Application of a new compact and cell dense continuous culture system for LAK cells generated from cryo-preserved lymphocytes

Recently, the adoptive immunotherapy using lymphokine activated killer (LAK) cells has been applied for various cancer patients. It is quite useful to generate LAK cells from cryo-preserved lymphocytes in order to make a rational treatment schedule. We made basic studies on the induction of LAK cells from frozen lymphocytes using our high yield continuous cell culture system. It was demonstrated that our culture bag can be used to proliferate frozen lymphocytes cultured in interleukin-2 (IL-2) to thirty four-fold the initial cell number in 14 days with comparable cytotoxicity to that of fresh lymphocytes cultured in the same system.     

Gamma-interferon enhances the cytotoxic activity of interleukin-2-induced peripheral blood lymphocyte (LAK) cells, tumor infiltrating lymphocytes (TIL), and effusion associated lymphocytes.

The effect of gamma-interferon (IFN-gamma) on the induction of interleukin-2 (IL-2) activated killer cell activity was studied: (I) in peripheral blood lymphocytes (LAK cells) from cancer patients and healthy donors, (II) in lymphocytes infiltrating solid tumors (TIL) from melanoma and breast cancer patients, and (III) in pleural effusion associated lymphocytes (EAL) from patients with lung adenocarcinoma. The coculture of LAK, TIL and pleural effusion mononuclear cells (MNC) with several doses of IFN-gamma (10, 50, 250, and 1250 U/ml) and a low dose of IL-2 (10 U/ml) for 5 days resulted in a synergistic effect on the cytotoxicity of these cells against several tumor cell lines. Furthermore there was a potentiation in the proliferation of MNC after a 5-day culture. The induction of lymphocyte cytotoxicity by a combination of IFN-gamma with low doses of IL-2 may be helpful in designing more effective cancer immunotherapeutic protocols with LAK, TIL or EAL.     

Activation and phenotyping of LAK generated by exposure to product of cells transformed by interleukin 2 cDNA.

It has been previously found that local administration of X63-m-IL-2 cells transformed by interleukin 2 (IL-2) cDNA and constitutively producing large quantities of IL-2 mediated regressions of murine plasmacytomas and 3-methyl-cholanthrene-induced sarcomas transplanted in syngeneic mice. Here we show that killer cells generated by cultivation of spleen cells in supernatants from X63-m-IL-2 cultures (LAK) or by co-cultivation of murine splenocytes with X63-m-IL-2 cells were cytolytic for natural killer (NK)-sensitive as well as NK-resistant target cells, including the IL-2-producing X63-m-IL-2 cells. Spleen cells cultured in X63-m-IL-2 supernatants or co-cultivated with X63-m-IL-2 cells yielded predominantly Thy 1.2+, CD3+, LFA-1+ lymphocytes. The in vitro results suggest that the LAK cells generated due to the IL-2 production by genetically engineered cells probably help to terminate the treatment by killing the IL-2 producers.     

Expression of an accessory cell phenotype by hairy cells during lymphocyte colony formation in agar culture

Human T lymphocytes require the cooperation of accessory cells to generate lymphocyte colonies in agar culture under PHA stimulation. Various hairy cell enriched fractions, as well as normal monocytes, have been found to be able to initiate colony formation by normal lymphocytes. Leukemic monocytes from CMML patients were also effective, but not the leukemic lymphocytes from CLL patients. The phenotype expressed by HC in agar colonies was further studied using cell surface and enzymatic markers. We have concluded that HC in agar culture in the presence of both normal T lymphocytes and PHA lose the B phenotype that they express in vivo and function like an accessory cell in contrast to normal or leukemic B lymphocytes.     

Cytotoxicity of interleukin 2-induced lymphokine-activated killer (LAK) cells against human leukemia and augmentation of killing by interferons and tumor necrosis factor.

Adoptive immunotherapy with interleukin 2-induced lymphokine-activated killer (LAK) cells or tumor-infiltrating lymphocytes (TIL) and the induction of anti-tumor responses by IL-2 alone having proven to be promising approaches in cancer therapy. The present study investigated the cytotoxicity of LAK cells towards human leukemia cells. LAK cells were generated by culturing peripheral blood mononuclear cells from normal donors for six days in the presence of recombinant interleukin 2 (IL-2). Cytotoxicity was evaluated using a standard 4-h chromium-release assay. A significant lysis of fresh uncultured leukemia cells by IL-2-activated killer cells could be detected in 77 of 150 leukemias examined. The mean Cr-release was 35.7 +/- 12.9% in the LAK cell-sensitive vs 9.9 +/- 5.9% in the resistant leukemias. With a view to the therapeutic utilization of the LAK-cell system, we attempted to improve the efficiency of its cytotoxic mechanisms. Combined application of IL-2 and interferon-alpha, interferon-gamma, or tumor necrosis factor-alpha in cultures for generation of activated killer cells significantly improved the effectiveness of cytotoxic mechanisms. Our results suggest that the performance of adoptive immunotherapy with ex vivo-activated LAK cells and the in vivo induction of cytotoxic immune responses by IL-2 alone or combined with different lymphokines or cytokines may be of value in treating human leukemia, especially when the tumor burden is low, e.g. during maintenance therapy or after bone marrow transplantation to eliminate minimal residual disease or in early relapse.     

