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Basal immunoglobulin signaling actively maintains developmental stage in immature B cells.

Tze LE, Schram BR, Lam KP, Hogquist KA, Hippen KL, Liu J, Shinton SA, Otipoby KL, Rodine PR, Vegoe AL, Kraus M, Hardy RR, Schlissel MS, Rajewsky K, Behrens TW - PLoS Biol. (2005)

Bottom Line: A subsequent functional light chain (LC) rearrangement then results in the surface expression of IgM at the immature B cell stage.Cells undergoing this reversal in development also showed evidence of new LC gene rearrangements, suggesting an important role for basal Ig signaling in the maintenance of LC allelic exclusion.These studies identify a previously unappreciated level of plasticity in the B cell developmental program, and have important implications for our understanding of central tolerance mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Center for Immunology, University of Minnesota Medical School, Minneapolis, Minnesota, USA.

ABSTRACT
In developing B lymphocytes, a successful V(D)J heavy chain (HC) immunoglobulin (Ig) rearrangement establishes HC allelic exclusion and signals pro-B cells to advance in development to the pre-B stage. A subsequent functional light chain (LC) rearrangement then results in the surface expression of IgM at the immature B cell stage. Here we show that interruption of basal IgM signaling in immature B cells, either by the inducible deletion of surface Ig via Cre-mediated excision or by incubating cells with the tyrosine kinase inhibitor herbimycin A or the phosphatidylinositol 3-kinase inhibitor wortmannin, led to a striking "back-differentiation" of cells to an earlier stage in B cell development, characterized by the expression of pro-B cell genes. Cells undergoing this reversal in development also showed evidence of new LC gene rearrangements, suggesting an important role for basal Ig signaling in the maintenance of LC allelic exclusion. These studies identify a previously unappreciated level of plasticity in the B cell developmental program, and have important implications for our understanding of central tolerance mechanisms.

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Gene Expression Phenotype of Cre-Mhi Cell Populations Is Intermediate between Ctrl-Mhi and Cre-Mlo Populations(A) Viable cell counts were performed in control B1-8f/3-83κ and B1-8f/3-83κ/Mx-Cre cell populations at the initiation of culture with medium alone (nil) or with IFNαβ (IFN) 1,000 units/ml, and daily for 3 d. Data are presented as percent of the day 0 cell numbers, and represent 4–7 experiments for each population. Standard errors were less than 10%, and are not shown.(B) BM cells were cultured for 5 d in IL-7, and were then labeled with CFSE and incubated with 1,000 units/ml IFNαβ or, as a proliferation control, 16 ng/ml IL-7 for an additional 3 d. Flow cytometric analysis of CFSE dye dilution of B220+ cells indicated no significant proliferation of IFN-treated cell populations. Numbers represent percent of gated cells.(C) A PCR-based assay was used to quantitate the extent of B1-8f deletion in Ctrl-Mhi, Cre-Mhi, and Cre-Mlo populations. Ctrl-Mhi cells contained 100% intact B1-8f alleles, Cre-Mlo cells were 100% deleted, and Cre-Mhi cells had 34 ± 16% average deletion of the B1-8f allele. Expression values for the Ctrl-Mhi transcripts (significantly different between Ctrl-Mhi and Cre-Mlo) were normalized to 1, and relative expression levels of transcripts up- (n = 184) and down-regulated (n = 143) in the three populations were calculated. In both groups of genes, Cre-Mhi cells showed intermediate levels of gene expression between Ctrl-Mhi and Cre-Mlo, indicating that Cre-Mlo cells originated from the Cre-Mhi population.
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pbio-0030082-g004: Gene Expression Phenotype of Cre-Mhi Cell Populations Is Intermediate between Ctrl-Mhi and Cre-Mlo Populations(A) Viable cell counts were performed in control B1-8f/3-83κ and B1-8f/3-83κ/Mx-Cre cell populations at the initiation of culture with medium alone (nil) or with IFNαβ (IFN) 1,000 units/ml, and daily for 3 d. Data are presented as percent of the day 0 cell numbers, and represent 4–7 experiments for each population. Standard errors were less than 10%, and are not shown.(B) BM cells were cultured for 5 d in IL-7, and were then labeled with CFSE and incubated with 1,000 units/ml IFNαβ or, as a proliferation control, 16 ng/ml IL-7 for an additional 3 d. Flow cytometric analysis of CFSE dye dilution of B220+ cells indicated no significant proliferation of IFN-treated cell populations. Numbers represent percent of gated cells.(C) A PCR-based assay was used to quantitate the extent of B1-8f deletion in Ctrl-Mhi, Cre-Mhi, and Cre-Mlo populations. Ctrl-Mhi cells contained 100% intact B1-8f alleles, Cre-Mlo cells were 100% deleted, and Cre-Mhi cells had 34 ± 16% average deletion of the B1-8f allele. Expression values for the Ctrl-Mhi transcripts (significantly different between Ctrl-Mhi and Cre-Mlo) were normalized to 1, and relative expression levels of transcripts up- (n = 184) and down-regulated (n = 143) in the three populations were calculated. In both groups of genes, Cre-Mhi cells showed intermediate levels of gene expression between Ctrl-Mhi and Cre-Mlo, indicating that Cre-Mlo cells originated from the Cre-Mhi population.

