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Since HA-1-specific T cells have been shown to make a significant contribution to the clinical responses in patients with relapsed leukemia, we investigated the feasibility of adoptive transfer of in vitro induced HA-1-specific CD8 positive T cells to patients with relapsed leukemia after allogeneic stem cell transplantation. The in vitro generation of clinical grade HA-1-specific T-cell lines from HA-1 negative donors was seen to be feasible and 3 patients were treated with HA-1-specific T-cell lines. No toxicity after infusion was observed. Although in one patient, during a period of stable disease, HA-1-specific T cells could be detected in the peripheral blood and bone marrow, these patients had no clear clinical response.

Patients with hematological malignancies can be successfully treated with HLA-matched T cell-depleted allogeneic stem cell transplantation (alloSCT) and subsequent donor lymphocyte infusions (DLIs). The efficacy of DLI is mediated by donor T cells recognizing minor histocompatibility antigens (mHags) on malignant recipient cells. Because HLA class II molecules are predominantly expressed on hematopoietic cells, mHag-specific CD4(+) T cells may selectively mediate graft versus leukemia (GvL) reactivity without graft versus host disease (GvHD). In this study, we used a recombinant bacteria cDNA library for the identification of the first autosomal HLA class II (HLA-DQB1*0603)-restricted mHag LB-PI4K2B-1S encoded by the broadly expressed phosphatidylinositol 4-kinase type II beta gene. A polyclonal CD4(+) T cell response against LB-PI4K2B-1S was demonstrated in a patient with relapsed chronic myeloid leukemia (CML) who responded to DLI after HLA-matched alloSCT. LB-PI4K2B-1S-specific CD4(+) T cells recognized and lysed the CD34(+) CML cells of the patient and other leukemic cells as well as high HLA-DQ-expressing normal hematopoietic cells. HLA-DQ expression on normal cells of nonhematopoietic origin was moderately up-regulated by IFN-gamma and not sufficient for T cell recognition. We hypothesize that LB-PI4K2B-1S-specific CD4(+) T cells contributed to the antitumor response by both directly eliminating malignant cells as effector cells and stimulating CD8(+) T cell immunity as helper cells.

Background and Objectives: Graft-versus-host-disease may be avoided and the likelihood of a graft-versus-leukemia reaction increased by infusion of in vitro generated, leukemia-reactive, cytotoxic T lymphocyte (CTL) lines as treatment for patients with relapsed leukemia after allogeneic stem cell transplantation, instead of donor lymphocyte infusion. The aim of this study phase I/II study was to assess the feasibility of large-scale in vitro generation of leukemia-reactive CTL for clinical use., Design and Methods: Using a modified limiting dilution culture system donor T cells were stimulated with HLA-identical leukemic antigen presenting cells. Feasibility experiments demonstrated that in 16 of 27 donor-recipient pairs tested a CTL line could be generated. Twelve of these 16 patients developed a relapse and for 11 of these 12 patients a CTL line was generated under Good Manufacturing Practice conditions., Results: The CTL lines showed moderate to high cytotoxic activity against original recipient leukemic cells in vitro. Eight patients with a relapse received from one to seven CTL lines. One patient entered a complete remission after CTL infusion only, one entered a complete remission after combined CTL infusion and donor lymphocyte infusion, two patients had temporarily stable disease, and in four patients no response was observed., Interpretation and Conclusions: Although the current procedure to generate these CTL lines is feasible, the strategy is logistically complex and time-consuming, and needs further improvement. Key words: cellular immunotherapy, CTL, leukemia, allogeneic stem cell transplantation.

T cells directed against hematopoietic-restricted minor histocompatibility antigens (mHags) may mediate graft-versus-leukemia (GVL) reactivity without graft-versus-host disease (GVHD). Recently, the HLA-A24-restricted mHag ACC-1 and the HLA-B44-restricted mHag ACC-2 encoded by separate polymorphisms within the BCL2A1 gene were characterized. Hematopoietic-restricted expression was suggested for these mHags. We demonstrate BCL2-related protein A1 (BCL2A1) mRNA expression in mesenchymal stromal cells (MSCs) that was up-regulated by the inflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and/or interferon gamma (IFN-gamma). Analysis of cytotoxicity and IFN-gamma production illustrated that ACC-2-specific T cells did not recognize untreated MSCs or IFN-gamma-treated MSCs but showed specific recognition and killing of MSCs treated with TNF-alpha plus IFN-gamma. We hypothesize that under steady-state circumstances BCL2A1-specific T cells may exhibit relative specificity for hematopoietic tissue, but reactivity against nonhematopoietic cells may occur when inflammatory infiltrates are present. Thus, the role of BCL2A1-specific T cells in differential induction of GVL reactivity and GVHD may depend on the presence of inflammatory responses that may occur during GVHD.