The protein-kinase-C inhibitor h7 blocks normal human lymphocyte stimulation and induces apoptosis of both normal lymphocytes and leukemia molt-4 cells

The isoquinoline sulfonamide (H7) is an inhibitor of protein kinase C (PKC) that also inhibits the activity of cyclic nucleotide-dependent protein kinases. The effect of H7 on mitogen stimulation (G0 to G1 transition) of normal human lymphocytes and on their subsequent progression through the cell cycle was investigated and compared with the effect of this inhibitor on proliferation of human lymphocytic leukemic MOLT-4 cells. At H7 concentrations of 10 and 50 muM, the transition of G0 lymphocytes to the cell cycle was suppressed by 45 and 98%, respectively. The cell cycle progression of stimulated lymphocytes was unaffected at 10 muM H7, whereas, at 50 muM, the overall rate of progression was reduced by 50% with no evidence of cell arrest at a specific phase of the cycle. Similar concentrations of H7 (45 muM) suppressed proliferation of MOLT-4 cells by 50%, though, in the latter case, cells underwent transition to higher DNA ploidy, most likely via endoreduplication. Thus, the G0 to G1 transition appears to be the event most sensitive to H7. Exposure of MOLT-4 cells to 100 muM H7 for 24 h induced extensive apoptosis: activation of an endogenous nuclease with preference to internucleosomal linker DNA sections resulted in DNA degradation (revealed by agarose gel electrophoresis and loss of DNA measured by flow cytometry), which was paralleled by intracellular proteolysis, while the integrity of the plasma membrane, mitochondria and lysosomes was preserved. Morphological examination of these apoptotic cells confirmed DNA degradation. However, the perinuclear and fine-granular localization of the remaining DNA and lack of typical chromatin condensation and nuclear fragmentation differed from the classical pattern of apoptosis observed in other cell systems, suggesting that some events of apoptosis (nuclear fragmentation) may be affected by H7. The observed effects are consistent with the possible role of H7 in inhibition of PKC or its direct effect on the ATP-binding domain of DNA topoisomerase II, which shares homology with the H7 binding sites on PKC and the cyclic nucleotide-dependent protein kinases.     

Induction of cytotoxic activity in human lymphocytes against autologous and allogeneic melanoma cells in vitro by culture with interleukin 2

The influence of interleukin 2 (IL2) on the cytotoxic activity of lymphocytes from patients with melanoma against autologous and a variety of allogeneic melanoma cells was studied. IL2 was produced from blood lymphocytes cultured for 24 h with phytohaemagglutinin (PHA) and purified by membrane chromatography to exclude PHA. Lymphocytes from 13 patients with melanoma at various clinical stages were cultured for 6 days with IL2 (2 U/ml) and then tested for cytotoxic activity against autologous melanoma cells, three allogeneic melanoma and three non-melanoma cells. Autologous cytotoxicity was generated by culture with IL2 alone and was not increased by culture with both IL2 and autologous tumour cells. Marked increases in cytotoxic activity were also generated against the allogeneic target cells and were maximal against the NK-insensitive Chang target cells. Similar degrees of cytotoxicity were induced by IL2 stimulation of lymphocytes from melanoma patients, patients with non-melanoma carcinoma and normal subjects against the allogeneic target cells. Cold target inhibition studies were carried out against IL2 induced autologous cytotoxicity in five patients. In four of five studies the autologous target cells inhibited more than the allogeneic target cells. There was no significant difference between the inhibition produced by allogeneic melanoma cells and that produced by non-melanoma cells. Similarly, in studies against allogeneic target cells, there was no significant difference in the inhibition produced by allogeneic melanoma compared to non-melanoma target cells. This applied irrespective of whether effector cells were from melanoma or non-melanoma subjects. These results suggest that lymphocytes from patients with melanoma are primed against autologous antigens in vivo and that provision of a second signal, IL2, in vitro can induce cytotoxicity against the autologous tumour. The cytotoxicity generated against the allogeneic target cells did not appear to have specificity to melanoma. Several results, such as the pattern of cytotoxicity against the target cells and changes in cell surface markers on the lymphocytes during culture, suggested that cytotoxicity was mediated by activated T cells rather than by natural killer cells. These findings appear to have important implications both in the understanding of tumour host relationships and for the use of IL2 in therapy.     

