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Combined heterozygous loss of Ebf1 and Pax5 allows for T-lineage conversion of B cell progenitors.

Ungerbäck J, Åhsberg J, Strid T, Somasundaram R, Sigvardsson M - J. Exp. Med. (2015)

Bottom Line: Whereas combined reduction of Pax5 and Ebf1 had minimal impact on the development of the earliest CD19(+) progenitors, these cells displayed an increased T cell potential in vivo and in vitro.This report stresses the importance of the levels of transcription factor expression during lymphocyte development, and suggests that Pax5 and Ebf1 collaborate to modulate the transcriptional response to Notch signaling.This provides an insight on how transcription factors like Ebf1 and Pax5 preserve cellular identity during differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical and Experimental Medicine, Experimental Hematopoiesis Unit, Faculty of Health Sciences, Linköping University, 58183 Linköping, Sweden.

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Combined heterozygous loss of Pax5 and Ebf1 results in Notch-dependent lineage plasticity in pro–B cells at the single cell level. (A) Representative FACS plots of sorted Wt and Pax5+/−Ebf1+/− pro–B cells after 14 d of T cell–inducing co-culture on OP9-DL1 stroma cells. (B) Cellular composition of clones from 10 Wt, Pax5+/−, Ebf1+/−, or Pax5+/Ebf1+/− (TH) or single Wt or Pax5+/−Ebf1+/− pro–B cells (Lin−B220+CD19+CD43highIgM−) incubated for 14 d on OP9-DL1 cells to stimulate T cell development, with or without the γ-secretase inhibitor DAPT. Total number of wells analyzed from co-culture of OP9-DL1 and 10 pro–B cells are Wt (51), Pax5+/− (14), Ebf1+/− (38), and Pax5+/−Ebf1+/− (86) and co-culture of OP9-DL1 and one pro–B cell are Wt (12) and Pax5+/−Ebf1+/− (15) collected from 2–3 independent experiments. Similarly, the number of wells analyzed after co-culture of OP9-DL1 and 10 pro–B cells in the presence of DAPT are Wt (21), Pax5+/− (2), Ebf1+/− (14), Pax5+/−Ebf1+/− (17). CD19 cells were scored as CD19+Thy1−CD3−, Thy1 cells as CD19−Thy1+CD3−, and CD3 as CD19−Thy1+CD3+. (C) The graphs display gene expression analyzed by Q-PCR in cultures derived from Wt (4 wells) and Pax5+/−Ebf1+/− (4 wells) after 14 d of T cell–inducing co-culture on OP9-DL1 stroma cells. Each square represents one well, analyzed in triplicate Q-PCR reactions. Mean of all wells is presented as a horizontal line. Statistical analysis was performed using unpaired Student’s t test. N.D = no samples in this group showed detectable expression after 45 cycles of PCR. (D) Representative FACS plot of Pax5+/−Ebf1+/−(TH) CD43highIgM− cells cultivated for 31 d on OP9-DL1 supplied with IL-7, kit ligand, and Flt3 ligand day 0–21. On days 22–31, IL-7 was substituted with IL-2 to further stimulate T cell