Intrinsic properties of human germinal center B cells set antigen affinity thresholds

Abstract

Protective antibody responses to vaccination or infection depend on affinity maturation, a process by which high-affinity germinal center (GC) B cells are selected on the basis of their ability to bind, gather, and present antigen to T follicular helper (T_fh) cells. Here, we show that human GC B cells have intrinsically higher-affinity thresholds for both B cell antigen receptor (BCR) signaling and antigen gathering as compared with naïve B cells and that these functions are mediated by distinct cellular structures and pathways that ultimately lead to antigen affinity- and T_fh cell-dependent differentiation to plasma cells. GC B cells bound antigen through highly dynamic, actin- and ezrin-rich pod-like structures that concentrated BCRs. The behavior of these structures was dictated by the intrinsic antigen affinity thresholds of GC B cells. Low-affinity antigens triggered continuous engagement and disengagement of membrane-associated antigens, whereas high-affinity antigens induced stable synapse formation. The pod-like structures also mediated affinity-dependent antigen internalization by unconventional pathways distinct from those of naïve B cells. Thus, intrinsic properties of human GC B cells set thresholds for affinity selection.

Figure 1

GC B Cells Engage Antigen through BCRs Concentrated in F-Actin and Ezrin-Rich Pod-like Structures. (A) DIC, IRM, and merged images of immune synapse of live naïve B cells placed on antigen-containing PLB and live LZ GC B cells placed on antigen-containing PLB or PLB with no antigen. (B) Kymographs of DIC images of naïve and LZ GC B cell immune synapse on antigen-containing PLB. (C) Membrane movement in the immune synapses of live naïve, LZ GC, and DZ GC B cells with time on antigen-containing PLB imaged by IRM. (D) STED super-resolution images of F-actin formed in immune synapses of naïve B cells and GC B cells placed on PLB that contained antigen. (E) Colocalization of F-actin and BCR in immune synapses of naïve and LZ GC B cells imaged by confocal microscopy on PLB that contained antigen. (F) (Left panel) Confocal microscopy images of immune synapses of naïve B cells and LZ GC B cells on antigen-coated PLB stained with Alexa Fluor 488 phalloidin for F-actin (green) and antibodies specific for ezrin (red). (Top right panel) Quantification of the MFI of ezrin and (Bottom right panel) colocalization of ezrin with F-actin (bottom) in the immune synapse of confocal images. (G) Bottom and orthogonal views of F-actin (green) and BCR (red) in naïve and LZ GC B cells imaged by confocal microscopy on antigen-containing PLB. (H) Side and top views of naïve and LZ GC B cells imaged by SEM on PLB without or with antigen. (I) Colocalization of pod-like structures and antigens in immune synapse of LZ GC B cells imaged by TIRFM on antigen-containing PLB. Scale bars are 5µm. ns>0.05, *P≤0.05, **P≤0.01, ***P≤0.001, and ****P≤0.0001 (unpaired t-test). Data are representative of two experiments (F: mean and s.e.m.).

Figure 2

Human GC B Cells Signal in Response to and Exert Pulling Forces on Membrane Bound Antigens through Pod-like Structures. (A) Immune synapses of naïve B cells and GC B cells imaged by TIRFM on PLB containing F(ab')₂ anti-λ/κ. (B) MFI of pPI3K and pSHP-1 and colocalization of BCR with pPI3K or pSHP-1 in the contact area of naïve and GC B cells placed on antigen-containing PLBs for 7 or 25 min. (C) (Top panels) Atto 647N FI (red) and Atto 550 (green) merged images. (Bottom panels) Sensor opening ratio (the ratio of Atto 647N FI to Atto 550 FI). (D) Quantification of sensor opening control sensors. (E) Colocalization of sensor opening locations and contact sites to the membrane with time imaged by TIRFM and IRM. Scale bars are 5µm. ns>0.05, *P≤0.05, **P≤0.01, ***P≤0.001, and ****P≤0.0001 (unpaired t-test). Data are representative of two experiments. (B and D: mean and s.e.m.)

