No abstract

The TNFR family member 4-1BB and its ligand 4-1BBL are involved in the costimulation of T-cells and tumor-derived soluble (s)4-1BBL may influence the interaction of malignant cells with the immune system. Here, we report that cell-surface-expressed (c)4-1BBL can be expressed on mononuclear blood cells from patients with acute myeloid leukemia (AML) (n = 35), myelodysplasia (n = 5) or non-Hodgkin lymphoma (n = 11) and can be coexpressed on varying proportions of lymphoid or myeloid malignant cells and on dendritic cells differentiated from AML-blasts. Direct correlations between c- and s4-1BBL were not found in the investigated cases. Up to now expression of 4-1BBL has not been described on primary myeloid malignant cells, but only on malignant cells of lymphoid or solid tumor origin or on tumor cell lines. With our work we further contribute to the understanding of the potential role of c/s4-1BBL in immune reactions and its influence on the interaction of tumor and immunoreactive cells.

The presentation of leukemic antigens can be improved in AML and MDS by in vitro conversion of leukemic cells in leukemia-derived DC (DCleu), thereby forming a platform for the generation of leukemia-specific cytotoxic lymphocytes (CTL). In preliminary analyses we could already define optimal serum-free culture conditions, generate and characterize DC from 55 MDS- and 99 AML-cases and characterize their T-cell activating function (Kufner S. 2005 I-III). DC/DCleu were quantified according to their surface DC /blast -marker profiles. However in 6/30% of cases with AML/MDS less than 10% of DC could be generated and only 50–60% of leukemic cells were convertible to DCleu. Therefore we tested 5 alternative methods to improve the harvest of DC/DCleu in 99 AML and 55 MDS-patients (1) ‘MCM-mimic’ (Lee, 2003), 2) ‘Ca-Ionophore’ (Houtenbos, 2003), 3) ‘Picibanil’ (Sato, 2003), 4) ‘Cytokines’ (Westers, 2003) and 5) ‘Poly I:C’ (Rouas, 2004)). 1. Although the percentual harvest of DC/DCleu was comparable in all of the methods (28–40% DC, with 45–65% of those DC being DCleu and about 47% of blasts being convertible to DCleu) not every method was successful in individual patients. 2. However we could demonstrate that DC can be generated from every sample with at least one of the following methods: MCM-mimic, Ca-Ionophore and Picibanil. 3. Highest DC/DCleu yield was seen in monocytoid differentiated FAB-types and was independent of the cytogenetic risk. 4. Many ‘DC-surface markers’ can be aready expressed on naive blasts in AML/MDS-patients before there conversion to DCleu. Moreover the ‘DC-marker expression’ is variable in individual patients and culture methods. In summary our data show 1. that the generation of DC/DCleu is possible independent from the karyotype in every patient under serum-free conditions with at least one of the 3 methods MCM-mimic, Ca-Ionophore and Picibanil. 2. The overall percentual harvest of DCleu from MNC-fractions after culture in the 5 methods compared is low. Possibly a combination of the optimal method with optimal ‘DC- and blast markers’ in every single case could improve the detectability and quantification of DC/DCleu and residual blasts. This contributes to improve the generability and quality of DCleu to stimulate and expand anti-leukemia-directed T-cells for the immunotherapy of AML and MDS.

