Follicular helper T cell differentiation requires continuous antigen presentation that is independent of unique B cell signaling

Abstract

Effective humoral immunity depends on the support of B cell responses by T follicular helper (Tfh) cells. Although it has been proposed that Tfh cell differentiation requires T-B interactions, the relative contribution of specific populations of Ag-presenting cells remains unknown. We employed three independent strategies that compromised interactions between CD4(+) T cells and activated B cells in vivo. Whereas the expansion of CD4(+) T cells was relatively unaffected, Tfh cell differentiation was completely blocked in all scenarios. Surprisingly, augmenting antigen presentation by non-B cells rescued Tfh cell differentiation, as determined by surface phenotype, gene expression, and germinal center localization. We conclude that although Ag presentation by responding B cells is typically required for the generation of Tfh cells, this does not result from the provision of a unique B cell-derived signal, but rather because responding B cells rapidly become the primary source of antigen.

Figure 1. SAP deficiency severely compromises the development of Tfh cells

WT or Sh2d1a^−/− (SAP KO) OT-II cells were transferred to congenic recipients that were then immunized with OVA plus Alum i.p. (A) the proportion of OT-II cells in the spleen was determined at various times (Mean ± SEM, n=5–14). (B–C) Cells were stained for CXCR5 and PD1 and the number of CXCR5^hiPD1^hi cells was determined (Mean ± SEM, n=5–14). (C) Contour plots show CXCR5 and PD1 expression on OT-II cells on days 3 (left panel) and 7 (right panel) post-immunization. Cells were stained for CCR7 (D) or ICOS (E). Histograms show CCR7 or ICOS expression on endogenous CD4⁺ T cells (grey) and OT-II cells (red) after 3 (upper panel) or 7 (lower panel) days. Graphs show relative change in expression of CCR7 and ICOS on OT-II cells compared to endogenous CD4⁺T cells (Mean ± SEM, n=7–8). (F) Immunofluorescence staining of spleen sections was carried out to determine the positioning of OT-II T cells (CD45.2⁺) within the follicle (IgD) and T cells zone (CD4) after 3 (left panel) or 7 days (right panel). (G) Further staining confirmed positioning within the GC (PNA) at day 7.

Figure 2. Rescuing germinal centre formation does not facilitate Tfh cell development from SAP-deficient CD4⁺ T cells

CD45.1-congenic mice were transferred with CD45.2⁺ OT-II cells. Mice received either Thy1.1⁻ WT OT-II or Thy1.1⁺ SAP KO OT-II alone or a combination of equal numbers of Thy1.1⁻ WT OT-II or Thy1.1⁺ SAP KO OT-II and were then immunized i.p with OVA plus Alum. The responses were examined 7 days later. (A) The proportion of IgG1⁺GL7⁺ (GC) B cells in recipient spleens. (B) The proportion of OT-II cells from each donor was determined – WT OT-II: filled bars, SAP KO OT-II: open bars. (C) The proportion of CXCR5^hiPD1^hi Tfh cells from each donor was determined. All graphs show Mean ± SEM, n=4 (D) Contour plots show expression of CXCR5 and PD1 on OT-II cells of each genotype.

Figure 3. Treatment with peptide can rescue Tfh cell development from SAP-deficient T cells

WT or SAP KO OT-II cells were transferred to congenic recipients that were then given OVA plus Alum i.p. at day 0. On day 3 some mice received additional OVA peptide i.v. and the mice then sacrificed on day 7. (A) The proportion of total OT-II cells in the spleen. (B) The proportion of CXCR5^hiPD1^hi Tfh OT-II cells was determined by (C) staining for CXCR5 and PD1 on OT-II cells. (D) Proportion of GC B cells as determined by staining for expression of GL7 and Fas. (E) Expression of CD62L and PD1 on OT-II cells. (F) Percentage of OT-II cells that are CD62L^lo. (G) Expression of CD127 and PD1 on OT-II cells. (H) Percentage of cells OT-II cells that are CD127^lo. All graphs show Mean±SEM, n=7–9, except (F) n=4–6. (I) Immunofluorescence staining of spleen sections was performed to determine the positioning of OT-II T cells (CD45.2⁺) within the follicle (IgD) and GC (PNA). (J-M) CD62L^hi, CD62L^loPD1^lo and CD62L^loPD1^hi populations of OT-II CD4⁺ T cells (CD4⁺B220⁻CD45.2⁺) were isolated by sorting. Expression of Bcl6 (K), Il21 (L) and Sh2d1a (M) in each of these populations was then determined by quantitative PCR. N.D. - Not done. The values represent the (Mean±SEM of 4 experiments for peptide boost, 3 experiments for non-boost).

