Dynamic signaling by T follicular helper cells during germinal center B cell selection

Abstract

T follicular helper (T(FH)) cells select high-affinity, antibody-producing B cells for clonal expansion in germinal centers (GCs), but the nature of their interaction is not well defined. Using intravital imaging, we found that selection is mediated by large but transient contacts between T(FH) and GC B cells presenting the highest levels of cognate peptide bound to major histocompatibility complex II. These interactions elicited transient and sustained increases in T(FH) intracellular free calcium (Ca(2+)) that were associated with T(FH) cell coexpression of the cytokines interleukin-4 and -21. However, increased intracellular Ca(2+) did not arrest TFH cell migration. Instead, T(FH) cells remained motile and continually scanned the surface of many GC B cells, forming short-lived contacts that induced selection through further repeated transient elevations in intracellular Ca(2+).

Fig. 1. Dynamics of T_FH and B cell interactions in the GC

(A) Timeline of the experimental protocol. i.p., intraperitonealy; s.c., subcutaneously. (B) GCs containing a 5:95 mixture of B1-8^hi Ly75^+/+ GFP⁺ B cells (cyan), B1-8^hi Ly75^−/− B cells (non-fluorescent) and OT-II DsRed⁺ T cells (red) were imaged by TPSLM after s.c. injection of αDEC-OVA or αDEC-CS as control. T-B contacts (green) were detected by red and green pixel co-localization. Collapsed z-stacks of 40 μm depth (in 5μm steps) are shown. Bottom panels depict T and B cell dynamics over time. Images correspond to movies S1–3. (C) Velocity analysis of OT-II T cells and B1-8^hi B cells in GCs. (D) T-B contact duration as measured by the lifetime of the T-B co-localized areas. Percentages indicate events lasting > 5 min, marked by dashed red box. (E) T-B conjugate velocity was measured as the velocity of the T-B co-localized area. (F) Contact size analysis as measured by T-B co-localized area. Each data point represents a single cell and red lines represent mean values. Data in C-F were pooled from 2-4 mice imaged in 2-4 independent experiments. * p < 0.0001; two-tailed Student’s t-test.

Fig. 2. T_FH cell interactions with selected and non-selected GC B cells

(A) GCs containing a ~5:5:90 mixture of B1-8^hi Ly75^+/+ CFP⁺ B cells (blue), B1-8^hi Ly75^−/− GFP⁺ (green), Ly75^−/− non-fluorescent B cells and OT-II DsRed⁺ T cells (red) were imaged by TPSLM after s.c. injection of αDEC-OVA. Contacts between OT-II T cells and either B1-8^hi Ly75^+/+ or Ly75^−/− B cells are shown in yellow and grey, respectively. 40 μm deep, collapsed z-stacks (5 μm steps) are shown. Arrowheads indicate B1-8^hi Ly75^+/+ and Ly75^−/− GC B cells interacting simultaneously with one OT-II T cell. (B) Quantitation of contact duration between OT-II T cells and B1-8^hi Ly75^+/+ or Ly75^−/− B cells. Each data point represents a single cell and red lines represent mean values. Percentages indicate events > 5 min, marked by dashed red box. (C) The number of B cell contacts (> 0.5 min, left and > 5 min, right) with OT-II T cells was normalized to the number of B cells in each group. Exp., experiment. Data in B-C were pooled from 4 independent experiments. * p < 0.0001; two-tailed Student’s t-test.

Fig. 3. T_FH cell Ca²⁺ signaling during B cell selection

(A) GCs containing a 5:95 mixture of B1-8^hi Ly75^+/+ CFP⁺ B cells (cyan), B1-8^hi Ly75^−/− B cells (non-fluorescent) and OT-II DsRed⁺ GCaMP3⁺ T cells (red, Ca²⁺ low; yellow, Ca²⁺ high) were imaged by TPLSM in untreated mice or after s.c. injection of αDEC-OVA. The bottom panels depict dynamic changes in GCaMP3 fluorescence in individual OT-II T cells over time. (B) Traces show changes in the GCaMP3/DsRed ratios over time for 6 single T cells in each condition.αI-A^b was injected intravenously after αDEC-OVA injection. (C) Scatter plots depict the GCaMP3/DsRed ratio versus instantaneous velocity as measured at successive 30 sec intervals. Each dot represents a single cell at a single time point. Average velocity (V) and GCaMP3/DsRed ratios (R) of 2-3 experiments are indicated in red and green, respectively. (D) GCaMP3/DsRed ratio fluctuations in single cells (expressed as variance) under control and αDEC-OVA conditions. (E) GCaMP3/DsRed spikes of 9 cells were synchronized. Traces of GCaMP3/DsRed ratios average and corresponding instantaneous velocities are shown. The synchronized spikes fall in the black rectangle. Error bars, SEM. (F) OT-II GCaMP3⁺ T cells and B1-8^hi Ly75^+/+ tdTomato⁺ B cells were imaged in GCs over time as in A. Images correspond to movie S11. (G) Traces show GCaMP3 mean fluorescence intensities for 5 OT-II T cells in contact with B1-8^hi Ly75^+/+ tdTomato⁺ GC B cells. (H) Initiation of contacts were synchronized in 4 OT-II GCaMP3⁺ T cells and the corresponding average of GCaMP3 fluorescence intensity was traced before and during the contact (au, arbitrary units). Data is representative of 2–3 mice imaged in 2–3 independent experiments. * p < 0.0001; two-tailed Student’s t-test.

Fig. 4. Multifunctional T_FH cells during B cell selection

(A-B) CD4⁺ T cells derived from OVA immunized Il4-IRES-GFP and Il21-IRES-Katushka double knock-in mice and a 5:95 ratio of B1-8^hi Ly75^+/+ and Ly75^-/- B cells were transferred into TCRβ deficient mice, before boosting with NP-OVA. After 7 days, mice were injected with αDEC-CS or αDEC-OVA and lymph nodes were analyzed 9 hours later. The proportions of cytokine-expressing cells among T_FH (CD8⁻, B220⁻, CD4⁺, CD44⁺, CD62L⁻, CXCR5^high and PD-1^high) subsets are indicated. (C-D) gMFI of IL-4 (C) or IL-21 (D) reporter expression. Each data point represents a single mouse and lines represent mean values. Pooled data from 3 experiments each with 3–4 mice per condition. * p = 0.021; ** p = 0.014 ***; p = 0.012; two-tailed Student’s t-test. NS, not significant.