A TaqI RFLP at the gas-2 locus.
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Animals↗
Biomedical subjects
G Parmiani
Publications and source records attributed to G Parmiani.
Differences between in vivo and in vitro activation of cancer patient lymphocytes by recombinant interleukin 2: possible role for lymphokine-activated killer cell infusion in the in vivo-induced activation.
In this study 15 consecutive melanoma patients were treated with two courses of bolus recombinant interleukin 2 (rIL2) and rIL2 plus in vitro-generated lymphokine-activated killers (LAK), respectively. The immunological monitoring performed after 4 days of rIL2 or rIL2 plus LAK, indicate that the in vivo peripheral blood lymphocyte (PBL), activation (spontaneous proliferation, tumor cytotoxicity, number of DR+ PBL, obtained after the second cycle of rIL2 plus LAK is significantly higher than after the first cycle of rIL2 alone. During the 5-day interval between the two courses, PBL activation returns to baseline levels and no evidence for increased sensitivity of PBL to rIL2 is present. To further confirm this, two additional patients were studied, in whom rIL2 was administered by continuous i.v. infusion. In these two patients the in vitro versus in vivo PBL activation could be directly and simultaneously compared by using in vitro the same concentration of rIL2 reached and maintained in the patients' sera. The PBL activation induced in vivo by a cycle of rIL2 alone was significantly less (about 10 times) than that obtained in vitro with a comparable rIL2 concentration. Thus, the infusion of in vitro highly activated PBL could explain the increased in vivo lymphocyte activation of the second cycle of rIL2 plus LAK over the first cycle of rIL2 alone.
Cell Line↗
Loss of heterozygosity of DQ alpha gene in human malignant melanoma.
DNAs from human melanoma cells, used at early in vitro passage, were evaluated by Southern blot analysis for somatic loss of heterozygosity at the DQ alpha gene. A total of 7 melanomas, 3 primary and 4 metastatic derived from 5 different patients were studied; in one case (pt 665) cell lines were derived from two anatomically different subcutaneous metastasis, whereas in a second case (pt 9923) both the primary tumor and a lymph node metastasis were available. Restriction length polymorphism (RFLP) analysis, performed on autologous peripheral blood lymphocyte (PBL) DNA digested with different enzymes, showed a pattern of bands compatible with the constitutional heterozygous typing at DQ alpha gene in 4 cases whereas 1 case revealed an homozygous typing. When melanoma DNAs were analysed, 1 out of the 4 informative cases (pt 1007) showed a loss of a diagnostic fragment for DQ alpha gene when digested with both Taq I and Bgl I enzymes. These results indicate that class II allelic losses detectable by RFLP can be found on malignant melanoma and add further complexity on the involvement of chromosome 6 whose cytogenetic abnormality are the most consistent in this human neoplasia.
DNA, Neoplasm↗
A phase II study of the administration of recombinant interleukin 2 (rIL-2) plus lymphokine activated killer (LAK) cells in stage IV melanoma patients.
From January 1987 to February 1988, 15 stage IV melanoma patients were treated with two courses of bolus injection of rIL-2 plus LAK cell infusions at the National Cancer Institute of Milan. The original treatment regimen included a first course of rIL-2 administration (400 micrograms/m2 bolus injection 3 times a day [TID] for 4 days) and a second course of rIL-2 administration (800 micrograms/m2 bolus injection TID for 7 days) separated by 4 consecutive daily leukaphereses. Autologous lymphokine activated killer (LAK) cells were reinfused into each patient on three occasions during the second period of rIL-2 administration. Due to the appearance of grade III-IV neurological, hepatic and cardiopulmonary toxicity, 7 patients discontinued dosing before the end of treatment, one patient desired to be withdrawn and one patient died from rapidly progressive disease, although complications of rIL-2 administration may have contributed to her death. Only 6 patients completed the schedule without evidence of major intolerance, even though the planned dose during the second course of rIL-2 was reduced to 400 micrograms/m2. The complete duration of treatment ranged from 11 to 19 days. The total dose of rIL-2 injected ranged from 12.6 to 30.4 mg. The number of infused LAK cells ranged from 15.5 x 10(9) to 60 x 10(9)/patient. Two of the 14 evaluable patients showed a minor anti-tumor response. In 5 patients new metastases in other sites were documented from 2 to 5 months after completion of dosing. No apparent association was found between progression of the disease (or the appearance of new metastases) and the total dose of rIL-2 injected, the number of LAK cells administered or the number of days of treatment. By December 1988, all patients had died of their disease in a period ranging from 3 to 14 months from the last injection of rIL-2. The lack of significant clinical responses in this study and the high toxicity of this treatment lead us to conclude that at least as far as melanoma patients are concerned, adoptive immunotherapy with rIL-2 plus LAK cells (as described here) is not a justifiable treatment option unless new evidence presents itself.
