the latter is probably not due to the emergence of CsA-resistant clones. In conclusion, this study demonstrates a low cloning efficiency in BMT patients associated with the well-known CD4/CD8 imbalance. A higher production of IL-2, an increased NK activity, but not the presence of CsA-resistant clones appear to differentiate patients with from patients without aGVHD.

Timed sequential chemotherapy following drug-induced kinetic recruitment in refractory ovarian cancer

Kinetic recruitment of cancer cells can seldom be monitored in human solid tumors. Repeated tumor sampling in ascitic ovarian cancer has been exploited to study tumor cell kinetic recruitment following treatment with the alkylating agent iphosphamide (IFX). The treatment schedule of the study was designed to administer the antimetabolic agents MTX-5FU at the time of the recruitment peak. Kinetic studies by the labelling index (LI) assay could be performed during and after the IFX treatment in four out of eight patients because of sampling difficulties. Experimental results showed that the IFX effectiveness reduced the proliferating cells, followed by cell kinetic recruitment from the resting pool. The antimetabolic treatment in concomitance with the proliferative peak (day 12) has been reduced with respect to the original schedule due to the heavy leukopenia occurring to the patients. It is likely that the reduced drug dosage might have contributed to the poor clinical response. However, the recruited cells exhibited an increased in vitro chemosensitivity to adriamycin in comparison to tumor cells studied before the IFX treatment.

In vitro hematoporphyrin (HPD) inhibitory effects on some immunological assays

Hematoporphyrin derivative (Hpd) is a fluorescent dye that is preferentially incorporated by tissues with a high mitotic index, such as tumor cells and blast cells. A cytotoxic effect is produced following light activation. Previous studies have shown a long lasting reversible inhibition of DNA synthesis in Hpd-treated cells that failed to stimulate allogeneic lymphocytes in either primary or in secondary MLR. In this study we report Hpd inhibitory effects on some immunological assays in vitro. Treatment with Hpd of cytotoxic effector cells resulted in inhibition of their lytic activity likely dependent on the loss of binding to target cells. In the same way Hpd treatment inactivated the lytic activity of NK cells. In contrast Hpd-treatment of target cells did not modify the above immunological reactions. Moreover Con A agglutinability, antibody dependent capping as well as E-rosettes were inhibited following an Hpd treatment of relevant cells. Since normal susceptibility to humoral and cell mediated lysis was exhibited by Hpd-treated cells it is unlikely that cell surface molecules were damaged. An inhibitory effect exerted by an Hpd treatment on cell surface movements might explain these findings.