Allogeneic human-leukocyte-antigen-matched stem cell transplantation is associated with a lower risk of relapse of leukaemia than autologous transplantation due to a T-cell-mediated graft-vs.-leukaemia effect. Replacement of patient haematopoiesis by donor haematopoiesis allows the application of donor-derived specifically targeted cellular immunotherapy for the treatment of leukaemia. Following allogeneic transplantation, donor-derived T cells recognizing minor histocompatibility antigens expressed on haematopoietic cells from the patient may result in eradication of all haematopoietic cells of recipient origin. Since after transplantation, normal haematopoiesis is of donor origin, these T-cell responses may result in establishment of full donor chimerism associated with elimination of the haematological malignancy. By targeting the immune response to minor histocompatibility antigens that are not expressed on non-haematopoietic tissues, graft-vs.-host reactions may be limited. Several methods can be used for in vitro selection of T-cell responses with high specificity for malignant cells, and in vitro manipulation of donor T cells including transfer of antigen-specific T-cell receptors may greatly enhance specificity and efficacy of donor-derived cellular immunotherapy of haematological malignancies.

T cells directed against minor histocompatibility antigens (mHags) might be responsible for eradication of hematological malignancies after allogeneic stem cell transplantation. We investigated whether transfer of T cell receptors (TCRs) directed against mHags, exclusively expressed on hematopoietic cells, could redirect virus-specific T cells toward antileukemic reactivity, without the loss of their original specificity. Generation of T cells with dual specificity may lead to survival of these TCR-transferred T cells for prolonged periods of time in vivo due to transactivation of the endogenous TCR of the tumor-reactive T cells by the latent presence of viral antigens. Furthermore, TCR transfer into restricted T cell populations, which are nonself reactive, will minimize the risk of autoimmunity. We demonstrate that cytomegalovirus (CMV)-specific T cells can be efficiently reprogrammed into leukemia-reactive T cells by transfer of TCRs directed against the mHag HA-2. HA-2-TCR-transferred CMV-specific T cells derived from human histocompatibility leukocyte antigen (HLA)-A2+ or HLA-A2- individuals exerted potent antileukemic as well as CMV reactivity, without signs of anti-HLA-A2 alloreactivity. The dual specificity of these mHag-specific, TCR-redirected virus-specific T cells opens new possibilities for the treatment of hematological malignancies of HLA-A2+ HA-2-expressing patients transplanted with HLA-A2-matched or -mismatched donors.

AraC resistance in vitro is explained by inactivation of dCK, while resistance to DNR is described by overexpression of multidrug efflux pumps like Pgp or MRP. Thus far, no correlation between resistance mechanisms in vitro and in patients with AML has been documented. We generated AraC and DNR double resistant cell lines to investigate resistance mechanisms of both agents. In these cell lines involvement of dCK was extensively investigated and Pgp expression and activity was determined. Our data implicate that similar resistance mechanisms like inactivation of dCK coincided by alternatively spliced dCK forms and overexpression of Pgp are induced in single-as well as in double resistant leukemic cell lines.