Adult↗
Generation and partial characterization of melanoma sublines resistant to lymphokine activated killer (LAK) cells. Relevance to doxorubicin resistance.
To see whether a tumor cell population may contain cells resistant to lymphokine-activated killer (LAK) lymphocytes, cells from a LAK-sensitive melanoma line (Me 665/2) were co-cultured with LAKs. Three sublines were obtained after 1, 2 or 3 immunoselection cycles. Immunoselected (IS) sublines show reduced proliferation, decreased reactivity to the monoclonal antibody (MAb) R24 and appeared morphologically more differentiated in comparison with the parental Me 665/2 line. A progressively reduced sensitivity to LAKs was observed in IS sublines with a more than 8-fold reduction in LAK susceptibility. A reduced complement (C)-mediated lysis was also observed in IS sublines. Since we have previously shown that LAK sensitivity of melanoma cells may be associated with Doxorubicin (Dx) resistance, the sensitivity to Dx was tested in these lines. An augmented sensitivity to Dx was noted in IS sublines as compared with Me 665/2. The differences in LAK susceptibility between the IS sublines and the parental Me 665/2 line remained stable for 2 weeks but declined and disappeared thereafter. These results indicate that (1) a LAK-sensitive tumor line may contain a subpopulation of cells which are significantly less lysed by LAKs; (2) a correlation between LAK sensitivity and susceptibility to C-mediated lysis is also present; and (3) increased sensitivity to Dx is evident in the IS sublines.
Antigens, Neoplasm↗
Cytotoxic T lymphocyte clones from peripheral blood and from tumor site detect intratumor heterogeneity of melanoma cells. Analysis of specificity and mechanisms of interaction.
CTL clones isolated from PBL or from tumor-infiltrating lymphocytes (TIL) of a melanoma patient (pt665) were screened for specificity on a panel including autologous tumor cells from two distinct metastases (Me665/1, Me665/2), autologous EBV-transformed B cells and 15 allogeneic cell lines of different histology. Each clone displayed a peculiar cytolytic activity ranging from lysis of most targets (PBL clone 4C4) to preferential reactivity on the two autologous metastases (TIL clone 8B3). Blocking and modulation experiments, revealed that the lysis of autologous-Tu cells by TIL clone 8B3, but not by PBL clone 4C4, could be inhibited by mAb to HLA-class I and to CD3 Ag or by CD3 complex modulation. Clone 8B3 was tested also on a panel of 25 tumor clones from Me665/2, revealing that only 4 neoplastic clones were lysed (2/4, 2/14, 2/17, and 2/51). Cold target competition experiments indicated that the uncloned autologous melanomas and one tumor clone (2/17), but no two other tumor clones (2/10, 2/15), could compete with one another for lysis by 8B3. Determination of melanin content of tumor clones from Me665/2 revealed that the four neoplastic clones recognized by 8B3 possessed much lower melanin levels than all the other 20 clones not lysed by this effector.
Antigens, Differentiation, T-Lymphocyte↗
Expression of retrovirus-related, cytotoxic T lymphocyte- and transplantation-defined antigens in NIH/3T3 transfectants after a single passage in nude mice.