Detection of suppressor lymphocytes in the blood of oncological patients by using a 3-component lymphocyte mixed culture

A 3 component mixed lymphocyte culture was used to identify the suppressor cell activity of lymphocytes in the blood of patients suffering from osteogenic sarcoma, osteoblastoclastoma and lung tumor. The lymphocytes of these patients suppressed the blastogenic reaction of lymphocytes from two unrelated donors. The suppressive activity of the lymphocytes form the patients with bone sarcoma was the strongest. In most cases, a direct correlation was found between the suppressive activities of lymphocytes and serum from the same patients.     

Generation of human LAK cells in tissue culture bags using recombinant IL-2 and serum free medium. Effects of pretreatment with phenylalanine-methylester

A technique for processing and culturing of human LAK cells using an automated closed system and tissue culture bags is described. To circumvent the inhibitory effects of monocytes on LAK cells the peripheral blood mononuclear cells (PBMC) were pretreated with phenylalanine-methylester (PheOMe). PBMC were obtained from healthy donors by leukapheresis of whole blood. After pretreatment with PheOMe and culturing with IL-2 for 96 h, 60% of the cells remained. PheOMe significantly reduced the number of monocytes (Leu-M3+ cells) from 20-12%. The lytic activity (against K562 and Daudi) of non-PheOMe-treated cells reached a plateau at 72-96 h while PheOMe-treated cells reached maximum activity at 96 h. The total lytic activity/tissue culture bag at 96 h of PheOMe-pretreated cells was significantly augmented in comparison to non-PheOMe-pretreated cells. The present technique allows rapid and simple generation of LAK cells without serum in sterile receptacles suitable for therapy. Additionally, the LAK cell efficacy was improved by reducing the inhibitory effects of monocytes.     

Kinetics and function of peritoneal-exudate cells during local IL-2 gene-therapy of cancer

The present study was designed to examine the kinetics and function of peritoneal exudate cells (PEC) during local interleukin 2 (IL-2) gene therapy of the X63-Ag8.653 plasmacytoma growing in the peritoneal cavity. BALB/c mice were inoculated i.p. on day 0 with a tumorigenic dose of the syngeneic plasmacytoma and on day 1 with non-tumorigenic plasmacytoma cells carrying an inserted IL-2 gene and producing constitutively IL-2. At regular time intervals the experimental mice were sacrificed and their peritoneal exudate cells were used for phenotypic analysis and Cr-51 microcytotoxicity assay. On the first day after i.p. inoculation of the genetically modified plasmacytoma cells the percentage of Thy 1.2+, CD3+, TCR alphabeta+ T lymphocytes and NK+ cells in the peritoneal fluid dramatically increased. The levels of the positive cells continually decreased until day 11, when the values of normal, healthy mice were obtained. The percentage of Thy 1.2+ and CD3+ cells remained at these, or slightly lower values, until the end of the observation period. A similar, though more slowly descending kinetics was seen in the CD5+ cell population, whereas the CD8+ cells, compared to the controls, exhibited only a short-term peak between days 3 and 5, and the values of TCR alphabeta+ and NK+ cells exhibited a second peak between days 25 and 48. The percentage of TCR gammadelta+ cells showed a permanent, moderately elevated plateau from day 1 till the end of the observation period. In control, untreated mice, inoculated i.p. with the X63-Ag8.653 plasmacytoma, the kinetics of peritoneal exudate cells was different. A moderate, permanent elevation of all of the T and NK cell subsets examined occured during the observation period. In addition, the percentage of TCR alphabeta+, TCR gammadelta+ and NK+ cells further increased continually from day 11 till the end of the observation period. The cytolytic activity of the peritoneal exudate cells was examined in vitro concurrently with FACS phenotyping. Free tumour-specific killer cells generated in the peritoneal fluid due to the local IL-2 gene therapy were found only on day 6, and these cells were cytolytic for both, the parental X63-Ag8.653 and the genetically modified X63-m-IL-2 plasmacytoma cells.     

Depressed responder and stimulator capacities of peripheral lymphocytes from patients with advanced breast cancer in the mixed lymphocyte culture.

