Zbtb1 prevents default myeloid differentiation of lymphoid-primed multipotent progenitors

Abstract

Zbtb1 is a transcription factor that prevents DNA damage and p53-mediated apoptosis in replicating immune progenitors, affecting lymphoid as well as myeloid development when hematopoietic progenitors are in competition in mixed bone marrow chimeras. However, Zbtb1-deficient mice do not have an apparent myeloid deficiency. We report here that Zbtb1-deficient lymphoid-primed multipotent progenitors (LMPPs) are biased to develop towards the myeloid fate in detriment of lymphoid development, contributing to the apparent unaffected myeloid development. Zbtb1 expression was maintained during lymphoid development of LMPP cells but downregulated during myeloid development. Deficiency of Zbtb1 in LMPP cells was sufficient to direct a myeloid fate in lymphoid-inducing conditions and in the absence of myeloid cytokines as shown by upregulation of a myeloid gene signature and the generation of myeloid cells in vitro. Finally, biased myeloid differentiation of Zbtb1-deficient LMPP cells was not due to increased p53-dependent apoptosis as it was not reverted by transgenic Bcl2 expression or p53 deficiency. Altogether, our results show that Zbtb1 expression prevents activation of a default myeloid program in LMPP cells, ensuring the generation of lymphoid cells.

Keywords: Immune response; Immunity; Immunology and Microbiology Section; Zbtb1; development; differentiation; lymphoid; myeloid.

Figure 1. ScanT mice have a proportionally increased myeloid compartment

A. FACs analysis of cell suspensions isolated from the indicated tissues. The numbers correspond to the proportion of events within the gates. B. Analysis of total cell numbers in the indicated tissues. C. Analysis of the proportion of CD11b⁺GR1⁺ obtained from the indicated tissues. D. Analysis of relative change in GFP levels from Zbtb1-GFP reporter mice. GFP MFI was measured in the indicated conditions before and after culture of cells in OP9-DL1 (T-cell condition); OP9 (B-cell condition) or OP9 plus myeloid cytokines (myeloid condition) for the indicated times. Represented data corresponds to the relative change MFI of Zbtb1-GFP/MFI of C57BL/6 at the indicated times. Data is representative of more than 3 independent experiments. p values corresponding to the significance using T-test are shown.

Figure 2. Zbtb1 prevents a default myeloid differentiation of LMPP in lymphoid inducing conditions

A. FACS analysis of cells obtained after co-cultured with OP9 or OP9-DL1 stromal cells in the presence of lymphoid (IL-7 and Flt3l) and absence of myeloid cytokines for 8 days to initiate lymphoid development. The numbers indicate the proportion of cells obtained within the gates. B. Representative Giemsa staining showing the morphology of cells obtained in the co-cultures represented in (A). C. FACs analysis of cells obtained after a 3-day co-culture of sorted LMPP cells. The numbers indicate the proportion of cells obtained within the gates. D. Gram plot showing the number of cells obtained during the co-cultures represented in (C). Each dot corresponds to data from a mouse. Horizontal bars represent the mean. E. Gram plot showing the number of myeloid colonies obtained after culture of sorted LMPP cells in methylcellulose in the presence of myeloid cytokines. Each dot corresponds to data from a mouse.

Figure 3. Zbtb1 represses the myeloid program in LMPP cells

A. Diagram showing the cell samples LMPP cells ex-vivo and after 18hs culture with OP9-DL1 in lymphoid inducing conditions, which were used for RNAseq assays. FACS analysis showing the phenotype of LMPP cells and percentage of annexin V+ cells after OP9-DL1 co-culture. The numbers indicate the proportion of cells within the gate. B. Gene Set Enrichment Analysis (GSEA) of pre-sorted genes showing differential expression between wild type and ScanT cells. The plots represent the gene enrichment observed in ScanT LMPP cells for genes expressed in the indicated immune subsets. C. Heatmap of selected myeloid signature genes represented in (B) showing the expression value in LMPP cells ex vivo and after culture. D. RT-PCR of selected genes to validate the RNAseq data. Data is representative of 3 independent experiments. p values corresponding to the significance using T-test are shown.

Figure 4. Protection from apoptosis or p53-deficiency does not rescue the myeloid bias of ScanT LMPP cells

A. FACs analysis of cells obtained after a 3-day co-culture of sorted LMPP cells with OP9-DL1 cells. The numbers indicate the proportion of cells obtained within the gates. B. Analysis of myeloid (CD11b⁺) cell numbers obtained after culture. Data is representative of 3 independent experiments. p values corresponding to the significance using T-test are shown.