Transfection of T24c-Ha-ras oncogene into NIH/3T3 fibroblasts resulted in the establishment of a transformed cell line (pT) that was tumorigenic when injected s.c. both into Swiss outbred nude mice and normal NIH inbred mice. The passage into nude mice, however, led to the development of a tumor variant (pT-nude) able to subsequently grow into sublethally x-irradiated but not into immunocompetent NIH mice. NIH mice immunized with this tumor variant developed a strong specific CTL response against the immunizing cell line, whereas the parental transformed pT cell line was not lysed. Clones were derived by limiting dilution from anti-pT-nude bulk population and were tested on a panel of transformed NIH/3T3 lines before and after their growth as tumor into nude mice. All of these lines were lysed by the Lyt-2+ CTL clones as a sole consequence of one in vivo passage into nude mice. The cross-reactive Ag were shown to be related to endogenous retroviral products as assessed by 1) immunoprecipitations of gp70, p15E, and p30 viral proteins in the nude variants but not in parental lines, and 2) by the ability of retroviruses from irradiated pT-nude cells to infect NIH/3T3 or pT lines making them susceptible to lysis by anti-pT-nude CTL clones. These results show that a single passage in nude mice can induce retrovirus-related, cell-surface Ag in transplanted neoplastic cells.
Animals↗
Osteonectin transcript and metastatic behavior in v-Ki-ras transformed fibroblasts.
Osteonectin is one of the major non-collagenous proteins of bone. However, its transcript has been found in many soft, extracellular matrix-producing tissues; an osteonectin-related protein was detected in tumor basement membrane. We have investigated the expression of osteonectin gene in fresh BALB/c fibroblasts transformed by v-Ki-ras. Transformed cells exhibited lower levels of RNA as compared with normal fibroblasts. The transformed cells were cloned after in vivo tumorigenic assay, and 4 clones were analyzed for osteonectin expression by Northern blots. Two of them were selected for high or low osteonectin expression and tested in vivo in spontaneous and artificial metastasis assays. High osteonectin expression was correlated with high lung colonization. When 10(5) cells were injected i.v., median colony value was 55 and 20 in higher expressor vs. lower expressor respectively (p less than 0.005). Spontaneous metastasis indicates a possible reverse correlation. Our data align osteonectin with other matrix-components and adhesion molecules in affecting potential metastatic spreading of transformed cells.
Animals↗
Influence of the donors' clinical status on in vitro and in vivo tumor-cytotoxic activation of interleukin-2-exposed lymphocytes and their circulation in different organs.
In-vitro-generated lymphokine-activated killer (LAK) cells of BALB/c mice, bearing the syngeneic colon carcinoma C-26 for 7 days, were as efficient as those from normal mice in lysing C-26 cells whereas LAK cells from 14-day tumor-bearing and 5- and 14-day tumor-resected animals had a lower C-26 cytotoxicity. The level of C-26 lysis returned to normal values 30 days after surgery. To identify the best source of LAK cells in vivo, groups of normal mice were treated with 10(4), 3 x 10(4) or 10(5) U/day of interleukin 2 (EL-2) for 7 days intraperitoneally (i.p.) or intravenously (i.v.) (3 x 10(4) dose only). The highest lysis on C-26 was obtained from peritoneal exudate cells of mice given 3 x 10(4) and 10(5) U whereas spleen cells were lytic only when taken from mice treated with 10(5) U IL-2. Peripheral blood lymphocytes lacked any cytotoxicity except for the group of mice which received IL-2 i.v. The kinetics of in vivo LAK activation in different organs showed a peak of anti-(C-26) lytic activity at day 5 in peritoneal exudate cells and spleen cells of mice given IL-2 for 5 days whereas administration of LAK cells alone had no effect: IL-2 plus LAK cells gave a lower peak of LAK activity as compared with IL-2 alone. A lower level of in vivo LAK activation was found in mice whose tumor was resected 5 days before; such impairment was evident even 14 days after surgery. Homing experiments were carried out with i.v. injected 51Cr-labelled LAK cells in normal or tumor-resected mice. In normal mice the highest radioactivity at 30 min was found in the lungs; liver and spleen also showed high radioactivity whereas blood had a negligible amount of radioactivity. Radioactivity declined rapidly in lungs (less than 10% after 24 h) while remaining at appreciable levels in the liver after 24 h and 48 h; spleen showed constant levels of 12%-15%. Homing of LAK cells was altered in mice receiving IL-2 i.p. for 5 days with slower and lower radioactivity peaks in the lung and higher levels in liver. In tumor-excised mice lower levels of radioactivity were found in lungs. These results show that: (a) alterations in LAK activity occur in early-tumor-resected and large-tumor-bearing animals; (b) the route of IL-2 administration is critical in LAK activation in vivo; (c) treatment with IL-2 modifies LAK homing.
