Adoptive transfer of Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) is effective prophylaxis and treatment of EBV-positive immunoblastic lymphoma in immunocompromised patients. In 50% of patients with Hodgkin's disease, the tumor cells are EBV antigen-positive and may therefore also be suitable targets for treatment with virus-specific CTLs. However, Hodgkin's disease may produce several inhibitory effects on immune induction and effector function in vivo, which may preclude the generation or effector function of CTLs reactive against EBV viral proteins, including those expressed by the tumor cells. We have investigated whether EBV-specific CTLs could be generated ex vivo from 13 patients with Hodgkin's disease: nine with active relapsed disease and four who were in clinical remission after a first or subsequent relapse. CTL lines were successfully generated from nine of 13 patients (five active disease, four remission). Although these lines had an abnormal pattern of expansion comparable to EBV-specific CTLs generated from normal donors, their phenotype was normal except for reduced expression of the zeta chain of the T-cell receptor (TCR). Their cytotoxicity was also compared to EBV-specific lines generated from normal donors and included activity against LMP2a, one of the three weakly immunogenic viral antigens expressed by Hodgkin's tumor cells. To assess the activity of the CTLs in vivo, they were gene-marked and infused into three patients with multiply relapsed disease. The CTLs persisted for more than 13 weeks postinfusion and retained their potent antiviral effects in vivo, thereby enhancing the patient immune response to EBV. This approach may therefore have value in the treatment of EBV-positive Hodgkin's disease.
It has been proposed that adoptive immunotherapy, for the treatment of relapsed AML, with cytotoxic T lymphocytes which show a relative specificity for the leukemic cells may have the advantage of maximizing the beneficial anti-leukemic effect whilst minimizing the probability of graft-versus-host disease. In this study we differentiated peripheral blood AML cells in vitro into functional dendritic cells (DCs), as demonstrated by cell morphology, immunophenotype and functional activity, in the presence of GM-CSF, IL-4, TNF-␣ and FLT3 ligand. Such DCs could be differentiated from 77% of AML patients, irrespective of their FAB classification and clinical status and, in all cases tested, the DCs were shown to derive from the leukemic clone by FISH analysis. Importantly, from Ͼ60% of AML patients, autologous T lymphocytes stimulated with these in vitro generated leukemic DCs displayed specific cytotoxic activity against AML blasts but low reactivity against autologous non-leukemic targets and HLA-matched normal PBMNCs therefore suggesting that the CTLs were AML-specific. The use of FLT3 ligand in our system resulted in a significantly higher number of leukemic DCs as compared to cultures from which FLT3 ligand was omitted which is obviously advantageous if large numbers of specific CTLs are to be generated in the shortest possible time. Leukemia (2001) 15, 246-255.
Although interferon alpha (IFN-alpha) is able to induce haematological remission in 60-80% of patients with chronic myeloid leukaemia (CML) in early chronic phase, major cytogenetic remissions are only achievable in 30-40%. Recent clinical data suggest that the addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) to IFN-alpha therapy can significantly improve the cytogenetic response in some patients, although the mechanism remains unknown. We hypothesized that the combination of GM-CSF and IFN-alpha induces the differentiation of dendritic cells, which subsequently stimulates a specific anti-leukaemic response. Monocytes from CML patients were cultured in GM-CSF and interleukin (IL)-4 (GM/IL-4)or in GM-CSF and IFN-alpha (GM/IFN-alpha). After 7 d, the number of cells exhibiting typical antigen-presenting cell (APC) morphology was equal in both groups, and fluorescence in situ hybridization (FISH) analysis confirmed that the APCs generated with GM/IFN-alpha were of leukaemic origin. Phenotypically, both sets of APCs expressed typical surface markers; however, CD86, CD83, CD11c, HLA-ABC and HLA-DR expression was significantly higher in the GM/IFN-alpha APCs, whereas CD1a expression was significantly lower. In mixed lymphocyte reactions (MLR), GM/IFN-alpha APCs stimulated the proliferation of allogeneic T cells significantly better than GM/IL-4 APCs. However, both groups of APCs stimulated autologous T-cell proliferation equally. Finally, we assessed the ability of GM/IFN-alpha APCs to induce a leukaemia-specific cytotoxic T-cell response. Some samples generated cytotoxic T lymphocytes (CTLs) that specifically lysed bcr-abl-positive target cells. These data show that the combination of GM-CSF and IFN-alpha, when used in vitro, induces the differentiation of malignant APCs with potent T-cell stimulatory capacity. Although there is no in vivo evidence to support these findings, it is possible that, when administered to CML patients, GM-CSF in combination with IFN-alpha results in the generation of highly stimulatory leukaemic APCs.
Leukemia cells may express tumor specific antigens in association with Class I and II major histocompatability complex (MHC) molecules. However, lack of expression of conventional costimulator molecules means that these cells tend to induce specific T-cell anergy rather than activation. CD40 ligand (CD40L) is a costimulator molecule that directly activates T cells and may promote antigen presentation by CD40-expressing cells, which include professional antigen presenting cells and B-acute lymphoblastic leukemia (ALL) cells from many patients. We determined whether transgenic expression of CD40L could enhance an antileukemia immune response using a CD40+ murine lymphoblastic (A20) leukemia and a CD40− myeloblastic (WEHI-3) leukemia in a tumor treatment model. Injection of otherwise nonimmunogenic A20 cells in the presence of CD40L induced an immune response active against preexisting A20 tumor at a distant site. Moreover, concomitant local secretion of transgenic interleukin-2 (IL-2) further amplified the antileukemic response induced and increased protection against preexisting tumor. In ex vivo studies, CD40 activation of A20 cells enhances the antigen presenting potential of A20 cells by upregulating expression of B7.1 (CD80), Class I and II MHC molecules, and increases expression of fas antigens. The importance of CD40 activation to the resulting antitumor response is further emphasized by the failure of transgenic CD40L to protect against the CD40− WEHI myeloblastic leukemia. Depletion studies showed the protective effects against A20 cells to be mediated by a combination of CD4+ and CD8+ T lymphocytes and by natural killer (NK) cells. These results suggest a means by which CD40+ leukemia cells may be rendered immunogenic in vivo.
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