mTOR-Dependent and Independent Survival Signaling by PI3K in B Lymphocytes

<div><p>Peripheral B lymphocyte survival requires the B cell receptor (BCR) and B cell activating factor (BAFF) binding to its receptor (BAFF-R). Deletion of the BCR, or its signal transducing chaperone Igβ, leads to rapid loss of mature B cells, indicating that signals initiated at the BCR are crucial for B cell survival. BAFF or BAFF-R deficiency also significantly reduces the numbers of mature B cells despite normal BCR expression. Together, these observations indicate that continued BCR and BAFF-R signaling are essential for the survival of mature resting B cells in the periphery. Here we demonstrate that tonic BCR signals up-regulate p100 (Nfkb2) as well as Mcl-1 protein expression at a post-transcriptional level via a PI3K-dependent pathway. p100 expression is mTOR-independent, whereas Mcl-1 expression is mTOR-dependent. BAFF treatment further elevated Mcl-1 levels by an mTOR-independent pathway, while consuming p100. Accordingly, Mcl-1 induction by BAFF is abrogated in <i>Nfkb2</i>^<i>-/-</i> B cells. We propose that the cumulative effects of the BCR and BAFF-R signaling pathways increase Mcl-1 levels beyond the threshold required for B cell survival.</p></div

Signaling pathway to BCR-induced Mcl-1 expression.

<p>(A, B) CD43^- splenic B cells from Bim^-/- mice were treated with anti-IgM F(ab’)₂ (15μg/ml) for various times in the presence or absence of Syk kinase inhibitor R406 (4μM), Bruton’s tyrosine kinase inhibitor Ibrutinib (PCI-32765) (20nM), or TORC1 inhibitor rapamycin (50nM). Whole cell extracts were fractionated by SDS-PAGE and Mcl-1 protein was analyzed by immunoblotting. β-actin was used as a loading control to normalize between samples. (C, D) CD43^- splenic B cells from BL6, BCAP^-/- (C) or CD19^-/- (D) mice were cultured for the indicated times with or without anti-IgM F(ab’)₂ (15μg/ml). Whole cell extracts were prepared and assayed for Mcl-1 levels by Western blot analysis. β-actin was used as a loading control to normalize between samples. Representative gels from 3 independent experiments are shown for A and B and 2 independent experiments for C and D. Cell viability profile of BCAP^-/- and CD19^-/- B cells are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146955#pone.0146955.s004" target="_blank">S4 Fig</a>.</p

PI3K-dependent <i>Nfkb2</i> transcription and p100 protein expression.

<p>CD43^- B cells from BL6 or Bim^-/- mice were incubated for various times with or without anti-IgM F(ab’)₂ fragment (15ug/ml) in the presence or absence of the PI3K inhibitors LY294002 (20μM) or ZSTK474 (ZS 0.1μM, 0.5μM, 1μM and 2.5μM). (A, C, D, F). Cytosolic (CE) or whole cell (WCE) extracts were fractionated by SDS-PAGE and p100 protein expression was analyzed by immunoblotting. β-actin was used to normalize between samples. (B, E) <i>Nfkb2</i> mRNA levels were determined by quantitative RT-PCR and <i>Nfkb2</i> mRNA expression was normalized to β-actin. mRNA fold-change (Y axis) was calculated relative to the levels in untreated cells at 0h. The average of 3 independent experiments is shown. Error bars represent the standard error of the mean between experiments with statistical comparison between untreated cells and LY treated or anti-IgM treated with anti-IgM+LY treated cells. P values were calculated using paired TTEST in Microsoft Office Excel (2013) with two tailed distribution. Ns = not significant. Cell viability profiles are noted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146955#pone.0146955.s003" target="_blank">S3 Fig</a>.</p

Role of PI3K pathway in Mcl-1 up-regulation by the BCR.

<p>CD43^- splenic B cells from BL6 (A-C) or Bim^-/- (D-F) mice were left untreated or treated with anti-IgM F(ab’)₂ (15μg/ml) for various times as indicated, in the presence or absence of the PI3K inhibitor LY294002 (20μM) or ZSTK474 (ZS, 1μM and 2.5μM). (A, C, D, F) Whole cell extracts were fractionated by SDS-PAGE and Mcl-1 expression has analyzed by immunoblotting. β-actin was used to normalize between samples. (B, E) Mcl-1 mRNA levels were determined by quantitative RT-PCR; β-actin served as a normalizing control between samples. mRNA fold-change (Y axis) was calculated relative to the mRNA levels in untreated cells at 0h. The average of 3 independent experiments is shown. Error bars represent the standard error of the mean between experiments. P values were calculated using paired TTEST in Microsoft Office Excel (2013) with two tailed distribution. Ns = not significant. Cell viability profile for all conditions are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146955#pone.0146955.s003" target="_blank">S3 Fig</a>.</p

Rapamycin-sensitive and insensitive pathways to Mcl-1 up-regulation by BAFF.