Animals↗
Lysis by interleukin 2-stimulated tumor-infiltrating lymphocytes of autologous and allogeneic tumor target cells.
Peripheral blood lymphocytes (PBL) and tumor-infiltrating lymphocytes (TIL) were isolated from six cancer patients and cultured in the presence of 100 units/ml of recombinant interleukin 2 (IL2). Both IL2-stimulated PBL (IL2-PBL) and IL2-stimulated TIL (IL2-TIL) lysed fresh and short-term cultured autologous tumor cells in four and six cases, respectively. In four out of six patients IL2-TIL showed a slightly higher tumor cytotoxicity than IL2-PBL without lysing autologous normal PBL or TIL. Like IL2-PBL, IL2-TIL also killed allogeneic fresh and cultured targets of different histotypes, suggesting a lack of autologous tumor cytotoxic specificity. TIL cultured for 3 weeks in IL2 maintained their killing activity against autologous and allogeneic tumor targets. Phenotypic analysis of uncultured TIL showed a predominance of CD3+ T cells (approximately 70%) with CD4+ (approximately 60%) and CD8+ (20%) lymphocyte subsets, whereas less than or equal to 3% of CD16+ natural killer cells were present. TIL but not PBL contained 12%-19% of lymphocytes which expressed activation markers such as DR and TAC. The culture of both TIL and PBL in IL2 for 2-3 weeks induced an increase in the percentage of CD8+ and a decrease in CD4+ and augmentation of Leu 19+, DR+, and TAC+ cells. These results indicate that IL2-TIL can lyse autologous tumor cells slightly better than IL2-PBL, although such an effect was also evident against allogeneic neoplastic targets.
Cell Line↗
Cancer patients' lymphocytes contain CD3+ CD4+ cells that proliferate in response to autologous tumor cells in the presence of exogenous low-dose interleukin-2 and autologous accessory cells.
To see whether cancer patients possess CD3+ CD4+ lymphocytes able to proliferate in response to autologous tumor cells (Auto-Tu), this lymphocyte subset was isolated either by positive or negative selection, both methods resulting in highly enriched CD4+ populations. Unseparated and isolated CD3+ CD4+ lymphocytes were then assayed for proliferating activity in the presence or absence of various amounts of Auto-Tu, with or without recombinant interleukin-2 (IL-2) (1.5-15 U/ml) and DR+ adherent cells or E- lymphocytes as autologous accessory cells (Auto-AC). Isolated CD3+ CD4+ lymphocytes were stimulated by Auto-Tu alone in only 1 out of 12 cases. CD3+ CD4+ cells failed to proliferate significantly in response to low doses of IL-2 alone but the addition of Auto-Tu caused stimulation in 8 out of 12 cases (67%). The further addition of Auto-AC to Auto-Tu + IL-2 resulted in enhanced response of isolated CD3+ CD4+ lymphocytes in 6 out of 8 cases tested. When reactivities to Auto-Tu in the presence of IL-2 and IL-2 + Auto-AC were considered together, positive responses of CD3+ CD4+ lymphocytes were seen in 11 out of 12 cases (92%). On the other hand, unseparated lymphocytes were stimulated by Auto-Tu alone in none out of 12 cases. Unseparated lymphocytes, however, responded to IL-2 in 11 out of 12 cases; such a response was increased by the addition of Auto-Tu in only 2 cases. Moreover, the IL-2 proliferation of unseparated lymphocytes was suppressed in 4 and in 3 out of 12 cases tested when Auto-Tu or Auto-Tu + Auto-AC were added respectively. These data indicate that lymphocytes of cancer patients contain CD3+ CD4+ cells that are usually unable to proliferate in response to Auto-Tu only. This proliferation, however, occurs when low doses of exogenous IL-2 are present and can be further amplified by the addition of Auto-AC. No response of CD4+ cells is observed in the presence of DR+ Auto-AC + IL-2 except in 2 out of 7 cases tested (28%), suggesting an Auto-Tu-restricted reactivity of CD3+ CD4+ lymphocytes in the majority of cases.