Peripheral lymphoid cells of patients with carcinoma of the breast were examined for reactivity (responders) against allogeneic lymphoid cells in the one-way mixed lymphocyte culture. The latter cells were also tested for response against the patients' lymphocytes (stimulators). The responder and stimulator capacities of lymphoid cells of patients with advanced cancer were found to be significantly reduced compared to cells from healthy subjects. Such defects were not observed in disease-free women previously treated for primary carcinoma of the breast. The results support the view that there is an abnormality of the lymphocyte-monocyte pool of cells in patients with advanced cancer.     

Human thymus/leukaemia-associated antigen (a low-molecular weight form of adenosine deaminase) and the phenotype of leukaemic cells

A water soluble human thymus/leukaemia-associated antigen (HThy-L) was recently identified as a low-molecular-weight form of adenosine deaminase (ADA). In the present study levels of the enzyme in normal and leukaemic cells and plasma have been assessed by an enzymatic method and/or radioimmunoassay (RIA) for HThy-L. In addition, the relationship between the levels of enzyme and the phenotype of leukaemic cells was evaluated. The findings can be summarised as follows: 1. There is a good correlation between levels of ADA detected by the RIA and the enzymatic method in normal and leukaemic cells but a poor correlation between levels in cells versus plasma in leukaemic patients. 2. All patients with T-cell acute lymphoblastic leukaemia (ALL) tested had high quantities of ADA in plasma and blast cells compared with normal blood or marrow cells. 3. Approximately 50% of patients with common ALL had increased quantities of ADA in leukaemic cells. 4. Patients with chronic myeloid leukaemia both in the stable phase of disease and in blast crisis generally had low quantities of ADA in leukaemic cells although some cases of blast crises (both ‘lymphoid’ and ‘myeloid’) had raised plasma levels. These data further support the view that ADA activity is highest in the early stages of T-lymphocyte maturation and in corresponding leukaemias.     

Macrophage-T cell interaction is essential for the induction of p75 interleukin 2 (IL-2) receptor and IL-2 responsiveness in human CD4+ T cells

Fresh human CD8+ T cells showed a strong proliferative response to a high concentration of interleukin 2 (IL-2) in the absence of macrophages. In contrast, CD4+ T cells revealed no significant IL-2 responsiveness in the absence of macrophages. However, if CD4+ T cells were cocultured with macrophages, they showed higher proliferative response to IL-2 than CD8+ T cells. In accordance with the magnitude of IL-2 responsiveness, freshly isolated CD8+ T cells expressed significant amounts of p75 IL-2 receptor, while fresh CD4+ T cells did not express p75 IL-2 receptor. The expression of p75 IL-2 receptor on CD4+ T cells was induced by coculture with macrophages. The macrophage-induced p75 IL-2 receptor acquisition was blocked by monoclonal antibody (mAb) against class II antigen. Moreover, the addition of anti-CD4 mAb or anti-class II mAb to the culture caused a great inhibition of IL-2 responsiveness of CD4+ T cells. These results strongly suggest that macrophage-T cell interaction through CD4 and/or class II molecules is essential for the expression of p75 IL-2 receptor and IL-2 responsiveness in human CD4+, but not CD8+ T cells.     

Autologous high-killing cytotoxic T lymphocytes against human lung cancer are induced using interleukin (IL)-1beta, IL-2, IL-4, and IL-6: possible involvement of dendritic cells.

Although CTLs bear main immune responses in human tumors, stable CTL clones against human lung cancer have rarely been generated. Our previous study demonstrated efficient autologous CTL induction in human gastric cancer and glioblastoma by cytokine combination of interleukin (IL)-1beta (167 IU/ml), IL-2 (67 IU/ml), IL-4 (67 IU/ml), and IL-6 (134 IU/ml). In this study, we demonstrated successful induction of autologous stable CTLs in five of six patients with lung adenocarcinoma from mixed-lymphocyte tumor culture using this cytokine combination. All CTLs revealed potent and specific killing activity against autologous target cells (over 75% in CD8+ CTLs and over 50% in CD4+ CTLs at an E:T ratio of 10 for 24 h). Using a series of antibodies, CD8+ CTLs showed to recognize tumor-specific antigens of lung cancer cells through HLA class I. In the separate experiments, failure of CTL induction from monocyte-depleted peripheral blood mononuclear cells and appearance of cells with characteristics of dendritic cells from adherent peripheral blood mononuclear cells in the culture of the same concentration of IL-1beta, IL-4, and IL-6 indicated that CTLs can be efficiently generated by this cytokine combination via possible dendritic cell induction. This is the first study of an efficient and reproducible in vitro CTL induction against human lung cancer.     

