PF368 DYSREGULATION OF BACH2 AND FOXP1 GENES IN T AND B CELLS IS MORE PRONOUNCED IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS COMPARED TO AGED‐MATCHED HEALTHY DONORS WITH AN IMPACT ON SURVIVAL
Authors: P. De Silva, B. Stamatopoulos, V.L. Dang Chi, S. Garaud, R. Francois, V. Thibaud, C. Solinas, A. Boisson, H. Duvillier, R. Rouas, P. Lewalle, K. Willard‐Gallo, D. Bron
Journal: HemaSphere (2019)

Background: We previously investigated BACH2/RPDM1 gene expression in B/T cells from chronic lymphocytic leukemia (CLL) patients and aged‐matched healthy donors (HD) to understand the immunosenescence with aging, which showed that BACH2/PRDM1 are strongly correlated with aging. Recent data show that candidate immune suppressor gene FOXP1 is an important regulator of CLL pathogenesis Aims: We further investigated the significance of BACH2, PRDM1 , FOXP1 and inhibitory molecule expression in T/B cells from CLL patients. Methods: Peripheral blood mononuclear cells were isolated from untreated CLL patients and age‐matched HD using Lymphoprep density gradient centrifugation. T/B cells were purified by magnetic isolation or FACS sorting . BACH2, PRDM1, FOXP1, PD1, PD‐L1 and TIM3 transcripts were quantified using RT‐qPCR. Nuclear labeling of BACH2/PRDM1 and FOXP1 was accomplished using fixed and permeabilized cells, pre‐labeled for membrane markers and acquired by flow cytometry. Prognostic value of BACH2 / PRDM1 and FOXP1 expression in purified CD19 + B cells was analyzed retrospectively in a cohort of CLL patients (n = 270), and correlated with clinical parameters. Results: BACH2 gene expression in HDs is significantly downregulated while PRDM1 gene expression increased in T and B cells according to age groups. BACH2 expression is further reduced in T/B cells from CLL patients compared to age‐matched HDs. Also, PRDM1 is significantly upregulated in T cells from CLL patients but not in leukemic‐B cells. We didn’t observe any age‐related changes in FOXP1 expression. However, among BACH2, FOXP1 and PRDM1 protein expression in lymphocytes from CLL patients, we observe a significant increase of BACH2 and FOXP1 protein expression in CD19 + B cells compared to T cell subsets (n = 21). We analyzed PD1, TIM3 transcript/protein expression in T cells and PD‐L1 expression in B cells to evaluate the exhaustion phenotype associated with CLL and aging. This analysis showed us that PD1 expression is significantly upregulated in CD4 + /CD8 + T cells in the older vs younger HDs (n = 60) and also in T cells from CLL patients (n = 41) when compared with age‐matched HDs both at transcript and protein level. High PD‐L1 mRNA and protein expression were also strongly correlated with increased age in HD B cells with a further increase detected in leukemic B cells. In addition, we found a strong inverse correlation between BACH2 and PD1 in CD4 + /CD8 + T cells; and between BACH2 and PDL1 in CD19 + B cells. TIM3 transcript/protein expression in T cells was very low in CLL patients and not detected in HD T cells. Prognostic value of BACH2/PRDM1 and FOXP1 expression in purified CD19 + B cells was analysed by RT‐qPCR retrospectively in a cohort of CLL patients, and correlated with clinical stage and IGHV mutation status. In this retrospectively analysed CLL cohort, increased BACH2 expression was correlated with improved treatment‐free survival (p = 0.0352). On the contrary, increased FOXP1 expression was correlated with worsened overall survival of CLL patients in the same cohort (p = 0.0165). However, there was no correlation between BACH2/FOXP1 and Binet stage or IGHV mutation status. We didn’t observe any significant correlations with PRDM1 in terms of survival of CLL patients. Summary/Conclusion: The decrease of BACH2 and increase of FOXP1 in leukemic‐B cells impact on CLL patients’ survival. PD1 and PD‐L1 expression in T/B cells from CLL patients and aged‐matched HDs are significantly correlated with aging‐associated exhaustion of immune cells. The role of BACH2 and FOXP1 in tumor microenvironment is thus further investigated.