Mixed autologous leukocytes reactions performed using matured CML dendritic cells and CDS or CD4 T-cells results in colony inhibition only with CD4 cultured with IL-12 throughout and is not leukemia specific

CD34+ cells were isolated from the initial aphérèses from 3 CML patients before any treatment. All these patient had the b3a2 junction and MHC complexes for which CTL generation had been described using peptides (A2, DR201 - A2, DR1101 -All, DR2). DCs were generated from cytaphereses from the same patients, harvested when they had normalized their counts to have a sufficient proportion of monocytes. DCs were generated by classical adherence enrichment followed by a 6 days culture in RPMI, autologous serum 2%, GM-CSF and IL-4. At day 6. CD40L was added to mature the DCs, which were harvested for the MLRs after 48 hrs. We first assess the functional capacity of CML DCs using immunophenotyping, allogeneic and autologous proliferative tests and specificic antigen (Influenza) presentation in short MLR followed by ELISPOT assays. We could conclude that these DCs were functionally normal. We then performed autologous MLRs: mature DCs and responder cells (ratiol/10) were cultured the first week in RPMI, autologous serum 10%, IL-12 and 1L-6. Three types of responders were used (CDS, CDS + CD4 and CD4). The mixture CD8/CD4 was used to test for a potential help] effect. In one half of the conditions, IL-12 was added throughout the 6 weeks culture which led to six different culture conditions. IL-12 was added to reverse potential tolerance. The cells were restimulated each week by the same DCs and cultured with IL2 and IL-7, +/- IL 12. At the end, T-cells from the mixed CD8/CD4 cultures were separated into CD4 and CDS cells, to assess the potential of CD8+ cells cultured in the presence of CD4+ cells. T-cells were then co-cultured with CD34-positive cells for 24 hrs with IL2, SCF, IL3 and GCS-F. They were then seeded in a soft agar CFU-GM inhibition assay. After 2 weeks, colonies were counted and individual colonies tested for BCR-ABL expression using RT-PCR. Results: a significant (50%) inhibition of the number of colonies was observed only when CD4+ cells, from the MLRs with IL-12 added throughout, were co-cultured with CD34+ cells. However, the proportion of Ph+/Ph- colonies was unchanged in comparison with the control. We conclude that in our experimental conditions, inhibition of colonies occurs only when CD4+ cells, cultured with IL-12 throughout the whole MLR, are used. This inhibition is not leukemic specific and seems to be directed against autologous hematological determinants. None of our patients being HLA A3 or B8 positive, the absence of CDS response should be interpreted with caution. However, our observations should lead to careful reflection when designing immunotherapy protocols in CML patients.SCOPUS: ar.j