Reduced c-myc expression levels limit follicular mature B cell cycling in response to TLR signals

Abstract

The splenic B cell compartment is comprised of two major, functionally distinct, mature B cell subsets, i.e., follicular mature (FM) and marginal zone (MZ) B cells. Whereas MZ B cells exhibit a robust proliferative response following stimulation with the TLR4 ligand LPS, FM B cells display markedly delayed and reduced levels of proliferation to the identical stimulus. The current study was designed to identify a potential mechanism(s) accounting for this differential responsiveness. In contrast to the delay in cell cycle entry, FM and MZ B cells exhibited nearly identical LPS-driven alterations in the expression level of cell surface activation markers. Furthermore, both the NF-kappaB and mTOR signaling cascades were similarly activated by LPS stimulation in FM vs MZ B cells, while inducible activation of ERK and AKT were nearly absent in both subsets. MZ B cells, however, exhibited higher basal levels of phospho-AKT and pS6, consistent with a preactivated status. Importantly, both basal and LPS activation-induced c-myc expression was markedly reduced in FM vs MZ B cells and enforced c-myc expression fully restored the defective proliferative response in FM B cells. These data support a model wherein TLR responses in FM B cells are tightly regulated by limiting c-myc levels, thereby providing an important checkpoint to control nonspecific FM B cell activation in the absence of cognate Ag.

Figure 1. Delayed cell cycle entry of FM B cells in response to LPS

A. Sort purified FM and MZ B cells were stimulated with media alone, or 10 µg/ml of anti-IgM or LPS for 48 hr, followed by measurement of ³H-thymidine incorporation. Data shown are representative of >5 independent experiments. B. Stimulation of sort purified, CFSE labeled FM and MZ B cells with varying doses of LPS (0.001 –10 µg/ml). Dilution of CFSE was analyzed at 48hr or 72 hrs as indicated. C–D. Cell cycle analysis of stimulated FM vs. MZp/MZ B cells. C. Example of staining and gating strategy using PyroninY and DAPI to assess FM B cells stimulated for 48 hr with either 10 µg/ml of anti-IgM or LPS as indicated. D. Cell cycle status at different time points in FM vs. MZp/MZ B cells following stimulation with anti-IgM, LPS or, 50 ng/ml BAFF, as indicated. Data shown are representative of one of 3 independent experiments.

Figure 2. FM B cells are highly sensitive to LPS stimulation

A–B. Expression of cell surface activation markers at 48 hr in response to varying doses of LPS on sort purified FM vs. MZp/MZ B cells. A. FACS histograms of stained cells. B. Example of gating strategy to identify non-proliferating vs. proliferating FM B cells (upper panel) and the dose-response curves for marker expression on FM vs. MZp/MZ B cells at 48 hr post stimulation. C. Expression of AID and BAFF-R transcript levels as determined by quantitative PCR in stimulated FM vs. MZ B cells at time points indicated. All data shown are representative of one of at least 3 independent experiments.

Figure 3. LPS stimulation promotes similar kinetics for both IκBα degradation and cRel and p65 nuclear translocation in FM and MZp/MZ B cells

A. IκBα degradation in sort purified FM vs. MZp/MZ B cells stimulated with 20 µg/ml LPS or anti-IgM, respectively. Actin was used as loading control. All data shown are representative of at least 3 independent experiments. B. Nuclear extracts from sort purified FM and MZp/MZ B cells stimulated for 3 hr with media alone, 20 µg/ml anti-IgM or LPS, respectively were probed with anti-c-rel and anti-p65 to determine nuclear translocation. HDAC1 was used as loading control. Data were quantified using the Licor system and the IκBα/actin, c-Rel/HDAC1 or p65/HDAC1 ratios were determined and shown as indicated. Data shown are representative of one of two independent experiments.

Figure 4. Calcium²⁺ flux and PI3K and mTOR signaling cascades following TLR4 engagement

A. Total CD43-depleted splenic B cells were loaded with Indo-1, surface stained and stimulated with 20 µg/ml anti-IgM or LPS (arrow). Ca²⁺ flux within each mature B cell subset was measured by flow cytometry. B. Phosphorylation of AKT was determined in FM vs. MZp/MZ B cells at 2 min post-stimulation with 20 µg/ml anti-IgM or LPS, respectively. C. Determination of basal levels of pAKT in unstimulated FM and MZ B cells by FACS (upper panel) and average of MFI from 3 independent experiments (lower panel) p=0.001. D. Phosphorylation of S6 in sort purified FM vs. MZp/MZ B cells stimulated with 20 µg/ml LPS for different periods of time as indicated. ERK was used as loading control. Data were quantified using the Licor system and the pS6/ERK determined

Figure 5. Activation of the mTOR pathway is crucial for B cell proliferation but not up-regulation of activation markers in response to LPS

A. Splenic B cells were pre-treated with Wortmannin, Rapamycin (both at 100 nM) or DMSO for 1hr and subsequently stimulated with 10 µg/ml anti-IgM or LPS. Expression of activation markers was determined after 24 hr. B. CFSE labeled splenic B cells were pre-treated with Wortmannin, Rapamycin or DMSO for 1hr and subsequently stimulated with 10 µg/ml anti-IgM or LPS, respectively. Dilution of CFSE was analyzed after 48hrs. Representative data from one out of 3 independent experiments is shown.

Figure 6. Basal c-myc levels are markedly lower in FM B cells, but similarly up-regulated in FM vs. MZp/MZ B cells in response to LPS

A. Sort purified FM and MZp/MZ B cells were stimulated with 20 µg/ml anti-IgM or LPS, respectively and expression of c-myc, A1, and Bcl-xL transcripts were determined by Q-PCR at indicated time points. B–C. ³H-thymidine incorporation and CFSE dilution assays for FM vs. MZp/MZ B cells isolated from wt vs. c-myc tg mice and stimulated with 10 µg/ml anti-IgM or different doses of LPS for 48 hr, respectively. D. Determination of c-myc protein levels in FM vs. MZp/MZ isolated from wt or c-myc tg mice by western blot analysis. ERK is used as a loading control. Western blot data were quantified using the Licor system and c-myc/ERK ratio determined. Data shown are from one of 3 independent experiments. E. c-myc transcript levels were determined in sort purified FM and MZp/MZ B cells from wt (n=3) and c-myc tg (n=4) mice by Q-PCR and data are shown as average with standard deviation. F. Splenic B cells were incubated with Wortmannin and Rapamycin (or DMSO) for 1hr prior to stimulation with 10µg/ml anti-IgM or LPS. Expression of c-myc was determined 3hr later by real-time PCR. Shown is the average of two independent experiments with SD.

Figure 7. c-myc tg FM B cells exhibit T cell- independent immune responses

A. Sort purified FM vs MZp/MZ B cells from wt and c-myc tg mice were stimulated with 20µg/ml LPS for 3 days and IgM in the supernatant was measured by ELISA. Data from 3 mice per group are shown. B–C. Wt vs. c-myc tg mice were injected intravenously with TNP-Ficoll and sacrificed 30min later. Splenocytes were isolated and stained with anti-TNP in addition to cell surface markers to detect TNP binding in different B cell subsets. B. Representative histograms of TNP binding with an unmanipulated mouse as control. C. MFI and relative fold change of MFI in TNP binding in each gated B cell subset with cells of an unmanipulated mouse set as 1. Shown is the mean of two animals with SD.