TFR Cells Express Functional CCR6 But It Is Dispensable for Their Development and Localization During Splenic Humoral Immune Responses

Abstract

Follicular T cells including T follicular helper (T_FH) and T follicular regulatory (T_FR) cells are essential in supporting and regulating the quality of antibody responses that develop in the germinal centre (GC). Follicular T cell migration during the propagation of antibody responses is largely attributed to the chemokine receptor CXCR5, however CXCR5 is reportedly redundant in migratory events prior to formation of the GC, and CXCR5-deficient T_FH and T_FR cells are still capable of localizing to GCs. Here we comprehensively assess chemokine receptor expression by follicular T cells during a model humoral immune response in the spleen. In addition to the known follicular T cell chemokine receptors Cxcr5 and Cxcr4, we show that follicular T cells express high levels of Ccr6, Ccr2 and Cxcr3 transcripts and we identify functional expression of CCR6 protein by both T_FH and T_FR cells. Notably, a greater proportion of T_FR cells expressed CCR6 compared to T_FH cells and gating on CCR6⁺CXCR5^hiPD-1^hi T cells strongly enriched for T_FR cells. Examination of Ccr6^-/- mice revealed that CCR6 is not essential for development of the GC response in the spleen, and mixed bone marrow chimera experiments found no evidence for an intrinsic requirement for CCR6 in T_FR cell development or localisation during splenic humoral responses. These findings point towards multiple functionally redundant chemotactic signals regulating T cell localisation in the GC.

Keywords: CCR6; T follicular helper (Tfh) cell; T follicular regulatory (Tfr) cells; chemokine receptor; germinal center T cell.

Figure 1

Chemokine receptor gene expression by follicular T cells. (A) Representative gating strategy for sorting follicular T cells (CD4⁺TCRβ⁺CXCR5^hiPD-1^hi) from day 5 SRBC i.p. immunized mice. Expression of (B) follicular T cell master transcription factor Bcl6, (C) known follicular T cell-tropic chemokine receptors, and (D) all other known classical chemokine receptors, between in naïve CD4 T cells (CD4⁺CD44^-CD62L⁺) and follicular T cells, relative to Gapdh. (B–D) Data pooled from two independent experiments with 3 mice pooled per time point ± SEM, two-tailed unpaired Student’s t test. *p < 0.05, **p < 0.01.

Figure 2

CCR6 expression is highest in T_FR cells amongst follicular T cell populations. (A, B) Representative gating strategy for CCR6⁺ T_FH cells (CD4⁺B220^-CXCR5^hiPD-1^hiFoxp3^-), T_FR cells (CD4⁺B220^-CXCR5^hiPD-1^hiFoxp3⁺), naïve CD4 T cells (CD4⁺B220^-CD44^loFoxp3^-) and natural T-regulatory cells (nTreg: CD4⁺B220^-CD44^midFoxp3⁺Nrp-1⁺) 6 days after SRBC immunization. (C) Geometrical mean fluorescence intensity (gMFI) of CCR6 and, (D) percentage of CCR6⁺ cells within populations from (A) and (B). (E) Representative gating strategy for T_FH (Foxp3^-) and T_FR cells (Foxp3⁺) within CCR6⁺ follicular T cells (CD4⁺B220^-CXCR5^hiPD-1^hiCCR6⁺). (F) Ratio of T_FH : T_FR cells within gating strategies from (A, E). (G) Frequency of CCR6⁺ T_FH and T_FR cells at indicated time points after i.p. NP-KLH/Alum immunization. (A–C, E, F) Data representative of two independent experiments, n=4 mice ± SEM, one-way ANOVA with Tukey’s multiple comparisons test, (G) n=4-5 mice per time-point ± SEM, two-way ANOVA with Sidak’s multiple comparison test. NS, Not significant, *p < 0.05, ***p < 0.001, ****p < 0.0001.