Figure 3

Human GC B Cells Have a High-Affinity Threshold for Antigen. Naïve and LZ GC B cells were placed on PLB that did not contain antigen (resting) or placed on PLB that contained either high-affinity or low-affinity anti-κ mAbs. (A) TIRFM images were obtained of cells stained for the BCR and pCD79A. (B-C) Quantification of the BCR (B) and pCD79A (C) in the contact area of the B cells with the PLB in TIRFM images. (D-F) Quantification of TIRF images of cells stained with pSyk (D), pBLNK (E) and pPLCγ2 (F) in the contact area with the PLB of naïve and LZ GC B cells activated by high and low-affinity antigen for 7 or 25 min. (G) DIC and IRM images of naïve and LZ GC B cells imaged by TIRFM on PLB containing either high-affinity or low-affinity anti-κ mAbs. The still images were taken between 3 to 12 min of the supplementary movie 6. (H) Kymographs of IRM images of naïve and LZ GC B cell immune synapse on low- or high-affinity antigen-containing PLB. Red arrow heads on low-affinity naïve B cell are extended membrane ruffles and on high-affinity LZ GC B cell are pod-like structures established stable contacts to antigen-containing PLB. (I) Quantitative analysis of pod-like structure movement in DIC images (number of tracks: n= 347 for high affinity early, n=887 for high affinity late, n=241 for low affinity early, and n=432 for low affinity late). Scale bars are 5µm. ns>0.05, *P≤0.05, **P≤0.01, ***P≤0.001, and ****P≤0.0001 (unpaired t-test). Data are representative of two experiments. (B-F: mean and s.e.m.)

Figure 4

Extraction and Trafficking of Membrane Associated Antigen Distinguishes GC B cells. (A) Antigen extraction by naïve and GC B cells placed for 2 h on PMS that contained either high-affinity or low-affinity anti-κ mAbs. Antigen-positive B cell populations were quantified by flow cytometry. (B) Transport of extracted antigens in naïve and LZ GC B cells placed on antigen-containing PMS. F-actin (green) and antigen (red). (C) Distance of MTOC in naïve and LZ GC B cells to antigen-containing PMS. F-actin (green), gamma-tubulin (cyan), antigen (red). (D) Localization and quantification of SNX9 or SNX18 in naïve and LZ GC B cells 45 min after activation on antigen-containing PMS imaged by confocal microscopy. F-actin (green), SNX9 and SNX18 (cyan), and antigen (red). MFI of SNX9 and SNX18 per each z-stack from the cell bottom to the top (n=25 per group). (E) Localization of SNX9 or SNX18 with MTOC in naïve and LZ GC B cells 45 min after activation on antigen-containing PMS. Alpha-tubulin (green), SNX9 and SNX18 (cyan), and antigen (red). (F) Localization pattern of LAMP-1 in naïve and LZ GC B cells (left) 30 min after activation on antigen-containing PMS. 3D colocalization of antigen with LAMP-1 in naïve and LZ GC B cells (right). LAMP-1 (cyan) and antigen (red). (G) Trafficking of extracted antigens to acidic intracellular compartments in naïve and LZ GC B cells with time on antigen-containing PMS. (H) Percentage of antigen-positive and pHrodo intensities (bottom left) of naïve B cells and LZ GC B cells which are similar in their antigen acquisition. ns>0.05, *P≤0.05, **P≤0.01, ***P≤0.001, and ****P≤0.0001 (unpaired t-test). Data are representative of two (B-F) or three experiments (A, G, and H). (A, C, D and F: mean and s.e.m.)

Figure 5

Human GC B Cells Express IRF-4 in Response to High-Affinity Antigen in Combination with Tfh Cell Help. (A-B) Relative mRNA expression level of IRF-4 in LZ GC B (A) and naive (B) cells after activation with antigens on PLB and/or Tfh like stimuli composed of anti-CD40 mAb, IL-21, and IL-4. ns>0.05, *P≤0.05, **P≤0.01, ***P≤0.001, and ****P≤0.0001 (paired t-test). (A-B: mean and s.e.m.)

Figure 6

The Differential Expression of Key Surface Markers may Further Contribute to Affinity Discrimination by Human GC B Cells. (A) Heat map: comparisons of differential surface molecule expression (B-C) Surface levels of CD29, CD49d, and VLA-4 active epitope in naïve and LZ GC B cells. (D) Binding ability of naïve and LZ GC B cells to VCAM-1 coated beads. ns>0.05, *P≤0.05, **P≤0.01, ***P≤0.001, and ****P≤0.0001 (unpaired t-test). Data are from one of five individuals (B) or data are from representative of three experiments (D) (C-D: mean and s.e.m.)