Fig 1.
LTβR expression in B lineage cells.
(A) Blimp1-GFP mice were immunized with NP-CGG/Alum, reimmunized with the same immunogen after 3 weeks and analyzed 4 days later. PCs were positively enriched from spleen and BM cells by CD138 PE magnetic bead-based selection and FACS sorted for qPCR analysis. Representative FACS plots of live Dump (CD4, CD8, F4/80, Ly6G)-B220+ cells with gates on TACI+CD98+ cells consisting of CD138+ Blimp1-GFPINT immature PCs or Blimp1-GFPHI mature PCs. (B) Ltbr gene expression relative to Hprt among total spleen cells, CD8-Gr-1-F4/80-Dead- B220+IgD+ FO B cells, B220+PNA+GL7+IgD- GC B and PCs. FO and GC B cells were FACS sorted from spleens of WT mice immunized with NP-CGG/Alum for 11 days. (C) LTβR protein expression by flow cytometry in LTβR-deficient (Ltbrfl/fl Mb1-cre+/-) and WT (Ltbr+/+ Mb1-cre+/-) PCs. Data shown in all panels are representative of two independent experiments. Pooled data from two independent experiments are shown (n = 4–6 mice per group for A-B and n = 3 for C). * p < 0.05, ** p < 0.01.
Fig 2.
LTβR expression in B cells is not required for PC responses to a T-dependent antigen.
(A) Diagram of experimental scheme. Chimeras were made by mixing CD45.2+ WT or Ltbr-/- BM cells with CD45.1+ wild-type cells at a 30:70 ratio and injecting into irradiated CD45.1+ recipients. After 8 weeks of reconstitution, mice were immunized with NP-CGG/Alum two times 21 days apart and analyzed 28 days after the booster immunization. (B) FACS plots of singlet cells with a gate on live Dump-B220+ cells (top row, left). The B220+ cells were further gated on IgD+GL7- FO B cells (top row, middle), with further gates on CD45.2+ or CD45.1+ cells (top row, right). Alternatively, B220+ cells were gated based on CD38-GL7+ GC B cells (middle row, left) with further gates on CD45.2+ or CD45.1+ cells (middle row, middle). NP+ GC B cells were identified as NP16-PE+ and decoy SA-PE-AF647- (middle row, right). Memory B cells were identified as CD38+GL7-CD95+IgD- (bottom row, left) with gates on CD45.2+ or CD45.1+ cells (bottom row, middle) or NP+ memory B cells (bottom row, right). (C) Chimerism based on the ratio of CD45.2+ WT or Ltbr-/- to CD45.1+ WT FO B cells (top). GC B cell ratio of CD45.2+ cells to CD45.1+ cells normalized to the FO B cell ratio (middle). Memory B cell ratio of CD45.2+ cells to CD45.1+ cells normalized to the FO B cell ratio (bottom). (D) Representative FACS plots of CD138-enriched BM cells from a CD45.2+ WT recipient chimera pre-gated on live Dump-B220loCD138+ cells. PCs were identified as B220- (left), TACI+CD98+ (top row, middle) with gates on CD45.2+ or CD45.1+ cells (top row, right). NP-CGG+ PCs were identified as NP-CGG-FITC+ (bottom row, left) with further gates on CD45.2+ or CD45.1+ cells (bottom row, right). (E) Ratio of CD45.2+ cells to CD45.1+ cells normalized to the FO B cell ratio for spleen PCs (top) or BM PCs (bottom). Data shown in all panels are representative of two independent experiments (n = 4–5 mice per group).
Fig 3.
LTβR expression in B cells is not required for PC responses to a T-independent antigen.
Ltbrfl/fl Mb1-cre+/- or control (Ltbrfl/fl or Ltbrfl/+ Mb1-cre+/-) mice were immunized with NP-Ficoll for 11 days. (A) Representative FACS plots of live Dump- CD138-enriched splenocytes from an unimmunized Ltbrfl/fl mouse and immunized mice (top row) with gates on TACI+CD98+ PCs. NP+ PCs cells were identified as NP16-PE+ and decoy SA-PE-AF647- (bottom row). (B) Numeration of total and NP+ PCs in spleens of immunized mice. Data shown in all panels are representative of two independent experiments (n = 4–5 mice per group).
Fig 4.
OE of LTβR increases PC numbers in vitro in response to LPS or anti-CD40 stimulation in the absence of ligand.
