Christian Scheffold

Survivin expression correlates with apoptosis resistance after lymphocyte activation and is found preferentially in memory T cells.

The prevention of apoptosis may be critical for immunological function. Survivin is a recently cloned member of the inhibitor of apoptosis protein family. We analyzed survivin expression before and after lymphocyte activation in isolated cell populations. Prior to activation, survivin was undetectable. After activation with IL-2 and OKT-3, CD3(+) cells expressed survivin. Next, we correlated survivin expression with Fas, FasL and the amount of apoptosis over time in culture. After activation, survivin was readily detected by day 2 and decreased thereafter. Prior to activation (day 0), Fas was present on 60% of the cells and on 100% by days 2-6. Peak FasL mRNA expression was at day 2. During peak survivin expression (days 2-4) the apoptotic fraction was low, but when survivin expression decreased the apoptotic fraction increased rapidly. Finally, we found that CD45RO(+) memory T cells showed a higher expression of survivin than did CD45RA(+) naive T cells after activation. These results suggest that survivin may contribute to T-cell survival early in T-cell responses as well as in memory immune responses.

Targeting of natural killer-like T immunologic effector cells against leukemia and lymphoma cells by reverse antibody-dependent cellular cytotoxicity.

Recently, highly efficient natural killer-like T immunologic effector cells called cytokine-induced killer (CIK) cells have been described. Most interestingly, CIK cells have been shown to eradicate established human lymphoma cells in a severe combined immunodeficient (SCID) mouse xenograft model in vivo. The current study was aimed at increasing the sensitivity of leukemia and lymphoma cells to CIK cells. In particular, the authors wanted to target CIK cells to leukemia and lymphoma cells via reverse antibody-dependent cellular cytotoxicity. Binding of an anti-CD3 monoclonal antibody to CIK cell cultures derived from patients with lymphoma was shown using flow cytometric analysis. For the target side, several B-cell lines were found to express CD19 on the cell surface. There was an impressive increase in sensitivity to CIK-mediated lysis of various lymphoma and leukemia cell lines by preincubation of the targets with a monoclonal antibody against CD3. This increase could be partially blocked by preincubation with anti-CD16 (Fc receptor III) and anti-CD32 (Fc receptor II) antibodies. These data suggest that the increase in cytotoxic activity is caused by Fc receptor-mediated antibody binding. Cytotoxic activity could be further increased by adding an anti-CD28 antibody in addition to anti-CD3. Finally, there was a further increase in sensitivity to CIK-mediated lysis of CD19+ malignant cells using the bispecific OKT3xHD37 antibody with specificity against CD3 and CD19. Interestingly, preincubation of malignant cells with an anti-CD3 monoclonal antibody followed by addition of the bispecific OKT3xHD37 antibody led to a further increase of cytotoxic sensitivity compared with the addition of the bispecific antibody alone. In conclusion, these data suggest that cytotoxic activity of immunologic effector cells can be increased not only by using the bispecific antibody OKT3xHD37 in vitro but also by preincubation of CD19+ leukemia and lymphoma cells with a monoclonal antibody against CD3. In addition, the immunostimulatory effect of the bispecific antibody OKT3xHD37 can be further increased by adding a monoclonal antibody against CD3.

Propagation of large numbers of cells of a human mixed-lineage T-lymphoid/myeloid

Summary. Previously, a subset of T cells co‐expressing the myeloid antigen CD33 has been described in patients with acute myelogenous leukaemia. However, normal lymphocytes have been viewed as not expressing the CD33 antigen. We have developed culture conditions which allow for the rapid expansion of CD3+CD33+ cells from patients with myeloid leukaemia as well as normal individuals. The protocol for cellular expansion includes the addition of interferon‐gamma on day 0, interleukin‐1, interleukin‐2 and a monoclonal antibody against CD 3 on day 1 to peripheral blood lymphocytes. Using this protocol, total cell number increased more than 600‐fold within 16 d of culture. Cells could be kept in culture for more than 6 months. Cells of the CD3+CD33+ phenotype increased to 15‐2 –4‐6% using this protocol after 16 d in culture. These cells have been characterized by flow cytometry and have been found to express the alpha, beta T‐cell receptor, co‐express the CD2, CD5, CD7 and HLA‐DR antigens and did not express CD14 or CD15 antigens. Cells of the CD3+CD33+ phenotype were unable to lyse tumour cells as determined in a 51Cr release assay. In patients with chronic myeloid leukaemia, CD3+CD33+ cells seem to be negative for expression of bcr/abl transcript in contrast to CD33 cells. Our data suggest that CD3+CD33+ cells do exist in peripheral blood from normal individuals.

Generation of melanoma-specific cytotoxic T lymphocytes for allogeneic immunotherapy.

