Myeloid lineage switch of Pax5 mutant but not wild-type B cell progenitors by C/EBPalpha and GATA factors

Abstract

The developmental potential of hematopoietic progenitors is restricted early on to either the erythromyeloid or lymphoid lineages. The broad developmental potential of Pax5(-/-) pro-B cells is in apparent conflict with such a strict separation, although these progenitors realize the myeloid and erythroid potential with lower efficiency compared to the lymphoid cell fates. Here we demonstrate that ectopic expression of the transcription factors C/EBPalpha, GATA1, GATA2 and GATA3 strongly promoted in vitro macrophage differentiation and myeloid colony formation of Pax5(-/-) pro-B cells. GATA2 and GATA3 expression also resulted in efficient engraftment and myeloid development of Pax5(-/-) pro-B cells in vivo. The myeloid transdifferentiation of Pax5(-/-) pro-B cells was accompanied by the rapid activation of myeloid genes and concomitant repression of B-lymphoid genes by C/EBPalpha and GATA factors. These data identify the Pax5(-/-) pro-B cells as lymphoid progenitors with a latent myeloid potential that can be efficiently activated by myeloid transcription factors. The same regulators were unable to induce a myeloid lineage switch in Pax5(+/+) pro-B cells, indicating that Pax5 dominates over myeloid transcription factors in B-lymphocytes.

Fig. 1. GATA factors rapidly induce myeloid differentiation of Pax5^–/– pro-B cells in vivo. (A) Retroviral vectors. Full-length cDNA coding for C/EBPα or GATA transcription factors (TF) was inserted into the parental vector MiCD2, which uses an internal ribosomal entry sequence (IRES) to express a human (h) CD2 protein lacking its intracellular domain. (B) Pax5^–/– pro-B cells were infected with the indicated retroviruses, and sorted hCD2⁺ cells were injected into sublethally irradiated RAG2^–/– mice. Twelve days after transfer, the donor cell contribution was investigated by flow cytometric analysis of the bone marrow and spleen. The expression of Gr-1 and Mac-1 is displayed for the hCD2⁺ cells of donor origin, and the percentages of cells in each gate or quadrant are indicated. Similar results were obtained for each retrovirus by analyzing four mice, which were generated in two separate transplantation experiments.

Fig. 2. GATA2 and GATA3 promote efficient engraftment and myeloid development of Pax5^–/– pro-B cells in the bone marrow. Following infection with the indicated retroviruses, hCD2⁺ Pax5^–/– pro-B cells were injected into sublethally irradiated RAG2^–/– mice. Twenty-one days after transfer, the bone marrow was analyzed by flow cytometry, and the expression of B220/c-Kit and Gr-1/Mac-1 is shown for the hCD2⁺ cells together with the percentages of the different cell populations. The relative ratio of hCD2⁺c-Kit⁺B220⁺ pro-B cells was determined by multiplying the percentage of hCD2⁺ cells with the percentage of c-Kit⁺B220⁺ cells (MiCD2, 8.7%; GATA1, 1.6%; GATA2, 12.6%; GATA3, 6%; C/EBPα, 4.1%). Similar results were obtained for each retrovirus by analyzing 5–9 mice, which were generated in 2–4 independent transplantation experiments. The absence of myeloid differentiation of GATA1 or C/EBPα virus-infected cells may indicate a strong selection for mutation of the GATA1 and C/EBPα proteins, which otherwise seem to interfere with the engraftment of Pax5^–/– pro-B cells in the bone marrow.

Fig. 3. Myeloid colony formation of Pax5^–/– pro-B cells upon expression of C/EBPα and GATA factors. (A) Plating efficiency in methyl cellulose. Infected hCD2⁺ Pax5^–/– pro-B cells were seeded in methylcellulose medium containing IL-3, IL-6, SCF and erythropoietin (Epo) or only SCF and Epo. The colonies were counted after 4–5 or 8 days, respectively, and are indicated as percentage of the input cells. The number (n) of independent experiments and the corresponding standard deviation are shown. (B) Gr-1/Mac-1 expression. Pax5^–/– cells infected with the GATA1 virus were incubated for 8 days in methylcellulose with the indicated cytokines and then collected from the entire Petri dish prior to flow cytometric analysis. (C–E) Cytospin preparation of May–Grünwald Giemsa-stained cells. Pax5^–/– cells infected with the GATA1 (C and E) or GATA2 (D) retrovirus were cultured in methylcellulose under the indicated cytokine conditions for 8 (C and D) or 10 (E) days followed by May–Grünwald Giemsa and benzidine staining. Erythroblasts (E) were identified as benzidine-stained cells (brown). (F and G) Increased cell survival upon GATA3 expression. Pax5^–/– pro-B cells were infected for 3 days with the GATA3 virus and then incubated for 2 days in the presence (F) or absence (G) of stromal ST2 cells and IL-7 prior to flow cytometric analysis.

Fig. 4. C/EBPα and GATA factors induce macrophage differentiation of Pax5^–/– pro-B cells under lymphoid culture conditions. (A and B) In vitro macrophage differentiation. The infection of Pax5^–/– pro-B cells was initiated at day 0 by co-culture with virus-producing GP+E-86 cells in IL-7 medium (for 3 days) followed by the subsequent propagation on stromal ST2 cells in the presence of IL-7. At day 5, expression of c-Kit was analyzed on infected hCD2⁺ (gray surface) and non-infected hCD2^– (black line) cells by flow cytometry, while the Mac-1 and F4/80 expression profiles at day 5 and 14 were displayed only for the infected hCD2⁺ cells. (C) Morphology and phagocytosis of in vitro differentiated cells. The morphological appearance and phagocytic activity of Pax5^–/– cells was revealed at day 7 and 14 after MiCD2 or C/EBPα virus infection by May–Grünwald Giemsa staining or incubation with FITC-labeled E.coli, respectively.

Fig. 5. C/EBPα and GATA factors fail to induce myeloid differentiation in wild-type pro-B cells. Bone marrow pro-B cells from wild-type mice were infected with the indicated retroviruses and cultured in the presence of IL-7 and stromal cells for 14 days prior to flow cytometric analysis.

Fig. 6. Gene expression changes in wild-type and Pax5^–/– pro-B cells upon expression of C/EBPα and GATA factors. Wild-type (+/+) and Pax5 mutant (–/–) pro-B cells were infected in IL-7 medium for 3 days on GP+E-86 cells producing the indicated retroviruses. Immediately after infection, the hCD2⁺ cells were sorted, and total RNA was prepared. Transcripts of the indicated genes were quantitated by RT–PCR, and the PCR products were visualized on agarose gels by ethidium bromide staining. Retroviral transcripts are indicated by asterisks.