A Step towards Reconstitution or Maintenance of Remission before or after Stem Cell Transplantation in AML/MDS by Ex Vivo Generation of Leukaemia-Derived DC (DCleu) and DC-Activated T-Cells: Role of the Quality and Quantity of DC/DCleu and Anti-Leukemia-Directed T-Cells To Predict the Course and Success of Immunotherapy.
Authors: Schmetzer Helga M. , Liepert Anja , Grabrucker Christine , Kremser Andreas , Loibl Julia , Schmid Christoph , Buhmann Raymund , Yang Ting , Kroell Tanja , Salih Helmut , Zitzelsberger Horst , Kolb Hans-Jochem 
Published: October 2019

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.