Figure 3

Follicular T cell populations migrate ex vivo to CCL20. (A–E) Transwell migration of splenocytes from day 6 SRBC immunized WT or Ccr6 ^-/- mice to increasing concentrations of CCL20. Cell populations were identified within total migrated splenocytes by flow cytometry and migration index was calculated relative to controls without chemokine. (A) Naïve CD4 T cells: CD4⁺B220^-CXCR5^-CD44^loFoxp3^-, (B) B cells: CD4^-B220⁺, (C) nTregs: CD4⁺B220^-CXCR5^-CD44^midFoxp3⁺Nrp-1⁺, (D) T_FH cells: CD4⁺B220^-CXCR5^hiPD-1^hiCD44^hiFoxp3^-, and (E) T_FR cells: CD4⁺B220^-CXCR5^hiPD-1^hiCD44^hiFoxp3⁺. Data pooled from two independent experiments, n=3-5 mice/strain ± SEM, two-tailed unpaired Student’s t test between strains at each concentration. *p < 0.05, **p < 0.01.

Figure 4

CCR6-deficient mice mount normal T_FH and T_FR cell responses. (A) Representative gating strategy of T_FH cells (CD4⁺B220^-CXCR5^hiPD-1^hiCD44^hiFoxp3^-) and T_FR cells (CD4⁺B220^-CXCR5^hiPD-1^hiCD44^hiFoxp3⁺) in WT and Ccr6 ^-/- mice four- and six-days post SRBC immunization. Frequency and number of (B) T_FH and (C) T_FR cells from (A). (D) Ratio of T_FH : T_FR cells four- and six-days post SRBC immunization in WT and Ccr6 ^-/- mice. (A–D) Data representative of 3 independent experiments, n=6-7 mice/time point ± SEM, two-tailed unpaired Student’s t test between strains at each time point.

Figure 5

CCR6-deficiency does not affect splenic Foxp3⁺ cell distribution during the peak of SRBC immunization. (A) Localisation of Foxp3⁺ cells in PFA and acetone fixed/permeabilized spleen sections from day 6 i.p. SRBC immunized WT and Ccr6 ^-/- mice. Sections were stained with antibodies against CD4 (red), IgD (blue) and Foxp3 (green). Based on CD4 and IgD staining, the following areas are outlined: follicles (blue), GCs (green), T-B border (magenta), and T-zone (red). Scale bar: 200µm. (B) Identification of Foxp3⁺ cells from (A), colour-coded based on localisation within the T-zone (red), T-B border (magenta), follicle (blue), or GC (green). (C) Average area (mm²) of delineated splenic compartments from (A). (D) Percentage and (E) number/mm² of Foxp3⁺ cells within each splenic niche, quantified from Figure 5.B. (A, B) Images representative of n=5 mice/strain, 2-6 images/mouse. (C–E) Each dot represents the average of technical replicates per biological replicate, n=5 mice/strain ± SEM. Two-tailed unpaired Student’s t test.

Figure 6

Cell-intrinsic CCR6 function is not required for the formation of T_FH and T_FR cell populations. Representative gating strategies of (A) naïve CD4 T cells and nTregs, and (B) T_FH and T_FR cells from day 6 SRBC immunized irradiated Ly5.1 hosts reconstituted with a 1:1 ratio of Ly5.1:CD45.2⁺ Ccr6 ^+/+ (WT, top row) or CD45.2⁺ Ccr6 ^-/- (bottom row) bone marrow. (C) Ratio of CD45.2⁺ T_FH cells:CD45.2⁺ naïve CD4 T cells in Ly5.1:WT and Ly5.1:Ccr6 ^-/- chimeras. (D) Ratio of CD45.2⁺ T_FR cells:CD45.2⁺ nTregs in Ly5.1:WT and Ly5.1:Ccr6 ^-/- chimeras. (A–D) n=6/chimera group, ± SEM, two-tailed unpaired Student’s t test.