development. The data are representative of three cultures from two experiments. (E) Dot plots representing the cellular content of cell cultures after transduction with either a pMIG (GFP) control or Ebf1 encoding retrovirus (Ebf1-pMIG) in Pax5+/Ebf1+/− (TH) pro–B cells after 14 d of co-culture with OP9-DL1. The data are collected from pro–B sorted from five different animals. Statistical analysis was performed using unpaired Student’s t test. **, P < 0.01; ****, P < 0.0001.
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fig2: Combined heterozygous loss of Pax5 and Ebf1 results in Notch-dependent lineage plasticity in pro–B cells at the single cell level. (A) Representative FACS plots of sorted Wt and Pax5+/−Ebf1+/− pro–B cells after 14 d of T cell–inducing co-culture on OP9-DL1 stroma cells. (B) Cellular composition of clones from 10 Wt, Pax5+/−, Ebf1+/−, or Pax5+/Ebf1+/− (TH) or single Wt or Pax5+/−Ebf1+/− pro–B cells (Lin−B220+CD19+CD43highIgM−) incubated for 14 d on OP9-DL1 cells to stimulate T cell development, with or without the γ-secretase inhibitor DAPT. Total number of wells analyzed from co-culture of OP9-DL1 and 10 pro–B cells are Wt (51), Pax5+/− (14), Ebf1+/− (38), and Pax5+/−Ebf1+/− (86) and co-culture of OP9-DL1 and one pro–B cell are Wt (12) and Pax5+/−Ebf1+/− (15) collected from 2–3 independent experiments. Similarly, the number of wells analyzed after co-culture of OP9-DL1 and 10 pro–B cells in the presence of DAPT are Wt (21), Pax5+/− (2), Ebf1+/− (14), Pax5+/−Ebf1+/− (17). CD19 cells were scored as CD19+Thy1−CD3−, Thy1 cells as CD19−Thy1+CD3−, and CD3 as CD19−Thy1+CD3+. (C) The graphs display gene expression analyzed by Q-PCR in cultures derived from Wt (4 wells) and Pax5+/−Ebf1+/− (4 wells) after 14 d of T cell–inducing co-culture on OP9-DL1 stroma cells. Each square represents one well, analyzed in triplicate Q-PCR reactions. Mean of all wells is presented as a horizontal line. Statistical analysis was performed using unpaired Student’s t test. N.D = no samples in this group showed detectable expression after 45 cycles of PCR. (D) Representative FACS plot of Pax5+/−Ebf1+/−(TH) CD43highIgM− cells cultivated for 31 d on OP9-DL1 supplied with IL-7, kit ligand, and Flt3 ligand day 0–21. On days 22–31, IL-7 was substituted with IL-2 to further stimulate T cell development. The data are representative of three cultures from two experiments. (E) Dot plots representing the cellular content of cell cultures after transduction with either a pMIG (GFP) control or Ebf1 encoding retrovirus (Ebf1-pMIG) in Pax5+/Ebf1+/− (TH) pro–B cells after 14 d of co-culture with OP9-DL1. The data are collected from pro–B sorted from five different animals. Statistical analysis was performed using unpaired Student’s t test. **, P < 0.01; ****, P < 0.0001.