Mentions: It was important to rule out the possibility that the back-differentiation observed in Cre-Mlo cells might be an artifact of selective expansion and/or survival of IgM− cells that were present at the initiation of the IFN cultures. Cell counts throughout the culture period revealed that the overall number of viable cells was not significantly different between control and BCR-deleting populations (Figure 4A). This was confirmed by similar annexin V and 7-amino-actinomycin D (7-AAD) flow cytometric staining profiles (box 4 (nd data not shown). CFSE [5(6)-carboxyfluorescein diacetate succinimidyl ester] labeling indicated that cells cultured with IFNαβ were not proliferating at significant levels (Figure 4B). We also isolated highly purified populations of IgM+ immature B cells prior to incubation with IFN, and found that these cells similarly underwent the back-differentiation response (Figure S1. Thus, these experiments ruled out a selective expansion of pre-existing IgM− cells in the IFN-treated B1-8f/3-83κ/Mx-Cre cultures.


Basal immunoglobulin signaling actively maintains developmental stage in immature B cells.

Tze LE, Schram BR, Lam KP, Hogquist KA, Hippen KL, Liu J, Shinton SA, Otipoby KL, Rodine PR, Vegoe AL, Kraus M, Hardy RR, Schlissel MS, Rajewsky K, Behrens TW - PLoS Biol. (2005)

Gene Expression Phenotype of Cre-Mhi Cell Populations Is Intermediate between Ctrl-Mhi and Cre-Mlo Populations(A) Viable cell counts were performed in control B1-8f/3-83κ and B1-8f/3-83κ/Mx-Cre cell populations at the initiation of culture with medium alone (nil) or with IFNαβ (IFN) 1,000 units/ml, and daily for 3 d. Data are presented as percent of the day 0 cell numbers, and represent 4–7 experiments for each population. Standard errors were less than 10%, and are not shown.(B) BM cells were cultured for 5 d in IL-7, and were then labeled with CFSE and incubated with 1,000 units/ml IFNαβ or, as a proliferation control, 16 ng/ml IL-7 for an additional 3 d. Flow cytometric analysis of CFSE dye dilution of B220+ cells indicated no significant proliferation of IFN-treated cell populations. Numbers represent percent of gated cells.(C) A PCR-based assay was used to quantitate the extent of B1-8f deletion in Ctrl-Mhi, Cre-Mhi, and Cre-Mlo populations. Ctrl-Mhi cells contained 100% intact B1-8f alleles, Cre-Mlo cells were 100% deleted, and Cre-Mhi cells had 34 ± 16% average deletion of the B1-8f allele. Expression values for the Ctrl-Mhi transcripts (significantly different between Ctrl-Mhi and Cre-Mlo) were normalized to 1, and relative expression levels of transcripts up- (n = 184) and down-regulated (n = 143) in the three populations were calculated. In both groups of genes, Cre-Mhi cells showed intermediate levels of gene expression between Ctrl-Mhi and Cre-Mlo, indicating that Cre-Mlo cells originated from the Cre-Mhi population.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1059451&req=5

