Propagation of cytotoxic effectors from chronic myeloid leukemia patients and cloning of cytotoxic T cells

Summary Cytotoxic cells (CTCs) generated from peripheral blood lymphocytes of 5 chronic myeloid leukemia (CML) patients in remission on stimulation with autologous leukemic cells and allogeneic lymphocytes (3-cell assay), were propagated in vitro in interleukin-2 (IL-2)-containing medium and periodic stimulation with autologous leukemic cells, for a period of 4 to 6 months. During this period, the cells were assessed for phenotype and for cytotoxic responses in a 4-h ⁵¹Cr release microcytotoxicity assay. The CTCs continued to show specific lysis of autologous leukemic cells and bone marrow (BM) cells. However, the nonspecific lysis of natural killer (NK) targets and the proportion of cells showing NK phenotype (HNK-1 antigen) increased progressively on cultivation in IL-2-containing medium. Therefore cells showing CD8 phenotype and specific cytotoxic function were segregated by cloning CTCs under the condition of limiting dilution in the presence of allogeneic feeder cells and IL-2-containing medium. Three cytotoxic T cell (CTL) clones expressing CD3+, CD8+, and HLA DR+ phenotypes were obtained from CTCs of 2 CML patients. These clonoid populations, maintained in IL-2-containing medium and periodic antigenic stimulation with autologous leukemic cells, showed specific lysis of autologous leukemic cells and BM cells even at lower (10:1) effector:target ratios. They did not kill K562 (erythroblastoid leukemic NK target cell line) cells and autologous phytohemagglutinin-induced blasts. These clones apparently functioned in an MHC-restricted manner as they did not lyse allogeneic CML cells which would also express a similar set of maturation antigens if sensitization was, as it appeared, against these antigens. Finally, interaction of autologous BM cells with CTL clones reduced the colony forming potential of BM cells only to the extent of 18%–30%. The results therefore indicate that such CTL clones can possibly be used in adoptive immunotherapy as they showed minimal BM toxicity.     

Suppressor cells in the effector phase of autologous cytotoxic reactions in cancer patients

Summary Cytotoxicity was induced in lymphocytes (CL) from 10 out of 15 patients by autologous mixed lymphocyte tumor cell culture and further cultivation with recombinant interleukin-2. In cells from 3 of the 10 patients, cytotoxicity was suppressed by more than 50% when autologous peripheral blood mononuclear cells (PBMC) from the patients with large tumors were added to the autologous killing system. The cells responsible for suppressing the cytotoxicity in the effector phase were adherent or nonadherent to plastic depending on the patient examined. The T cell fraction from 1 patient significantly suppressed the cytotoxic activity, and this suppression was seen only in the autologous system. On the other hand, plastic adherent cells but not T cells from PBMC of 2 subjects suppressed the cytotoxic activity of CL. The reason why the main cell population suppressing the CL activity differed among the patients is unclear. However, the findings that the suppression was mostly abrogated following resection of the tumor mass suggested that suppressor cells, either of macrophage lineage or T cells, are induced in patients with a large tumor mass. This speculation is supported by the finding that the PBMC from a patient with tumor recurrence regained the suppressive activity.     

In vitro generation of murine myeloid dendritic cells from CD34-positive precursors

Dendritic cells (DCs) link the innate and adaptive immune system. Currently, murine DCs for cell biology investigations are developed from MHC class II-negative bone marrow (BM) precursor cells, non-depleted BM cells or BM monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Here we demonstrate an isolation procedure of functionally intact myeloid CD11c⁺ CD11b⁺ DCs derived from murine CD34-positive precursors. DCs derived from CD34⁺ cells show functional internalization, maturation, cytokine secretion, MHC-restricted antigen presentation, and MHCII retrograde transport of antigens from the lysosomes to the cell surface. In comparison to the established method, the advantages of this isolation procedure are a shorter cultivation period, a superior transfection efficiency, the yield of a purer and more homogeneous population of immature DCs, and less consumption of cell culture medium and GM-CSF. The new isolation procedure and the functional quality of CD34⁺ cell-derived murine myeloid DCs make them ideally suited for immunology and cell biology studies.     