Mentions: The finding that CD19+ progenitors from Pax5+/−Ebf1+/− mice generated T-lineage cells in vivo suggest that even though these cells express CD19, they are not stably committed to B-lineage cell fate. To investigate the lineage fidelity of B cell progenitors generated with reduced levels of Pax5 and Ebf1 in more detail, we sorted pro–B cells and seeded them on OP9-DL1 stroma cells, generating conditions permissive for T-lineage development of noncommitted progenitors (Schmitt and Zuñiga-Pflucker, 2002). Seeding 10 Wt pro–B cells per well, we detected growth in 32% of the wells. The overall cloning frequency of Pax5+/− cells was 7%, a decrease as compared with Wt cells (P < 0.0001), whereas ∼24% of the wells seeded with Ebf1+/− cells generated colonies, suggesting that the cloning frequency was reduced by loss of one allele of Pax5. Pax5+/−Ebf1+/− cells generated colonies in almost 50% of the wells. Upon analysis of the cellular content of the cultures, 100% of the colonies generated from Wt progenitors contained only CD19+ cells, whereas wells seeded with Pax5+/− or Ebf1+/− cells generated a low frequency of colonies with CD19− cells expressing Thy1.2 (Fig. 2, A and B). Analysis of the content in wells seeded with pro–B cells from Pax5+/−Ebf1+/− mice revealed that the majority of these cultures were composed of a mixture of cells, including CD19−Thy1.2+ and CD3+ cells (Fig. 2, A and B), supporting an increased T cell potential in Pax5+/−Ebf1+/− as compared with Wt (P < 0.0001) or single heterozygous mice. To verify plasticity at the single-cell level, we seeded single Wt or Pax5+/−Ebf1+/− pro–B cells on OP9-DL1 stromal cells. Analysis of the cellular content of the single cell cultures revealed that although all the Wt cells generated CD19+ cells, a majority of the Pax5+/−Ebf1+/− cells generated mixed colonies (Fig. 2 B), revealing plasticity at the single-cell level. Inclusion of the γ-secretase inhibitor DAPT in the cell cultures inhibited the formation of CD19−Thy1.2+ cells, confirming a need of Notch signaling to initiate the phenotypic change (Fig. 2 B). We could also verify that the plasticity was critically dependent on the Ebf1 dose in the pro–B cells because retroviral transduction of Pax5+/−Ebf1+/− pro–B cells with an Ebf1 encoding retrovirus blocked the formation of CD19−Thy1.2+ cells (Fig. 2 E). Even though high expression of Thy1.2 and CD3 normally serves as reliable markers for cells that have entered the T-lineage pathway, we extended our analysis by performing a Q-PCR analysis of colonies containing either CD19+ or Thy1.2+ cells generated from Wt pro–B cells or from Pax5+/−Ebf1+/− pro–B cells, respectively. Although the expression of B-lineage genes such as CD19 and Pax5 was decreased as compared with Wt cells cultured under the same conditions (Fig. 2 C), analysis of the levels of T cell–associated transcripts including CD3e, Pre-Tα, Lck, and CD27 suggested that these genes were all expressed in the generated Thy1.2+ cells, verifying that the cells had initiated development toward T-lineage. Culturing Wt or Pax5+/−Ebf1+/− pro–B cells on OP9-DL1 cells for 21 d, followed by replacement of IL-7 with IL-2 for 10 additional days, resulted in cell death among the Wt cells, whereas the Pax5+/−Ebf1+/− cells generated offspring expressing TCRβ on the surface (Fig. 2 D).