Donor lymphocyte infusion (DLI) into patients with a relapse of their leukemia or multiple myeloma after allogeneic stem cell transplantation (alloSCT) has been shown to be a successful treatment approach. The hematopoiesis-restricted minor histocompatibility antigens (mHAgs) HA-1 or HA-2 expressed on malignant cells of the recipient may serve as target antigens for alloreactive donor T cells. Recently we treated three mHAg HA-1- and/or HA-2-positive patients with a relapse of their disease after alloSCT with DLI from their mHAg HA-1- and/or HA-2-negative donors. Using HLA-A2HA-1 and HA-2 peptide tetrameric complexes we showed the emergence of HA-1- and HA-2-specific CD8(+) T cells in the blood of the recipients 5-7 weeks after DLI. The appearance of these tetramer-positive cells was followed immediately by a complete remission of the disease and restoration of 100% donor chimerism in each of the patients. Furthermore, cloned tetramer-positive T cells isolated during the clinical response specifically recognized HA-1 and HA-2 expressing malignant progenitor cells of the recipient and inhibited the growth of leukemic precursor cells in vitro. Thus, HA-1- and HA-2-specific cytotoxic T lymphocytes emerging in the blood of patients after DLI demonstrate graft-versus-leukemia or myeloma reactivity resulting in a durable remission. This finding implies that in vitro generated HA-1- and HA-2-specific cytotoxic T lymphocytes could be used as adoptive immunotherapy to treat hematological malignancies relapsing after alloSCT.

Accelerated telomere shortening has been proposed as a possible long-term risk of allogeneic bone marrow transplantation (allo-BMT). In this study we monitored telomere length in white blood cells (WBC), granulocytes, and naïve and memory CD4+ T lymphocytes in recipients of allo-BMT at long-term follow-up. Peripheral blood was collected from 10 allo-BMT recipients and donors at a median interval of 18 years after allo-BMT. Telomere length was determined using Southern blot analysis. Similar to results previously reported at short-term follow-up, a small difference in telomere length (0.1-0.3 kb) between recipients and donors was detected in WBC, granulocytes and naïve CD4+ T cells. Our data therefore provide no evidence for sustained telomere shortening in leucocytes, and render the possibility of long-term haematopoietic graft failure unlikely. In addition, we observed two phenomena that may be related to involution of the thymus. First, the number of naïve CD4+ T cells in the blood was significantly lower in recipients (0.4 x 10(9)/l) than in donors (0.7 x 10(9)/l) (P < 0.05). Second, telomeres in memory CD4+ T cells from recipients were on average 0.6 kb shorter than those from donors (P = 0.01). The latter may be related to the reported rapid peripheral expansion of memory T cells immediately after transplantation.

The assays for the detection of unlabeled 1-beta-D-arabinofuranosylcytosine (cytosine arabinoside, Ara-C) incorporation into DNA was simplified. The procedure includes DNA isolation from leukemic cells, quantification of DNA concentrations, breakdown by enzymatic digestion of DNA to nucleosides and a radioimmunoassay (RIA) using an antibody against Ara-C. Different techniques for quantification of DNA concentrations are compared. A fluorimetric technique using Hoechst 33258 is preferred because it is the most specific method. Comparison of this RIA assay with measurement of [3H]-Ara-C/DNA formation under similar conditions in HL-60 cells showed a correlation of 0.99. Ara-C incorporation into DNA of leukemic cells was studied using two rat-leukemia cell lines, one of which is sensitive to Ara-C and the other is an Ara-C-resistant wild type: BNML-Cl/0 and BNML-Cl/Ara-C, respectively. The results showed that Ara-C is incorporated when the cells are incubated at concentrations equal to or higher than the Ara-C concentration that induces 50% growth inhibition after 48 h incubation (IC50). This implies that at lower Ara-C concentration, i.e. levels that do not induce cytotoxicity, Ara-C is not incorporated into DNA. Similar results were obtained with human HL-60 myeloid leukemia cells. The detection limit of this assay is 2 pmol/ml Ara-C; therefore, the assay is more sensitive than measurement of Ara-C triphosphate (Ara-CTP), the only metabolite that can be measured in leukemic cells from patients after in vivo Ara-C administration. On the basis of in vitro studies, the finding of detectable Ara-C/DNA levels in vivo is expected to correlate with cytotoxicity; whether or not the Ara-C/DNA level itself is informative remains to be evaluated. [ABSTRACT FROM AUTHOR]