<p>(A-D) CD43^- splenic B cells from BL6 or Bim^-/- mice were treated with BAFF (200ng/ml) for various times in the presence or absence of the PI3K inhibitor LY294002 (20μM) or rapamycin (50nM) as indicated. anti-IgM F(ab’)₂ (15ug/ml) was included in some experiments as indicated. Whole cell extract (WCE) (A, D) and nuclear extracts (NE) (B) were fractionated by SDS-PAGE and assayed for Mcl-1 or p52 proteins by immunoblotting. β-actin was used to normalize between samples. Gel in part B shows representative Western blot from four independent experiments. p52 levels were quantified as described in the methods section, and the average of four independent experiments is shown in part C. The level of p52 at 0h was assigned the value of 1 (Y axis). (E, F) CD43^- splenic B cells from BL6 and Nfkb2^-/- mice were left untreated or treated with BAFF (200ng/ml) or anti-IgM F(ab’)₂ (15μg/ml) for the indicated times. Whole cell extract (WCE) (E) was fractionated by SDS-PAGE and assayed for Mcl-1 protein by immunoblotting and vinculin was used to normalize between samples. (F) Mcl-1 mRNA levels were determined by quantitative RT-PCR normalized to β-actin. The average of 2 independent experiments is shown. P values were calculated using paired TTEST in Microsoft Office Excel (2013) with two tailed distribution. Ns = not significant.</p

Signaling pathway to BCR-induced p100 expression.

<p>(A-C) CD43^- splenic B cells from Bim^-/- mice were treated with anti-IgM F(ab’)₂ (15μg/ml) for various times in the presence or absence of Syk kinase inhibitor R406 (4μM), Bruton’s tyrosine kinase inhibitor Ibrutinib (PCI-32765) (20nM), or TORC1 inhibitor rapamycin (50nM). Whole cell extracts were fractionated by SDS-PAGE and p100 proteins were analyzed by immunoblotting. β-actin was used as a loading control to normalize between samples. (D, E) CD43^- splenic B cells from BL6, BCAP^-/- (D) or CD19^-/- (E) mice were cultured for the indicated times with or without anti-IgM F(ab’)₂ (15μg/ml). Whole cell extracts were prepared and assayed for p100 protein levels by Western blot analysis. β-actin was used as a loading control to normalize between samples. Representative gels from 3 independent experiments are shown. Cell viability is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146955#pone.0146955.s004" target="_blank">S4 Fig</a>.</p

Established Thymic Epithelial Progenitor/Stem Cell-Like Cell Lines Differentiate into Mature Thymic Epithelial Cells and Support T Cell Development

<div><p>Common thymic epithelial progenitor/stem cells (TEPCs) differentiate into cortical and medullary thymic epithelial cells (TECs), which are required for the development and selection of thymocytes. Mature TEC lines have been widely established. However, the establishment of TEPC lines is rarely reported. Here we describe the establishment of thymic epithelial stomal cell lines, named TSCs, from fetal thymus. TSCs express some of the markers present on tissue progenitor/stem cells such as Sca-1. Gene expression profiling verifies the thymic identity of TSCs. RANK stimulation of these cells induces expression of autoimmune regulator (Aire) and Aire-dependent tissue-restricted antigens (TRAs) in TSCs <i>in vitro</i>. TSCs could be differentiated into medullary thymic epithelial cell-like cells with exogenously expressed NF-κB subunits RelB and p52. Importantly, upon transplantation under the kidney capsules of nude mice, TSCs are able to differentiate into mature TEC-like cells that can support some limited development of T cells <i>in vivo</i>. These findings suggest that the TSC lines we established bear some characteristics of TEPC cells and are able to differentiate into functional TEC-like cells <i>in vitro</i> and <i>in vivo</i>. The cloned TEPC-like cell lines may provide useful tools to study the differentiation of mature TEC cells from precursors.</p> </div

Established TSC cells express markers of non-hematopoietic stem cells.

<p>(a) Representative spindle-like morphology of TSC clone 2 established from C57BL/6 E14.5 thymus repeated subculture and limiting dilution cloning.(b) Flow cytometric analysis of WT TSC with antibodies to Sca-1, CD29, CD44, CD45, CD73, CD105, CD133, CD80, MHC class I and II.</p

TSCs display thymus identity.

<p>(a) RNAs were extracted from TSC2, 1307-6.1.7 and mTEC8 cells, and transcripts were detected by RT-PCR for the expression of indicated genes. (b) Immunoblot analysis of CBX4, delta Np63, TAp63 and DNMT3a in extracts of TSC2, mTEC1 and mTEC8 cells. GAPDH was used as a loading control.</p

TSCs differentiate into Aire-expressing TECs <i>in vitro</i>.

<p>(a) Immunoblot analysis of Aire, delta Np63, DNMT3a, c-Myc, p52 and. RelB in extracts of TSCs stably overexpressed with p52 and RelB for 11 days. (b) Immunofluorescence analysis for UEA-1 and K8 in TSCs stably overexpressed with p52 and RelB for 11 days.</p