Combined heterozygous loss of Ebf1 and Pax5 allows for T-lineage conversion of B cell progenitors.

Ungerbäck J, Åhsberg J, Strid T, Somasundaram R, Sigvardsson M - J. Exp. Med. (2015)

Combined heterozygous loss of Pax5 and Ebf1 results in Notch-dependent lineage plasticity in pro–B cells at the single cell level. (A) Representative FACS plots of sorted Wt and Pax5+/−Ebf1+/− pro–B cells after 14 d of T cell–inducing co-culture on OP9-DL1 stroma cells. (B) Cellular composition of clones from 10 Wt, Pax5+/−, Ebf1+/−, or Pax5+/Ebf1+/− (TH) or single Wt or Pax5+/−Ebf1+/− pro–B cells (Lin−B220+CD19+CD43highIgM−) incubated for 14 d on OP9-DL1 cells to stimulate T cell development, with or without the γ-secretase inhibitor DAPT. Total number of wells analyzed from co-culture of OP9-DL1 and 10 pro–B cells are Wt (51), Pax5+/− (14), Ebf1+/− (38), and Pax5+/−Ebf1+/− (86) and co-culture of OP9-DL1 and one pro–B cell are Wt (12) and Pax5+/−Ebf1+/− (15) collected from 2–3 independent experiments. Similarly, the number of wells analyzed after co-culture of OP9-DL1 and 10 pro–B cells in the presence of DAPT are Wt (21), Pax5+/− (2), Ebf1+/− (14), Pax5+/−Ebf1+/− (17). CD19 cells were scored as CD19+Thy1−CD3−, Thy1 cells as CD19−Thy1+CD3−, and CD3 as CD19−Thy1+CD3+. (C) The graphs display gene expression analyzed by Q-PCR in cultures derived from Wt (4 wells) and Pax5+/−Ebf1+/− (4 wells) after 14 d of T cell–inducing co-culture on OP9-DL1 stroma cells. Each square represents one well, analyzed in triplicate Q-PCR reactions. Mean of all wells is presented as a horizontal line. Statistical analysis was performed using unpaired Student’s t test. N.D = no samples in this group showed detectable expression after 45 cycles of PCR. (D) Representative FACS plot of Pax5+/−Ebf1+/−(TH) CD43highIgM− cells cultivated for 31 d on OP9-DL1 supplied with IL-7, kit ligand, and Flt3 ligand day 0–21. On days 22–31, IL-7 was substituted with IL-2 to further stimulate T cell development. The data are representative of three cultures from two experiments. (E) Dot plots representing the cellular content of cell cultures after transduction with either a pMIG (GFP) control or Ebf1 encoding retrovirus (Ebf1-pMIG) in Pax5+/Ebf1+/− (TH) pro–B cells after 14 d of co-culture with OP9-DL1. The data are collected from pro–B sorted from five different animals. Statistical analysis was performed using unpaired Student’s t test. **, P < 0.01; ****, P < 0.0001.
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fig2: Combined heterozygous loss of Pax5 and Ebf1 results in Notch-dependent lineage plasticity in pro–B cells at the single cell level. (A) Representative FACS plots of sorted Wt and Pax5+/−Ebf1+/− pro–B cells after 14 d of T cell–inducing co-culture on OP9-DL1 stroma cells. (B) Cellular composition of clones from 10 Wt, Pax5+/−, Ebf1+/−, or Pax5+/Ebf1+/− (TH) or single Wt or Pax5+/−Ebf1+/− pro–B cells (Lin−B220+CD19+CD43highIgM−) incubated for 14 d on OP9-DL1 cells to stimulate T cell development, with or without the γ-secretase inhibitor DAPT. Total number of wells analyzed from co-culture of OP9-DL1 and 10 pro–B cells are Wt (51), Pax5+/− (14), Ebf1+/− (38), and Pax5+/−Ebf1+/− (86) and co-culture of OP9-DL1 and one pro–B cell are Wt (12) and Pax5+/−Ebf1+/− (15) collected from 2–3 independent experiments. Similarly, the number of wells analyzed after co-culture of OP9-DL1 and 10 pro–B cells in the presence of DAPT are Wt (21), Pax5+/− (2), Ebf1+/− (14), Pax5+/−Ebf1+/− (17). CD19 cells were scored as CD19+Thy1−CD3−, Thy1 cells as CD19−Thy1+CD3−, and CD3 as CD19−Thy1+CD3+. (C) The graphs display gene expression analyzed by Q-PCR in cultures derived from Wt (4 wells) and Pax5+/−Ebf1+/− (4 wells) after 14 d of T cell–inducing co-culture on OP9-DL1 stroma cells. Each square represents one well, analyzed in triplicate Q-PCR reactions. Mean of all wells is presented as a horizontal line. Statistical analysis was performed using unpaired Student’s t test. N.D = no samples in this group showed detectable expression after 45 cycles of PCR. (D) Representative FACS plot of Pax5+/−Ebf1+/−(TH) CD43highIgM− cells cultivated for 31 d on OP9-DL1 supplied with IL-7, kit ligand, and Flt3 ligand day 0–21. On days 22–31, IL-7 was substituted with IL-2 to further stimulate T cell development. The data are representative of three cultures from two experiments. (E) Dot plots representing the cellular content of cell cultures after transduction with either a pMIG (GFP) control or Ebf1 encoding retrovirus (Ebf1-pMIG) in Pax5+/Ebf1+/− (TH) pro–B cells after 14 d of co-culture with OP9-DL1. The data are collected from pro–B sorted from five different animals. Statistical analysis was performed using unpaired Student’s t test. **, P < 0.01; ****, P < 0.0001.