(A) Diagram of experimental scheme. Chimeras were made by retroviral transduction of CD45.2+ CD19-Cre+/- BM with Empty-MSCV-Thy1.1 or loxp-EGFP-loxp-LTβR-MSCV-Thy1.1 retrovirus and transfer into irradiated CD45.1+ recipients. After reconstitution, B cells were isolated from the spleen for in vitro culture. (B) Representative FACS plots of CD45.2+B220- splenocytes in chimeras stimulated with or anti-CD40 with IL-4, IL-5, and IL-21 for 3 days. Transduction efficiency based on Thy1.1 expression on FO B cells was 43–93% among the mice analyzed. Blocking of the LTβR ligands was assessed through the addition of human LTβR-Fc blocking antibody. (C) FACS plots of live Dump-B220+CD45.2+ B cells with gates on Thy1.1+ cells and further gates on CD98+CD138+ PCs. (D) Percentages of CD98+CD138+ PCs among Thy1.1+ (left) and Thy1.1- (right) B cells as gated in B. Numbers of CD98+CD138+ Thy1.1+ PCs (middle). (E) LTβR protein expression by flow cytometry of WT B cells stimulated with LPS for 3 days and retrovirally transduced with Empty-MSCV-Thy1.1 or LTβR-MSCV-Thy1.1 vectors. (F) Frequency of CD98+CD138+ PCs among Thy1.1+ transduced WT or Ltb-/- B cells stimulated with LPS media for 3 days. Data shown in all panels are representative of two independent experiments (n = 2–3 mice per group). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Fig 5.
LTβR OE promotes PC accumulation in vivo.
(A) Diagram of experimental scheme. Chimeras were made by retroviral transduction of CD45.2+ WT BM with Empty-MSCV-Thy1.1 or LTβR-MSCV-Thy1.1 (LTβR OE) retrovirus and transfer into irradiated CD45.1+ recipients. After reconstitution, mice were immunized with NP-CGG/Alum two times 21 days apart. After 26 days from the booster immunization, spleen and BM were analyzed. (B) Representative flow plots representing Dump-B220+CD45.2+CD45.1-IgD+ spleen FO B cells with gates on Thy1.1+ cells. (C) Thy1.1 frequency of FO B cells. (D) Thy1.1 frequency of spleen GC B cells normalized to Thy1.1 frequency of FO B cells in matched mice. (E) FACS plots of Dump-CD138+CD45.2+ cells with gates on B220-, TACI+CD98+ PCs with further gates on Thy1.1 cells among total spleen PCs or (F) NP+ spleen PCs. NP+ PCs were identified as NP16-PE+ and decoy SA-PE-AF647-. (G) Number of total spleen or BM PCs (left) and NP frequency of PCs (right). (H) Thy1.1 frequency of spleen or BM PCs normalized to Thy1.1 frequency of FO B cells in matched mice. Data shown in all panels are representative of two independent experiments (n = 6–8 mice per group). * p < 0.05, ** p < 0.01.
Fig 6.
LTβR OE PCs have greater transcription of Nfkb2, Bcl2, and Mcl1.
B cell specific LTβR OE or control chimeras were generated as in Fig 4. Dump-CD138+B220-TACI+CD98+CD45.2+ Thy1.1+ or Thy1.1- PCs were sorted from spleens. qPCR analysis was conducted to determine the transcription levels of Nfkb2, Bcl2, and Mcl1. Control samples consist of Thy1.1+ or Thy1.1- PCs from Empty-MSCV-Thy1.1 chimeras and Thy1.1- PCs from loxp-EGFP-loxp-Ltbr-MSCV-Thy1.1 chimeras. In some instances, cells from multiple mice were combined into one sample to obtain sufficient cells for analysis. Pooled data from two independent experiments are shown. * p < 0.05, ** p < 0.01.
Fig 7.
Model for LTβR over-expression (OE) effect on PC accumulation.
In WT PCs, TACI and BCMA are highly expressed while LTβR expression is very low. Under conditions where LTβR is OE, there is an overall increase in signaling via NF-kB leading to augmented Bcl2-family protein expression and reduced apoptosis, and to increases in other NF-kB target genes that augment PC accumulation. While TACI and BCMA signaling is ligand (BAFF, APRIL) dependent, the OE LTβR may signal in a ligand independent manner. Chronic LTβR OE in PCs due to gene amplification may allow marked over accumulation of PCs, increasing the chances of additional genetic variants emerging and progression to MM.