Figure 4. B cell activation via CD40 is not required for Tfh development

Chimeras were generated as described in Experimental Procedures (see also Figure S4A) using 80:20 mix of µMT:Cd40^−/− BM (to generate mice with B cells lacking CD40) or B6: Cd40^−/− BM (controls). Thy1.1⁺ WT OT-II cells were transferred into the chimeras, which were then given OVA plus Alum i.p. at day 0. On day 3 some mice received additional OVA peptide i.v. and the mice were sacrificed on day 7 for analysis. (A) Reconstitution was determined by staining B cells (B220⁺) and DC (CD11c⁺) for CD40 expression. (B) The proportion of total OT-II cells in the spleen. (C) The proportion of CXCR5^hiPD1^hi Tfh OT-II cells was determined by (D) staining for CXCR5 and PD1 on OT-II cells. (E) Proportion of GC B cells as assessed by staining for expression of GL7 and Fas. (F) Percentage of cells OT-II cells that are CD127^lo. All plots show Mean ± SEM, n=7–14.

Figure 5. B cell Ag-presentation is not required for Tfh development

Chimeras were generated as described in Experimental Procedures using 80:20 mix of µMT:MHCII^−/− BM (to generate mice with B cells lacking MHC class II) or B6:MHCII^−/− BM (controls). Thy1.1⁺ WT OT-II cells were transferred into the chimeras, which were then given OVA plus Alum i.p. at day 0. On day 3 some mice received additional OVA peptide i.v. and the mice were sacrificed on day 7 for analysis. (A) Reconstitution was determined by staining B cells (B220⁺) and DC (CD11c⁺) for MHCII expression. (B) The proportion of total OT-II cells in the spleen. (C) The proportion of CXCR5^hiPD1^hi Tfh OT-II cells was determined by (D) staining for CXCR5 and PD1 on OT-II cells. (E) Proportion of GC B cells as assessed by staining for expression of GL7 and Fas. (F) Percentage of cells OT-II cells that are CD127^lo. All plots show Mean ± SEM, n=6–8.

Figure 6. Peptide boost prolongs Ag-presentation by DC

Mice were given Alum or OVA plus Alum i.p. at day 0. At day 4 mice some mice received additional OVA peptide i.v. On day 6 the mice were sacrificed and spleens removed. CD11c⁺ cells were isolated by cell sorting and were placed in culture with CFSE-labeled OT-II cells to detect presentation of OVA peptide. Cultures were harvested 3 days later and OT-II cells identified by staining for CD4. (A) Representative CFSE profiles of OT-II cells. (B) Plots show percentage of cells that are in division (Mean ± range of two experiments).

Figure 7. Effect of peptide boost on B cell responses

WT congenic mice received WT or SAP KO OT-II cells in combination with WT SW_HEL cells. Mice were immunized i.p. with OVA plus Alum and i.v. with HEL^2X-OVA_pep. Some mice received additional peptide at Day3 and 6. Mice were sacrificed at day 10 and spleens removed. (A) The proportions of total OT-II cells and (B) CXCR5^hiPD1^hi Tfh OT-II cells were determined. (C) Splenocytes were stained for CD45.1 and CD45.2 to detect donor cells and the proportion of total HEL-binders was determined by staining with HEL^WT. (D) The titers of anti-HEL IgG1 in the serum were measured by ELISA against HEL^WT (E) The proportion of IgG1-switched cells of high affinity was determined by staining for with HEL^3X. Plots show donor SW_HEL cells (gated using CD45.1 and CD45.2) that are IgG1⁺. Numbers show proportion of cells that bind HEL^3X with high affinity (i.e. cells that have undergone affinity maturation). (F) Graphs show proportion of IgG1⁺ SW_HEL cells that had undergone affinity maturation (numbers are expressed relative to the average of non-boosted controls in each experiment). (G) The degree of affinity matured anti-HEL in the serum was determined by coating plates with HEL^4X. All plots show mean ± SEM of at least 12 animals combined from multiple experiments.