Germinal center B cell development has distinctly regulated stages completed by disengagement from T cell help
- Yale University School of Medicine, United States
- Yale School of Medicine, United States
- Western University, Canada
- Third Military Medical University, China
- Nanjing Normal University, China
- Okayama University, Japan
Figures
Figure 1 with 3 supplements
GC precursors co-express counter-regulatory factors BCL6, RelB and IRF4.
(A) Diagram of the experimental protocol. The GFP+ NP-specific B cells were transferred into non-responding C57BL recipients, which were subsequently immunized by footpad with NP-CGG in CFA. …
Figure 1—figure supplement 1
RelB is selectively expressed in Ag-specific responding B cells in a CD40-dependent manner during early immune responses.
GFP+ NP-specific B cells were transferred into WT C57BL recipients, followed by footpad immunization of NP-CGG in CFA. Shown are representative immunofluorescence histology of LN sections (n = 3 …
Figure 1—figure supplement 2
RelB+ cells are identified as having nuclear but not cytosolic distribution of RelB.
RelB+ cells identified and summarized in Figure 1 (B) (C) and (D) show intracellular staining of RelB, which co-localizes mostly with nuclear BCL6 and IRF4. (A) Representaive intracellular RelB …
Figure 1—figure supplement 3
Absence of BCL6 staining in Ag-specific B cells of mice treated with anti-CD40L.
GFP+ NP-specific B cells were transferred into WT C57BL recipients, followed by footpad immunization of NP-CGG in CFA. Mice recieved either 30 μg hamster Ig isotype control or blocking anti-CD40L …
Figure 2
BCL6int RelB+ IRF4+ Ag-specific B cells emerge early during immune responses and have partial GC phenotypes.
(A–E) Flow cytometric analysis of draining LN Ag-specific B cells. GFP+ NP-specific B cells were transferred into C57BL/6 recipients, which were subsequently immunized by footpad with NP-CGG in CFA. …
Figure 3
Multiple activated B cell subsets have elevated Fas and PNA-binding levels, including BCL6int IRF4+ cells.
GFP+ NP-specific B cells were transferred into C57BL/6 recipients, followed by f.p. immunization of NP-CGG in CFA. Popliteal draining LNs were stained for flow cytometric analysis. (A–B) …
Figure 4 with 2 supplements
The coordinated transition of GC precursor to BCL6hi GC B cells is associated with cell division.
(A) Diagram of the experimental protocol. CFSE-labeled NP-specific B cells were transferred into CD45.1+ congenic recipients, followed by f.p. immunization with NP-CGG in CFA. Popliteal draining LNs …
Figure 4—figure supplement 1
BCL6lo IRF4hi antigen-specific B cell emerge and expand through cell divisions with kinetics that differ from GC precursors or GC B cells.
Shown are representative plots of BCL6 expression relative to CFSE dilution. BCL6lo IRF4+ cells (light blue dots), GC precursors (red dots) and GC B cells (blue dots) are overlaid onto total …
Figure 4—figure supplement 2
GC precursors are in active cell cycle and not apoptotic.
(A–B) Flow cytometric analysis for cell cycle and apoptosis. NP-specific B cells were transferred into C57BL/6 recipients, followed by f.p. immunization of NP-CGG in CFA. 3.5 days p.i, popliteal …
Figure 5
A smaller immunogen dose promotes the transition of precursors to GC B cells.
(A) Diagram of the experimental protocol. CFSE-labeled NP-specific B cells were transferred into CD45.1+ congenic recipients, followed by f.p. immunization of NP-CGG of various doses (either 1.5 μg …
Figure 6
Potentiating T cell help at the initiation stage discourages the transition of GC precursors to GC B cells, and diverts their fate towards plasma cells.
(A) Diagram of the experimental protocol. 3 days after the initial immunization, recipient mice received an additional injection of either soluble NP-CGG, agonistic anti-CD40 (clone: FGK), PBS or …
Figure 7 with 1 supplement
Disruption of T/B interaction differentially impacts GC precursors and GC B cells.
(A–F) Diagram of the experimental protocol (A). By day 3 post-immunization, 20 μg αCD4 or αCD40L was further injected locally to delete CD4 T cells or block CD40 ligation on B cells. Popliteal …
Figure 7—figure supplement 1
In contrast to the initial transition of pre-GC to BCL6hi GC B cells, T cell help is required for the furthered maintenance or expansion of GC B cells.
(A) Diagram of the experimental protocol. (B) Percent GC B cells among transferred B cells 2 days after depletionof CD4 T cells or blockade of CD40L. Two independent experiments were carried with n =…
Figure 8 with 2 supplements
Deprivation of T-cell derived stimuli is required for the maturation of BCL6hi GC B cells in vitro.
(A) Schematic of the culture system using primary murine B cells. (B) Overlay of BCL6 expression in live B cells under different culture conditions as indicated. (C) Contour plots of BCL6 expression …
Figure 8—figure supplement 1
Flow cytometric analysis of BCL6 protein and bcl6 mRNA.
After fixation and intracellular staining with antibody, cultured cells were further hybridized with probes for murine bcl6 to allow for the detection of both BCL6 protein and bcl6 RNA at the single …
Figure 8—figure supplement 2
BCL6int precursors, not BCL6lo cells, give rise to BCL6hi GC B cells in vitro.
(A) Bcl6yfp/+ B cells were used for in vitro cell cultures. YFP-Bcl6 hi and lo cells in live B cells from day 3 culture with a continuous exposure of aCD40+IL-4/21 were gated based on control …
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