pbio-0030082-g004: Gene Expression Phenotype of Cre-Mhi Cell Populations Is Intermediate between Ctrl-Mhi and Cre-Mlo Populations(A) Viable cell counts were performed in control B1-8f/3-83κ and B1-8f/3-83κ/Mx-Cre cell populations at the initiation of culture with medium alone (nil) or with IFNαβ (IFN) 1,000 units/ml, and daily for 3 d. Data are presented as percent of the day 0 cell numbers, and represent 4–7 experiments for each population. Standard errors were less than 10%, and are not shown.(B) BM cells were cultured for 5 d in IL-7, and were then labeled with CFSE and incubated with 1,000 units/ml IFNαβ or, as a proliferation control, 16 ng/ml IL-7 for an additional 3 d. Flow cytometric analysis of CFSE dye dilution of B220+ cells indicated no significant proliferation of IFN-treated cell populations. Numbers represent percent of gated cells.(C) A PCR-based assay was used to quantitate the extent of B1-8f deletion in Ctrl-Mhi, Cre-Mhi, and Cre-Mlo populations. Ctrl-Mhi cells contained 100% intact B1-8f alleles, Cre-Mlo cells were 100% deleted, and Cre-Mhi cells had 34 ± 16% average deletion of the B1-8f allele. Expression values for the Ctrl-Mhi transcripts (significantly different between Ctrl-Mhi and Cre-Mlo) were normalized to 1, and relative expression levels of transcripts up- (n = 184) and down-regulated (n = 143) in the three populations were calculated. In both groups of genes, Cre-Mhi cells showed intermediate levels of gene expression between Ctrl-Mhi and Cre-Mlo, indicating that Cre-Mlo cells originated from the Cre-Mhi population.
Mentions: It was important to rule out the possibility that the back-differentiation observed in Cre-Mlo cells might be an artifact of selective expansion and/or survival of IgM− cells that were present at the initiation of the IFN cultures. Cell counts throughout the culture period revealed that the overall number of viable cells was not significantly different between control and BCR-deleting populations (Figure 4A). This was confirmed by similar annexin V and 7-amino-actinomycin D (7-AAD) flow cytometric staining profiles (box 4 (nd data not shown). CFSE [5(6)-carboxyfluorescein diacetate succinimidyl ester] labeling indicated that cells cultured with IFNαβ were not proliferating at significant levels (Figure 4B). We also isolated highly purified populations of IgM+ immature B cells prior to incubation with IFN, and found that these cells similarly underwent the back-differentiation response (Figure S1. Thus, these experiments ruled out a selective expansion of pre-existing IgM− cells in the IFN-treated B1-8f/3-83κ/Mx-Cre cultures.

Bottom Line: A subsequent functional light chain (LC) rearrangement then results in the surface expression of IgM at the immature B cell stage.Cells undergoing this reversal in development also showed evidence of new LC gene rearrangements, suggesting an important role for basal Ig signaling in the maintenance of LC allelic exclusion.These studies identify a previously unappreciated level of plasticity in the B cell developmental program, and have important implications for our understanding of central tolerance mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Center for Immunology, University of Minnesota Medical School, Minneapolis, Minnesota, USA.

ABSTRACT
In developing B lymphocytes, a successful V(D)J heavy chain (HC) immunoglobulin (Ig) rearrangement establishes HC allelic exclusion and signals pro-B cells to advance in development to the pre-B stage. A subsequent functional light chain (LC) rearrangement then results in the surface expression of IgM at the immature B cell stage. Here we show that interruption of basal IgM signaling in immature B cells, either by the inducible deletion of surface Ig via Cre-mediated excision or by incubating cells with the tyrosine kinase inhibitor herbimycin A or the phosphatidylinositol 3-kinase inhibitor wortmannin, led to a striking "back-differentiation" of cells to an earlier stage in B cell development, characterized by the expression of pro-B cell genes. Cells undergoing this reversal in development also showed evidence of new LC gene rearrangements, suggesting an important role for basal Ig signaling in the maintenance of LC allelic exclusion. These studies identify a previously unappreciated level of plasticity in the B cell developmental program, and have important implications for our understanding of central tolerance mechanisms.

Show MeSH