To exploit alloreactive T-cell responses known as the graft-versus-tumor effect, allogeneic hematopoietic stem cell transplantation is being explored as experimental therapy in selected solid tumors, including metastatic melanoma. However, donor T-cell responses are often delayed and associated with severe graft-versus-host disease. Posttransplant adoptive immunotherapy using tumor-specific cytotoxic T lymphocytes (CTL) of donor origin might provide immediate graft-versus-tumor effects but not graft-versus-host disease. Therefore, the aim of the current study was to define in vitro conditions for the expansion of allogeneic major histocompatibility complex–matched CTLs targeting melanoma-associated antigens (MAA). The CTLs were generated from peripheral blood mononuclear cells (PBMCs) of HLA-A*0201+ healthy donors by repetitive stimulations with HLA-A*0201–restricted MAA-derived peptides. Melanoma reactivity, as determined by lysis of peptide-pulsed T2 cells and HLA-A2+/Ag+ melanoma cells, was detected using in vitro expanded CTL targeting MAA peptides AAGIGILTV(MT27–35), IMDQVPFSV(G209–2M), and YMDGTMSQV(T368–376). In contrast, FLWGPRALV(MAGE3271–279) and VLPDVFIRCV(GnT-Vnt38–67) induced peptide-specific recognition of T2 target cells only, whereas ITDQVPFSV(G209–217), KTWGQYWQV(G9154), MLLAVLYCL(T1–9), and tumor lysate could not induce specific CTLs. Specific cytolytic activity was accompanied by interferon-&ggr; secretion. Peptide-pulsed dendritic cells were required only for the initial stimulation of CTLs and could be substituted by PBMCs during restimulations. The median expansion rate of CTL was five to six times, regardless of whether dendritic cells or PBMCs were used after the initial stimulation. The results delineate the conditions for effective ex vivo expansion of melanoma-specific CTLs from PBMCs of healthy donors to be used as an adjunct in allogeneic cell therapy of metastatic melanoma.

Increased Sensitivity of Myeloid Leukemia Cell Lines: Potential of Lovastatin as Bone-Marrow-Purging Agent

Lovastatin reduces the isoprenylation of p21ras via suppression of mevalonic acid generation. Lovastatin has been shown to reduce tumor cell proliferation in a dose-dependent manner. Here, the potential of lovastatin for purging leukemia cells from bone marrow was investigated using the myeloblastic cell lines K562 and KG-1 as a model system, derived from an erythroleukemia and an acute myelogenous leukemia, respectively. Optimal purging conditions were determined using an MTT proliferation and a leukemia colony assay. Elimination of leukemia cells was time- and dose-dependent. Depletion of K562 was 2.5 logs for 100 μM of lovastatin at 72 h of incubation. Compared to another purging agent, 100 μg/ml mafosfamide had an activity comparable to 100 mM lovastatin. Interestingly, KG-1 acute myelogenous leukemia cells were even more sensitive to lovastatin than K562 cells. In clonogenic assays, 100 μM of lovastatin resulted in a 3- to 4-log reduction of K562 colonies. Lovastatin had a progressive effect on normal hematopoietic progenitor cells. At a concentration of 100 μM of lovastatin, CFU-GM colonies were reduced by 1–2 logs. In conclusion, a differential effect on leukemia and normal progenitor cells could be detected in a clonogenic assay. These results suggest that lovastatin deserves further study as an agent for ex vivo marrow purging.

Potential of Autologous Immunologic Effector Cells for Prediction of Progression of Disease in Patients with Chronic Myelogenous Leukemia

In autologous bone marrow transplantation, immunologic effector cells such as lymphokine activated killer (LAK) cells may be useful for purging of bone marrow since these cells might have an additional in vivo effect on tumor cells in contrast to other purging protocols. Recently, immunologic effector cells termed cytokine-induced killer (CIK) cells have been shown to be more useful than LAK cells for purging of autologous BM in the context of autologous BMT. Here, we show that the expression of bcr/abl in CIK cells generated from patients with CML correlates with progression of disease in individual patients. In addition, progression of disease from chronic phase to accelerated phase could be predicted in two patients by studying the expression of bcr/abl in CIK cells generated from CML patients. Thus, it might be possible to use CIK cell generation for the prediction of progression of disease in CML patients.

Induction of Apoptosis in B Lymphoma Cells by Activation with CD40L

Accessible online at: www.karger.com/journals/aha CD40 is one of the key molecules involved in the survival, growth and differentiation of B lymphocytes [1]. It is expressed on immature and mature B cells, dendritic cells, thymic epithelial cells and some carcinoma cells. Cross-linking of the CD40 receptor on Bcl-2-negative germinal center cells upregulates their Bcl-2 expression leading to prevention of apoptosis. The ligand CD40L (CD154) is mainly expressed in activated CD4-positive T cells and is one of the main costimulatury signals by which specific antigen-reactive T cells offer help to B cells presenting that antigen in the context of MHC class II [2]. Ligation of CD40 by its ligand, CD40L, results in inhibition of apoptosis, proliferation, differentiation and expressing of activation antigens including Fas [3]. The activation of CD40 molecules on normal B cells spontaneously induces CD95 expression on the cell surface. The survival or apoptosis of B cells in such situations seems to depend on the stage of activation, cytolytic factors and the degree of signal expression [4]. Activation of CD40 evokes a cytotoxic T cell response that eradicates lymphoma [5, 6]. Since activation of CD40 can evoke a cytotoxic T cell response that eradicates lymphoma, we tested the effect of activation of B lymphoma cells with fibroblasts transfected with CD40L. Cocultivation of B lymphoma cells with transfected fibroblasts led to a significant increase in CD40L and CD80 costimulatory molecule expression on the OCI-Ly8-LAM53 B lymphoma cell line. Similarly, several B lymphoma cell lines showed induction of apoptosis and decrease in proliferation rate.

Hochdosistherapie und Stammzelltransplantation

In den 50 Jahren seit den ersten klinischen Transplantationsversuchen hat sich die hamatopoetische Stammzelltransplantation zu einem wichtigen Element der modernen Tumortherapie und der Behandlung nichtmaligner Stammzell- und Immundefekte sowie bestimmter hereditarer Stoffwechselerkrankungen entwickelt. Neue Indikationsgebiete werden erschlossen und Anwendungsgrenzen, bedingt durch Alter, Vorbehandlung und Komorbiditat der Patienten, verschieben sich. Moglich wurde dieser Fortschritt durch eine Vielzahl paralleler Entwicklungen auf den Gebieten der Transplantationsimmunologie und Stammzellbiologie sowie der immunsuppressiven und supportiven Therapie.