In vitro generation of cytotoxic lymphocytes against radiation and radiation leukemia virus-induced tumors

Summary Cytotoxic T lymphocytes (CTL) to syngeneic radiation- or radiation leukemia virus (RadLV)-induced tumors were generated in vitro in mixed lymphocytetumor cultures (MLTC) using splenocytes of mice primed in vivo with inactivated tumor cells. Effective sensitization was obtained with virus-producer cell lines, while cells of a virus-nonproducer line did not sensitize.The CTL could lyse syngeneic, but not allogeneic, tumor cells of established lines producing C-type virus and therefore expressing membrane-associated viral antigenicity.Susceptibility of primary leukemias to cell-mediated lysis could not be tested due to a very high spontaneous ⁵¹ Cr release shortly after labeling. In a cold target competition assay, however, the RadLV-induced, but not the X-radiation-induced primary tumor cells inhibited the cytotoxic reactivity. This inhibition was correlated with the level of viral antigen expression on the inhibiting cells, which was high in the RadLV-induced and low in the radiation-induced primary tumors.These results suggest that antitumor CTL generated under conventional MLTC conditions are largely stimulated by and directed at virus-related antigens not necessarily associated with the malignant state of the cell.     

In vitro growth of erythropoietic progenitor cells in long-term remission of acute leukemia

In vitro growth of CFU-e and BFU-e in bone marrow and of circulating BFU-e in a group of adult long-term survivors of acute leukemia has been evaluated. Six patients with acute nonlymphoblastic and three patients with acute lymphoblastic leukemia in first continuous remission for more than four years (range 4-12 years) and without maintenance therapy for at least one year were studied. BFU-e and CFU-e growth in patients' bone marrow was not statistically different from a control group of 12 healthy adult volunteers. However, proliferation of BFU-e in peripheral blood of patients was significantly reduced (p less than 0.001). This growth pattern was found in both lymphoblastic and myeloblastic leukemia.     

In vitro cell adhesion and integrin expression by calcium ionophore-treated mononuclear cells from patients with acute myeloid leukemia

As shown elsewhere, cultured acute myeloid leukaemia blasts acquire certain characteristics of dendritic cells upon stimulation with cytokines and calcium ionophore. The ability of leukaemia-derived dendritic-like cells to express immune costimulatory molecules and dendritic cell marker CD83 has been extensively investigated. Although migratory capacity is a major attribute of dendritic cells, the ability of in vitro modified blasts for adhesion, chemotaxis and homing remain elusive. In the present paper, we show that after stimulation with calcium ionophore acute myeloid leukaemia blasts as well as normal dendritic cell precursors demonstrate increased capacity of binding fibronectin and denatured collagen. The expression pattern of integrins on dendritic-like leukaemic cells in general closely resembles that of monocyte-derived dendritic cells, however, variation in cell properties isolated from blood of individual patients are observed.     

In vitro culture studies of blood and marrow cells in chronic myeloid leukemia at different phases of the disease

The in vitro culture growth of peripheral blood (PB) and bone marrow (BM) cells were studied simultaneously from 100 adult patients with chronic myeloid leukemia at different phases. Sixty-five patients were investigated at initial diagnosis, 30 patients in control phase, and 41 patients in blast phase. In untreated chronic phase, the relative concentrations of granulocyte-macrophage progenitor cells (CFU-GM) in BM were not significantly different from those of normal controls, but there was generally a marked increase in circulating CFU-GM. The 6 Ph1-negative patients did not show different growth characteristics. We were unable to correlate the CFU-GM number to any of the hematologic parameters as well as to the response to busulfan therapy. Pretreated patients with excessive cluster formation did not necessarily indicate impending blast crisis. In hematologic remission, the numbers of CFU-GM in both BM and PB were well within the ranges of normal controls. Culture results in blast phase revealed a spectrum of abnormal growth. In myeloid crisis, 14/29 BM and 12/29 PB samples showed increased colony and cluster formations which were composed predominantly of immature cells with variable degeneration. Marrow cells in lymphoid crisis produced low numbers of both colonies and clusters in 5 out of 8 patients, while blood cells from 8 out of 10 patients formed large amount of colonies of normal morphology. This study indicates that the in vitro CFU-GM assay may have diagnostic utility in differentiating lymphoid crisis from myeloid crisis.     