The presentation of leukemic antigens can be improved in AML and MDS by in vitro conversion of leukemic cells in leukemia-derived DC (DCleu), thereby forming a platform for the generation of leukemia-specific cytotoxic lymphocytes (CTL). In preliminary analyses with 140 AML and 60 MDS-cases we could already define optimal serum-free culture conditions to generate DC/DCleu.(Kufner 2005 I–III). Now we want to predict or correlate the clinical response to a DC/CTL-based immunotherapy by detailed analyses of the ex vivo generated/activated DC/DCleu and T-cells: 1)By a combination of 3 different DC-generating methods (‘MCM-mimic’, Lee 2003; ‘Ca-Ionophore’, Houtenbos 2003; ‘Picibanil’, Sato 2003) we can generate DC/DCleu in every case of AML/MDS, independently from FAB-type or karyotype. DC/DCleu are quantified according to their surface DC/blast-marker profiles. On average 42–45%/39–66% DC in AML/MDS could be generated with 48–54%/39–51% mature (CD83+) and 31–34%/23–31% migratory (CCR7+) DC. 45–65% of DC were ‘DCleu’; on average 47% of blasts are convertible to DCleu.. 2) In AML-patients who had presented with a relapse after SCT we could correlate a better ex vivo convertibility of blasts to DCleu with the patients’ in vivo response to a GM-CSF/Donor-lymphocyte Infusion (DLI)-therapy of their relapse after SCT (33% vs 7% to DCleu convertible blasts in ‘non-responders’). 3) A ‘Mixed lymphocyte culture’ (MLC) of autologous AML-patients’ or allogeneic donor-T-cells showed an on average higher proliferation and stimulation of DC-primed compared to MNC-primed T-cells: Upregulation of CD80/CD86-CD28;CD40-CD154;CD137L-CD137; moreover DC-priming yielded higher proportions of CD4+ cells, CD3+CD45RO+ memory cells CCR4+ T-cells (+59%, +52%, +91%) compared to MNC-primed T-cells (+35%, +13%, +44%) and a higher leukaemia-cytolytic activity (average 62%) compared to MNC-stimulated CTL (average 26%). 4) A detailed analysis of data showed great individual variations depending on the quality and composition of DC and T-cells: a) non-DC-primed autologous or allogeneic T-cells an lead to an increase of naive blasts after 3h incubation with blasts b) in cases with an ineffective DC-mediated ex vivo lysis of naïve blasts lower proportions of mature DC (29% vs 63%), DCleu (41% vs 68%) or a reduced T-cell proliferation or even loss of CD4/CD8/memory T-cells were seen. In summary our data show 1. that DC/DCleu can be generated in every single AML/MDS-case. 2. Grade of ex-vivo generability of DC/DCleu correlates with the in vivo response to a GM-CSF/DLI-relapse therapy. 3. Composition and quality of DC and autologous or donor T-cells after DC-priming provides informations about the activability and quality of CTLs in individual patients. We conclude, that ex vivo analysis of the DC/anti-leukemic T-cell-activability is necessary to develop and select promising anti-leukemia-directed T-cells for the immunotherapy of AML and MDS.

The presentation of leukemic antigens can be improved by in vitro conversion of leukemic cells in leukemia-derived DC (DCleu), thereby forming a platform for the generation of leukemia-specific cytotoxic lymphocytes (CTL). DC/ DCleu can be quantified by combination of suitable blast and DC-antigens (Schmetzer 2007). Now we want to enlight the role of the quality of DC/ DCleu and (DC-trained) T-cells to mediate leukemia-cytotoxic reactions ex vivo or to predict or correlate the clinical response to a DC/DLI-based immunotherapy in vivo. Methods: DC were generated with the best of 3 DC-generating methods(‘MCM-mimic’, Lee 2003;’Ca-Ionophore’, Houtenbos 2003; ‘Picibanil’, Sato 2003; Kufner S. 2005 I-III) and used to train T-cells in a ‘Mixed lymphocyte culture’ (MLC) for 10 days in the presence of IL-2 and restimulated with patient-derived DC every 3 days. Co-expression of T-cell-antigens on T-cells was measured before and after MLC. The antileukemic cytotoxic activity of DC-trained (or blast trained or untrained) T-cells against naïve blasts was quantified. We could show, that DC can be generated in every case of AML. In 65% of the cases T-cells gained a leukaemia-lytic activity after 24h training with DC, in 35% an increase of blasts was seen. The T-cell training efficacy with DC was superior to a blast training given rise to specific leukaemia-cytotoxic cells. A comparison of cases with a gain of lytic T-cell activity (n=11)with those without a lytic activity (n=6) showed 78 vs 51% DCleu, 55 vs 34% mature and 32 vs 18% migratory DC and 50vs40% proliferating T-cells, 53 vs 46% memory T-cells, 68vs56% CD4 and 38 vs 60% CD8 pos T-cells. Moreover we could evaluate cut-off values: 90% of DC-trained T-cells could gain a lytic activity if > 65% DCleu were in the MLR. In AML-patients who had presented with a relapse after SCT we could demonstrate a better ex vivo convertibility of blasts to DCleu if patients had successfully responded to a GM-CSF/DLI-based therapy of their relapse after SCT compared to cases with no response (72 vs 36% blasts convertible to DCleu; 44 vs 29% generable DC). Summary: The generation of DC/DCleu is possible in every AML/MDS-patient. Ex vivo convertibility of blasts to DCleu could predict a clinical response to a GM-CSF/DLI-based therapy or indirectly prove, that GM-CSF in vivo could contribute to produce DC/DCleu in vivo. A successful DC-training of T-cells is associated with high matureDC/ DCleu counts and high rates of proliferating, CD4 and Memory-T-cells. The lytic activity of DC-trained T-cells is predictable by quantities of DCleu generable in individual cases. So the generability of DC/DCleu and of DC/MNC-trained T-cells could contribute to predict the clinical course of the disease and could help to create specific anti-leukemic T-cells for immunotherapy of AML.