Objective: B–lineage acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia in lymphatic blastic phase in adults have poor prognoses despite intensive chemotherapy. Novel targeted treatment modalities emerge, but their evaluation requires relevant in vitro models of lymphoblastic leukemia. Presently available cell lines do not fully represent this heterogeneous disease. Available in vitro culturing protocols do not support long–term proliferation of primary cells. We therefore aimed to develop a culture system that allows long–term proliferation of primary human B–lineage lymphoblastic leukemia. Materials and Methods: Primary lymphoblastic leukemia cells were cultured in a defined serum–free medium, in the absence or presence of human hematopoietic growth factors or serum. Results: In the defined serum–free medium, cells from 12 of 34 cases immediately proliferated in vitro. In the absence of hematopoietic growth factors and serum these cases proliferated for more than 1 year without signs of exhaustion. The culturing system supported different subtypes of lymphoblastic leukemia. Two chronic myeloid leukemia in lymphatic blastic phase, four bcr/abl–positive ALL, one etv6/abl–positive ALL, 2 e2a–pbx1−positive ALL, and one t(9;11)–positive ALL could be long–term expanded, as well as two ALL that displayed nontypical cytogenetics. Not all bcr/abl– or e2a–pbx1−positive ALL proliferated in vitro, demonstrating heterogeneity within these subtypes. The proliferating bcr/abl– and etv6/abl–positive cells displayed sensitivity to imatinib, demonstrating that their proliferation depended on the activity of these oncoproteins. Conclusion: The serum–free culturing system may be a valuable instrument in the study of ALL cell biology, as well as in the evaluation of novel targeted therapeutics. [Copyright &y& Elsevier]

Acute lymphoblastic leukaemia (ALL) in adults is a relatively rare neoplasm with a curability rate around 30% at 5 years. This consideration makes it imperative to dissect further the biological mechanisms of disease, in order to selectively implement an hitherto unsatisfactory success rate. The recognition of discrete ALL subtypes (some of which deserve specific therapeutic approaches, like T-lineage ALL (T-ALL) and mature B-lineage ALL (B-ALL)) is possible through an accurate combination of cytomorphology, immunophenotytpe and cytogenetic assays and has been a major result of clinical research studies conducted over the past 20 years. Two–three major prognostic groups are now easily identifiable, with a survival probability ranging from <10 to 20% (Philadelphia-positive ALL) to about 50–60% (low-risk T-ALL and selected patients with B-lineage ALL). These issues are extensively reviewed and form the basis of current knowledge. The second major point relates to the emerging importance of studies that reveal a dysregulated gene activity and its clinical counterpart. It is now clear that prognostication is a complex matter ranging from patient-related issues to cytogenetics to molecular biology, including the evaluation of minimal residual disease (MRD) and possibly gene array tests. On these bases, the role of a correct, highly personalised therapeutic choice will soon become fundamental. Therapeutic progress may be obtainable through a careful integration of chemotherapy, stem cell transplantation, and the new targeted treatments with highly specific metabolic inhibitors and humanised monoclonal antibodies. [Copyright &y& Elsevier]

: ObjectiveUnresponsiveness to chemotherapy is a major problem in the treatment of leukemia, which can be caused by unresponsiveness of noncycling cells to cell cycle-dependent cytotoxic agents. Targeted toxins consisting of a targeting and activating cytokine (granulocyte-macrophage colony-stimulating factor [GM-CSF]) and diphtheria toxin (DT) can be used to overcome this kind of resistance of leukemic cells. In this study we manipulated the cell cycle and proliferative status of leukemic cells, explored the effect on sensitivity to DT, and determined the ability of DT388GMCSF fusion proteins to activate and subsequently kill leukemic cells.: Materials and MethodsWe used the GM-CSF–dependent myeloid leukemic cell line AML-193 as a model. GM-CSF or granulocyte colony-stimulating factor (G-CSF) was used to manipulate the cell cycle and proliferative state of AML-193 cells. Cell death was quantified by 51Cr release assays. The results obtained in the AML-193 cell line model were confirmed using primary leukemic blasts.: ResultsSimilar to treatment with chemotherapy and immunotherapy, leukemic cells in resting G0 phase were relatively resistant to DT-induced cell death. Synchronized recruitment of leukemic cells into activated phases of the cell cycle by low concentrations of GM-CSF or G-CSF resulted in significant increased DT sensitivity. DT388GMCSF fusion proteins specifically targeted GM-CSF receptor-expressing cells, resulting in recruitment of leukemic cells from G0 phase of the cell cycle and subsequent kill of these cells.: ConclusionLeukemic cells in G0 phase, which are resistant to conventional chemotherapy, Fas-induced immunotherapy, and DT alone, can be synchronically activated and subsequently killed by DT388GMCSF fusion proteins. [Copyright &y& Elsevier]