Acriflavine targets oncogenic STAT5 signaling in myeloid leukemia cells

Acriflavine (ACF) is an antiseptic with anticancer properties, blocking the growth of solid and haematopoietic tumour cells. Moreover, this compound has been also shown to overcome the resistance of cancer cells to chemotherapeutic agents. ACF has been shown to target hypoxia‐inducible factors (HIFs) activity, which are key effectors of hypoxia‐mediated chemoresistance. In this study, we showed that ACF inhibits the growth and survival of chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML) cell lines in normoxic conditions. We further demonstrated that ACF down‐regulates STAT5 expression in CML and AML cells but activates STAT3 in CML cells in a HIF‐independent manner. In addition, we demonstrated that ACF suppresses the resistance of CML cells to tyrosine kinase inhibitors, such as imatinib. Our data suggest that the dual effect of ACF might be exploited to eradicate de novo or acquired resistance of myeloid leukaemia cells to chemotherapy.     

Tyrosine kinase inhibitor Thiotanib targets Bcr-Abl and induces apoptosis and autophagy in human chronic myeloid leukemia cells

Chronic myeloid leukemia (CML) is characterized by abnormal Bcr and Abl genes and enhanced tyrosine kinase activity. Anti-CML therapy has been much improved along with the applications of tyrosine kinase inhibitors (TKIs) which selectively target Bcr-Abl and have a cytotoxic effect on CML. Recently, four-membered heterocycles as “compact modules” have attracted much interest in drug discovery. Grafting these small four-membered heterocycles onto a molecular scaffold could probably provide compounds that retain notable activity and populate chemical space otherwise not previously accessed. Accordingly, a novel TKI, Thiotanib, has been designed and synthesized. It selectively targets Bcr-Abl, inducing growth inhibition, cell cycle arrest, and apoptosis of CML cells. Meanwhile, the compound Thiotanib could also induce autophagy in CML cells. Interestingly, inhibition of autophagy promotes Thiotanib-induced apoptosis with no further activation of caspase 3, while inhibition of caspases did not affect the cell survival of CML cells. Moreover, the compound Thiotanib could inhibit phosphorylation of Akt and mTOR, increase beclin-1 and Vps34, and block the formation of the Bcl-2 and Beclin-1 complex. This indicates the probable pathway of autophagy initiation. Our results highlight a new approach for TKI reforming and further provide an indication of the efficacy enhancement of TKIs in combination with autophagy inhibitors.     

In vitro generation of germ cells from murine embryonic stem cells

The demonstration of germ cell and haploid gamete development from embryonic stem cells (ESCs) in vitro has engendered a unique set of possibilities for the study of germ cell development and the associated epigenetic phenomenon. The process of embryoid body (EB) differentiation, like teratoma formation, signifies a spontaneous differentiation of ESCs into cells of all three germ layers, and it is from these differentiating aggregates of cells that putative primordial germ cells (PGCs) and more mature gametes can be identified and isolated. The differentiation system presented here requires the differentiation of murine ESCs into EBs and the subsequent isolation of PGCs as well as haploid male gametes from EBs at various stages of differentiation. It serves as a platform for studying the poorly understood process of germ cell allocation, imprint erasure and gamete formation, with 4-6 weeks being required to isolate PGCs as well as haploid cells.     

