CD40L is transferred to antigen-presenting B cells during delivery of T-cell help

Abstract

The delivery of T-cell help to B cells is antigen-specific, MHC-restricted, and CD40L (CD154) dependent. It has been thought that when a T cell recognizes an antigen-presenting B cell, CD40L expressed on the T-cell surface engages with CD40 on the surface of B cells as long as the cells remain conjugated. By adding fluorescently labeled anti-CD40L antibody during overnight incubation of antigen-presenting B cells with antigen-specific T cells, we discovered that CD40L does not remain on the surface of the T cell, but it is transferred to and endocytosed by B cells receiving T-cell help. In the presence of anti-CD40L antibody, transferred CD40L is nearly absent on bystander B cells that are not presenting antigen, and the bystander cells do not become activated. Because transfer of CD40L to B cells correlates with B-cell activation, we speculate that persistence of helper T-cell-derived CD40L on or in B cells could permit sustained CD40 signaling enabling survival and proliferation of antigen-presenting B cells following brief interactions with helper T cells in vivo in germinal centers.

Keywords: CD154; CD40 ligand; CD40L; Immunological synapse; Membrane transfer; T-cell help.

Figure 1. Antigen-specific, CD40-dependent transfer of CD40L to antigen-presenting B cells

(A) The gating scheme for the in vitro overnight CD40L transfer assay is shown. (B, C, D, E) CD40L KO antigen-pulsed (Ag+), CD40 KO Ag+, and CD40L KO Ag- B cell populations were incubated with Th1 cells overnight, then fixed, permeabilized, and stained with anti-CD40L-PE antibody. The control histogram (solid grey) represents intracellular staining with a hamster IgG-PE antibody of CD40LKO Ag+ B cells incubated with Ag- B cells and Th1 cells. The experiment with CD40L KO Ag+ and CD40 KO Ag- B cells was performed twice. The experiment with CD40L KO Ag+ and CD40L KO Ag- B cells was performed 3 times with similar results.

Figure 2. Enhanced detection of CD40L transfer in the presence of fluorescent anti-CD40L antibody

(A, C) Each contour plot shows the percentage of B cells that have acquired CD40L following overnight incubation with Th1 cells in the presence of fluorescently labeled anti-CD40L antibody in a representative experiment. (B) The graph shows the results from seven independent experiments (mean +/- SD). p values were calculated using an unpaired, two-tailed Mann-Whitney test: Ag+ vs. Ag- p = 0.0006, Ag+ vs. CD40KO Ag+ p = 0.0061, Ag- vs. CD40KO Ag+ p = 0.0061. ** p ≤ 0.01, *** p ≤ 0.001. (D, E) Contour plots and graphs show the effects of CD40, FcR deficiency, and blocking anti-FcR antibody on detection of CD40L transfer. The graph shows the percentage of B cells bearing transferred CD40L in five (D top panels) or three individual experiments (D bottom panels, E) (mean +/- SD).

Figure 3. De novo synthesis of CD40L is not required for Ag-specific CD40L transfer

(A) Histograms depict T cell surface CD40L after overnight culture with Ag+ B cells and anti-CD40L in the absence or presence of CsA. (B, C) Contour plots show representative transfer of CD40L to B cells in the absence of antigen (no Ag) and to bystander B cells (Ag-) and antigen-pulsed B cells (Ag+) within the same well following overnight incubation with Th1 cells and CsA. (D) Contour plots show representative transfer of CD40L to the bystander and Ag+ B cells during a two-hour incubation with Th1 cells and CsA. Results are representative of three independent experiments.

Figure 4. Transferred CD40L is found intracellularly and on the surface of antigen-pulsed B cells following overnight incubation with Th1 cells in the presence of anti-CD40L

(A, B) Two representative images of Ag+ B cells (green (CD19+) and blue (CTV)) and bystander B cells (green only (CD19+)) together with T cells are shown with and without the differential interference contrast overlay following overnight incubation. The images in A and B show a frame through the middle of the cells from videos (Supporting Information Video 1 and Video 2) created from 2.5 μM z stacks. Intracellular CD40L (allophycocyanin-labeled anti-CD40L, shown in red) is detected in the Ag+ B cell in A and an example of surface CD40L is shown in B. (C) The graph shows the percentage Ag+ and Ag- B cells positive for the presence of CD40L in two individual experiments. (D) The pie charts show the relative numbers of B cells positive for surface, intracellular, or both surface and intracellular CD40L. There were 281 B cells analyzed in Experiment 1 (128 Ag+ and 153 Ag-) and 193 B cells in Experiment 2 (88 Ag+ and 105 Ag-).

Figure 5. Antigen-specific CD40L transfer and help, unlike bystander help, is resistant to blocking by anti-CD40L antibody

(A) Representative contour plot of CD40L transfer and ICAM-1 upregulation is shown for a single experiment in the antigen-pulsed B cells. Graph shows ICAM geometric mean fluorescent intensity values for CD40L positive and negative Ag-pulsed B cells after overnight incubation with Th1 cells in the presence of fluorescently labeled anti-CD40L antibody. p value was calculated using an unpaired, two-tailed Mann-Whitney test: p = 0.002 (B, C, D, E, F) Th1 and B cells were added to the top and bottom chambers of the Transwells® as depicted in the schematics in the presence (B) or absence (C, D, E, F) of fluorescently labeled anti-CD40L antibody. CD40L transfer (B) or ICAM-1 upregulation (C, D, E, F) was measured on the B cell populations in both the top and bottom chambers, and CD40L contour plots (B) or ICAM-1 histograms (C, D, E, F) are shown. The filled grey histogram on each panel is the control without antigen in the top chamber. This experiment is representative of two (B) or three (C, D, E, F) independent experiments. (G) Representative histograms of ICAM-1 upregulation on Ag+, Ag-, and no antigen control B cells after overnight incubation with 5CC7 Th1 cells. The graph shows the fold increase in ICAM-1 mean fluorescence intensity for three individual experiments (mean +/- SD) relative to the no Ag control B cell. (H, I left panel) Th1 cells were incubated with antigen-pulsed (Ag+) and bystander (Ag-) B cells in the presence or absence of neutralizing anti-CD40L antibody (10 μg/ml) and activation was assessed by ICAM upregulation. (I right panel) The graphs show the effects of different concentrations of anti-CD40L on activation of Ag+ and Ag- B cells. The left and right graphs are representative of three or two independent experiments, respectively. (J, K) Differentially labeled (CTV or CFSE) antigen-pulsed (Ag+) and CD40KO Ag+ along with Ag- B cells were incubated with Th1 cells, and proliferation was assessed after 48 hours by dilution of these dyes in the presence and absence of anti-CD40L (0.1 μg/ml). The percent of divided cells is labeled on each histogram and is representative of two independent experiments.