Mentions: The finding that CD19+ progenitors from Pax5+/−Ebf1+/− mice generated T-lineage cells in vivo suggest that even though these cells express CD19, they are not stably committed to B-lineage cell fate. To investigate the lineage fidelity of B cell progenitors generated with reduced levels of Pax5 and Ebf1 in more detail, we sorted pro–B cells and seeded them on OP9-DL1 stroma cells, generating conditions permissive for T-lineage development of noncommitted progenitors (Schmitt and Zuñiga-Pflucker, 2002). Seeding 10 Wt pro–B cells per well, we detected growth in 32% of the wells. The overall cloning frequency of Pax5+/− cells was 7%, a decrease as compared with Wt cells (P < 0.0001), whereas ∼24% of the wells seeded with Ebf1+/− cells generated colonies, suggesting that the cloning frequency was reduced by loss of one allele of Pax5. Pax5+/−Ebf1+/− cells generated colonies in almost 50% of the wells. Upon analysis of the cellular content of the cultures, 100% of the colonies generated from Wt progenitors contained only CD19+ cells, whereas wells seeded with Pax5+/− or Ebf1+/− cells generated a low frequency of colonies with CD19− cells expressing Thy1.2 (Fig. 2, A and B). Analysis of the content in wells seeded with pro–B cells from Pax5+/−Ebf1+/− mice revealed that the majority of these cultures were composed of a mixture of cells, including CD19−Thy1.2+ and CD3+ cells (Fig. 2, A and B), supporting an increased T cell potential in Pax5+/−Ebf1+/− as compared with Wt (P < 0.0001) or single heterozygous mice. To verify plasticity at the single-cell level, we seeded single Wt or Pax5+/−Ebf1+/− pro–B cells on OP9-DL1 stromal cells. Analysis of the cellular content of the single cell cultures revealed that although all the Wt cells generated CD19+ cells, a majority of the Pax5+/−Ebf1+/− cells generated mixed colonies (Fig. 2 B), revealing plasticity at the single-cell level. Inclusion of the γ-secretase inhibitor DAPT in the cell cultures inhibited the formation of CD19−Thy1.2+ cells, confirming a need of Notch signaling to initiate the phenotypic change (Fig. 2 B). We could also verify that the plasticity was critically dependent on the Ebf1 dose in the pro–B cells because retroviral transduction of Pax5+/−Ebf1+/− pro–B cells with an Ebf1 encoding retrovirus blocked the formation of CD19−Thy1.2+ cells (Fig. 2 E). Even though high expression of Thy1.2 and CD3 normally serves as reliable markers for cells that have entered the T-lineage pathway, we extended our analysis by performing a Q-PCR analysis of colonies containing either CD19+ or Thy1.2+ cells generated from Wt pro–B cells or from Pax5+/−Ebf1+/− pro–B cells, respectively. Although the expression of B-lineage genes such as CD19 and Pax5 was decreased as compared with Wt cells cultured under the same conditions (Fig. 2 C), analysis of the levels of T cell–associated transcripts including CD3e, Pre-Tα, Lck, and CD27 suggested that these genes were all expressed in the generated Thy1.2+ cells, verifying that the cells had initiated development toward T-lineage. Culturing Wt or Pax5+/−Ebf1+/− pro–B cells on OP9-DL1 cells for 21 d, followed by replacement of IL-7 with IL-2 for 10 additional days, resulted in cell death among the Wt cells, whereas the Pax5+/−Ebf1+/− cells generated offspring expressing TCRβ on the surface (Fig. 2 D).

Bottom Line: Whereas combined reduction of Pax5 and Ebf1 had minimal impact on the development of the earliest CD19(+) progenitors, these cells displayed an increased T cell potential in vivo and in vitro.This report stresses the importance of the levels of transcription factor expression during lymphocyte development, and suggests that Pax5 and Ebf1 collaborate to modulate the transcriptional response to Notch signaling.This provides an insight on how transcription factors like Ebf1 and Pax5 preserve cellular identity during differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical and Experimental Medicine, Experimental Hematopoiesis Unit, Faculty of Health Sciences, Linköping University, 58183 Linköping, Sweden.

Show MeSH
Related in: MedlinePlus