Cellular immune responses to autologous chronic myelogenous leukaemia cells in vitro

 Using a modification of the autologous mixed lymphocyte/tumour cell culture (MLTC), it is demonstrated here that lymphocytes from chronic-phase myelogenous leukaemia (CML) patients (n = 58), but not from their HLA-identical siblings, proliferated upon coculture with autologous tumour cells. However, in most cases, the level of proliferation measured was low (stimulation index <3, n = 37). This was most likely related to the amount of interleukin-10 (IL-10) released into the culture medium by the CML cells, because addition of neutralizing anti-IL-10 serum to MLTC markedly enhanced proliferative responses. In addition, supplementation of media with IL-1α further enhanced proliferative responses and a combination of anti-IL-10 serum and IL-1α was more effective than either agent alone. Only HLA-DR-matched CML cells, but not HLA-DR-mismatched CML cells or matched or mismatched PBMC restimulated proliferation of IL-2-dependent T cell lines derived from MLTC supplemented with IL-1α and anti-IL-10 serum. The responding cells under these conditions were predominantly CD4⁺ and secreted IL-2, and interferon γ; some secreted IL-4, but none secreted IL-10. These data therefore suggest the existence of an HLA-DR-restricted DTH/Th1-type of tumour-specific immunity in CML patients, which may be down-regulated in vitro by excessive secretion of IL-10 together with depressed secretion of IL-1. Received: 9 November 1995 / Accepted: 8 February 1996     

Cell-mediated immunity to friend virus-induced leukemia. IV. In vitro generation of primary and secondary cell-mediated cytotoxic responses.

Primary and secondary cell-mediated cytotoxic responses to FBL-3 cells, a syngeneic Friend virus-induced leukemia in C57BL/6 mice, could be generated by in vitro techniques as tested by the 125IUdR release assay. The specificity of the cytotoxic reactions appeared to be directed against the Friend type-specific antigen and the FMR (Friend, Moloney, Rauscher) antigen which were also the major antigens for transplantation immunity to FBL-3. In comparison to the primary cytotoxic response, the secondary cytotoxic response was accelerated (detected at an earlier time after sensitization), enhanced (gave much higher levels of cytotoxicity), was also longer lasting, and could be induced by a wide dose range of tumor cells. The secondary response could only be induced with lymphocytes obtained from regressors that were resistant to FBL-3 challenge; lymphocytes from mice with progressive tumor growth had no detectable secondary response. It was found that both induction phase and the effector phase of cytotoxic responses were T cell dependent. The characteristics of these reactions were thus very similar to those obtained with in vivo immunization or challenge, providing a good correlation with in vivo tumor immunity.     

In vitro response of cells from three patients with hairy cell leukemia to a recombinant leukocyte interferon

Mononuclear cells from the peripheral blood of two patients and from the spleen of one patient, all of whom had hairy cell leukemia, were cultured with a recombinant human leukocyte interferon (RD alpha 2-IFN). The IFN was added at concentrations of 10, 100, 1,000, and 10,000 IU/ml, and the cells were cultured for 1, 3, and 7 days. A cytocidal effect of IFN was observed only on cultured cells from the spleen at day 7. Electron-microscopic observations demonstrated that RD alpha 2-IFN induced the formation of tubuloreticular structures (TRSs) and annulate lamellae (ALs) in hairy cells, as well as in co-isolated non-leukemic cells, from all three patients. Ultrastructural examination revealed a close proximity between ALs and TRSs in co-isolated non-leukemic cells. A variability with respect to the induction of TRSs in hairy cells was observed among the three patients. In two of the three patients, the percentage of hairy cells with TRSs increased with the duration of incubation and with the dose of IFN. In the third patient, few hairy cells showed TRSs after 7 days of incubation with IFN. Our findings indicate that leukemic hairy cells may be heterogenous in their response to IFN.