Antigen-Presenting Cells↗
P-glycoprotein gene amplification and expression in multidrug-resistant murine P388 and B16 cell lines.
P-glycoprotein gene (mdrl) amplification and expression were examined in murine leukaemia P388/DX and melanoma B16VDXR cell lines, which exhibit a high level of resistance to a selecting agent, doxorubicin, and express a multidrug-resistant phenotype because they are cross-resistant to multiple cytotoxic drugs. The multidrug-resistant phenotype was obtained in different conditions of selection (in vivo and in vitro for P388/DX and B16VDXR, respectively). In both multidrug-resistant cell lines, an increased expression of P-glycoprotein gene (5 kb transcript detected in Northern blots) was observed and the level of P-glycoprotein mRNA correlated with the degree of resistance. In addition, high molecular weight mRNAs homologous to mdrl gene sequence were consistently detected only in P388/DX cells. Overexpression was associated with a high level of gene amplification only in resistant melanoma cells, whereas it occurred in P388/DX cells with a marginal increase in gene copy number. These results, suggesting that different genetic mechanisms could be responsible for P-glycoprotein overexpression, emphasise the complexity of genetic regulation that may affect tumour cell sensitivity to cytotoxic agents.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
In vitro lymphocyte response to autologous melanoma: clues in designing new adoptive immunotherapy protocols.
Recent studies indicate that different types of antitumour lymphocytes (T and non-T) can be generated in vitro and in vivo after exposure to recombinant interleukin-2 (rIL-2). In vitro it is possible to selectively expand the CD3 cells by using anti-CD3 monoclonal antibody and rIL-2. Adherence can select a minor subpopulation of lymphokine-activated killer (LAK) cells (A-LAK) endowed with a higher lytic activity. We have investigated whether melanoma-specific T-lymphocytes can be obtained to be used for therapy or endogenously expanded in vivo by the appropriate use of limited amounts of rIL-2. Clonal analysis of lymphocytes obtained either from tumour lesions or from blood indicated the existence of melanoma-restricted T-lymphocytes, both with cytotoxic or helper function. Though a minority, these cells can be generated and expanded with low amounts of rIL-2 (3-30 Cetus U/ml) in the presence of autologous melanoma and B-cells. The use of rIL-1 and rIL-4, together with rIL-2 at certain phases of T-lymphocyte growth, can help in expanding more selectively the melanoma-specific cells. These T-cells are major histocompatibility complex-restricted in their killing but they also need to see adhesion molecules (ICAM-1) on the target cells. New clinical protocols can thus be conceived on the basis of some of these results.
Humans↗
Potentiation of adoptive immunotherapy by cis-diamminedichloroplatinum(II), but not by doxorubicin, on a disseminated mouse lymphoma and its association with reduction of tumor burden.
