Program: Oral and Poster Abstracts
Session: 631. Chronic Myeloid Leukemia: Biology and Pathophysiology, excluding Therapy: Poster III
METHODS. G-MDSC (CD11b+CD33+CD14-HLADR- cells) were analyzed in peripheral blood (PB) of 20 healthy donors (HD) and 30 CML patients at diagnosis by cytofluorimetric analysis. Immuno-suppressive activity was tested through incubation of G-MDSC with autologous CFSE-labeled T cells and stimulation with phytohaemagglutinin (PHA). Controls included a positive T cell proliferation control (T cells plus PHA) and a negative one (T cells only). After three days, T cell proliferation was analyzed by flow cytometry. For G-MDSC generation, human peripheral blood mononucleated cells (PBMC) from HD were cultured alone and with MSC of CML (n=10) or HD (n=8) (1:100 ratio). After one week, G-MDSC were isolated using anti-CD66b magnetic microbeads and the phenotype was confirmed by cytofluorimetric analysis. Expression of ARG1, NOS2, PTGS2, TNFα, TGFβ, IL6, IL10, IL1β was also evaluated using real time PCR.
RESULTS. Percentage of cells with a G-MDSC phenotype was greater in PB obtained from CML patients than HD (82.5±9.6% vs 56,2±5.4%, p<0.0001). G-MDSC were able to inhibit T cell proliferation compared to positive control (25±5% vs 48±7.6%, p=0.0057). To investigate if CML MSC may be involved in G-MDSC generation, we incubated HD PBMC with CML or HD MSC for one week. After magnetic isolation, we found that only CML MSC-educated G-MDSC acquired immune-suppressive ability, inhibiting T cell proliferation compared to G-MDSCs control (isolated from PBMC cultured in medium alone) (32±12% vs 63±5.9%, p=0.003). On the contrary, HD MSC-educated G-MDSC did not show any suppressive effect. We also found that CML MSC-educated G-MDSC expressed higher level of the following immune modulatory factors: TNFα (20.8±19.3, p=0.006), IL1β (47.3±25.2, p=0.001), PTGS2 (20.7±10.9, p=0.002) and IL6 (33.8±13.9, p=0.004) compared to HD MSC-educated G-MDSCs (arbitrarily 2-ΔΔCt value: 1). MSC WE also observed ane an up-regulation of PTGS2 (19±4.4, p=0.04), TGFβ (6±3, p=0.01) and IL6 (5±2.8, p=0.04) in CML MSCs at time 0 with a great variability among the patients (calculated value of 2-ΔΔCt in HD MSC was 1). After 48 h of co-culture with PBMC, CML MSC showed statistically significant up-regulation of ARG1 (23.5±11.9, p=0.02), TGFβ (4.8±3, p=0.04), IL10 (5.6±2.8, p=0.03) and IL6 (54.3±23, p=0.02) expression, suggesting that multiple mechanisms are involved in MDSC induction by CML MSC.
CONCLUSION. Our work demonstrates that CML MSCs are able to activate MDSCs favoring cancer immune evasion in CML patients.
Disclosures: Palumbo: Novartis: Honoraria , Other: Advisory Board .
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