CCR6 regulates the function of alloreactive and regulatory CD4+ T cells during acute graft-versus-host disease

The chemokine receptor CCR6 is expressed by CD4+ T cell effector/memory and regulatory effector/memory (TREM) subsets. Here we show that CCR6 modulates graft-versus-host-disease (GVHD) responses in both alloreactive CD4+ T effector cells and regulatory T (Treg) cells. Mortality and morbidity due to acute GVHD were drastically reduced and delayed when na?ve T cells were derived from CCR6-deficient donor mice. This deficiency also affected the suppressive ability of Treg cells in GVHD. CCR6-/- Treg cells were able to suppress T cell proliferation in vitro, but their in vivo capacity to downregulate target tissue damage induced by na?ve wild type (WT) T cells was impaired. The data demonstrate a requirement for CCR6 in CD4+ T cell function in GVHD, in both effector and regulatory cell subsets.     

Human adipose tissue from normal and tumoral breast regulates the behavior of mammary epithelial cells

Introduction

Stromal-epithelial interactions mediate both breast development and breast cancer progression. In the present work, we evaluated the effects of conditioned media (CMs) of human adipose tissue explants from normal (hATN) and tumor (hATT) breast on proliferation, adhesion, migration and metalloproteases activity on tumor (MCF-7 and IBH-7) and non-tumor (MCF-10A) human breast epithelial cell lines.

Materials and methods

Human adipose tissues were obtained from patients and the conditioned medium from hATN and hATT collected after 24 h of incubation. MCF-10A, MCF-7 and IBH-7 cells were grown and incubated with CMs and proliferation and adhesion, as well as migration ability and metalloprotease activity, of epithelial cells after exposing cell cultures to hATN- or hATT-CMs were quantified. The statistical significance between different experimental conditions was evaluated by one-way ANOVA. Tukey′s post hoc tests were performed.

Results

Tumor and non-tumor breast epithelial cells significantly increased their proliferation activity after 24 h of treatment with hATT-CMs compared to control-CMs. Furthermore, cellular adhesion of these two tumor cell lines was significantly lower with hATT-CMs than with hATN-CMs. Therefore, hATT-CMs seem to induce significantly lower expression or less activity of the components involved in cellular adhesion than hATN-CMs. In addition, hATT-CMs induced pro-MMP-9 and MMP-9 activity and increased the migration of MCF-7 and IBH-7 cells compared to hATN-CMs.

Conclusions

We conclude that the microenvironment of the tumor interacts in a dynamic way with the mutated epithelium. This evidence leads to the possibility to modify the tumor behavior/phenotype through the regulation or modification of its microenvironment. We developed a model in which we obtained CMs from adipose tissue explants completely, either from normal or tumor breast. In this way, we studied the contribution of soluble factors independently of the possible effects of direct cell contact.     

Studies on the in vivo production of a lymphokine activity, interleukin 3 (IL-3) elaborated by lymphocytes and a myeloid leukaemic line in vitro and the fate of IL-3 dependent cell lines.

Interleukin 3 (IL-3) is produced constitutively by WEHI-3b leukaemic cells and stimulated lymphoid cell populations in vitro. We have investigated the in vivo production of IL-3 in mice rendered leukaemic with WEHI-3b cells and mice stimulated by acute graft versus host disease (GVHD). In leukaemic mice, IL-3 was not found in serum or sonicates of 18-day spleens or bone marrow, although cells from the leukaemic organs were fully competent to elaborate IL-3 in vitro. Further, elaboration of IL-3 by WEHI cells in vitro was not affected by co-culture with normal haemopoietic cells. However, intracellular IL-3 was detected in leukaemic nodules isolated from the liver. Inhibitors specific for IL-3 were not found, although liver-cell conditioned medium and leukaemic nodule sonicates contained potent non-specific inhibitors of cell growth. At 21 days, intracellular IL-3 was also present in spleens and correlated with the presence of non-specific inhibitors. In GVHD, no evidence for IL-3 elaboration in vivo was found, nor did lymphoid populations affected by GVHD spontaneously elaborate it in vitro; however, their competence to produce it was unaffected, as IL-3 was elaborated on subsequent mitogen stimulation in vitro. We also investigated the recovery and circulation of in vitro 111Indium-labelled IL-3 dependent cells after injection in vivo and the half-life of semi-purified IL-3. Dependent cells were not recovered after injection into irradiated recipients, although the cells recirculated for at least 24 hours. Inability to recover dependent cells was explicable on general cytotoxicity which masked potential recovery. The serum half-life of injected partially purified material with IL-3 activity was short (less than 30 min). We conclude that the elaboration of IL-3 by leukaemic WEHI-3b is not an in vitro artifact and these results are discussed in relationship to other growth factors and the leukaemic state, and the origin of IL-3 dependent lines.