The therapeutic effect of a combination therapy employing syngeneic anti-tumor immune T lymphocytes and either doxorubicin (DX) or cis-diamminedichloroplatinum II (DDP) was tested on BALB/c mice bearing a disseminated, weakly immunogenic lymphoma (YC8) which metastasizes mainly to the liver. Therapy with specific tumor-immune lymphocytes alone cure 80-100% mice bearing 3-day established tumors. The same effector cells, however, were less effective in mice at a more advanced stage of the disease (day 5) and ineffective when treatment was further delayed (day 7). Chemotherapeutic treatment alone with DX or DDP given i.p. at the maximal tolerated doses was less effective than immune lymphocytes on 3-day tumors since cures were very seldom observed, but both drugs prolonged survival time even when administered on day 7. While DX did not enhance the antitumor effect of immune lymphocytes on 5-day tumors, the association of DDP with immunotherapy by giving the 2 modalities sequentially according to the two sequences (i.e., chemotherapy before immunotherapy and vice versa) with a 2-day interval, was significantly more effective than each treatment alone. Like mice cured by immunotherapy alone, most of the animals cured by chemo-immunotherapy developed a systemic transplantation immunity to the tumor as revealed by the specific rejection of a second lethal tumor challenge 90 days after the first tumor inoculum. The higher activity of DDP compared with DX in potentiating the effect of immunotherapy was shown to be associated with a greater reduction of tumor burden in the liver, whereas the 2 drugs gave a similar reduction of tumor burden in other organs, as determined from bioassay of tumor-bearing organs of chemotherapy-treated mice. These results indicate that combination of chemotherapy with immunotherapy may improve the effects of each treatment alone, and that the synergistic effect is associated with the reduction of the tumor load in the main organ (liver) of tumor dissemination.
Animals↗
A new procedure for large scale production and freezing of lymphokine activated killer (LAK) cells to be used in adoptive immunotherapy of cancer.
A new procedure for activation of peripheral blood lymphocytes (PBL) with recombinant interleukin 2 (rIL2) is described. PBL obtained by leukapheresis were subjected to NH4Cl (ACK) treatment to clear erythrocyte contamination; Ficoll separation was not performed. PBL were subsequently seeded in 10-floor multitrays (Cell FactoryTM, CF), gasified and incubated at 37 degrees C for 3-4 days in a humidified 5% CO2 atmosphere. This procedure achieved an activation (evaluated as cytotoxicity and proliferation) comparable with that obtained by culturing PBL in small flasks. Optimal activation of PBL was achieved in CF even in the presence of granulocyte contamination of up to 40%. It was also possible to freeze, thaw and recover most of the frozen cells and their cytotoxic activity. With this procedure therefore large quantities of lymphokine activated killer cells (LAK) can be easily produced to be used in adoptive immunotherapy trials.
Blood Preservation↗
Differential lysis of melanoma clones by autologous recombinant interleukin 2-activated lymphocytes. Relationship with spontaneous resistance to doxorubicin (Dx).
To investigate whether human melanoma cells intrinsically resistant to autologous LAKs do exist, and whether a relationship between the level of lysis of LAKs and spontaneous drug resistance can be identified at the clonal level, we studied 44 clones obtained from a metastatic melanoma lesion. The antigenic phenotype of clones revealed a marked heterogeneity in the expression of HLA antigens of classes I and II. The clones were subsequently tested for sensitivity to autologous LAK and for spontaneous resistance to Dx. No clone resistant to autologous LAK was found, although a considerable range of lysis was noted with a normal frequency distribution. Growth in agar of the 2 clones in which lysis was least pronounced (6 and 26) was completely inhibited after co-culture with LAKs, indicating a lack of absolute resistance to these effectors. Spontaneous resistance to Dx, evaluated as ID50, revealed instead that the majority of clones had a low ID50. The frequency distribution of clones showed a left-skewed curve. The percentage of specific 51Cr-release and the ID50 for Dx could be correlated in 25 clones by linear regression. Sensitivity to LAK did not correlate with HLA classes I or II or melanoma-associated antigen expression. These results support the contention that increased LAK sensitivity of tumor cells is associated with drug resistance.
Antigens, Neoplasm↗
Strain polymorphism and tentative mapping of mouse ornithine decarboxylase.
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Animals↗
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.
Animals↗