Identification of novel markers for mouse CD4(+) T follicular helper cells

Abstract

CD4(+) T follicular helper (TFH) cells are central for generation of long-term B-cell immunity. A defining phenotypic attribute of TFH cells is the expression of the chemokine R CXCR5, and TFH cells are typically identified by co-expression of CXCR5 together with other markers such as PD-1, ICOS, and Bcl-6. Herein, we report high-level expression of the nutrient transporter folate R 4 (FR4) on TFH cells in acute viral infection. Distinct from the expression profile of conventional TFH markers, FR4 was highly expressed by naive CD4(+) T cells, was downregulated after activation and subsequently re-expressed on TFH cells. Furthermore, FR4 expression was maintained, albeit at lower levels, on memory TFH cells. Comparative gene expression profiling of FR4(hi) versus FR4(lo) Ag-specific CD4(+) effector T cells revealed a molecular signature consistent with TFH and TH1 subsets, respectively. Interestingly, genes involved in the purine metabolic pathway, including the ecto-enzyme CD73, were enriched in TFH cells compared with TH1 cells, and phenotypic analysis confirmed expression of CD73 on TFH cells. As there is now considerable interest in developing vaccines that would induce optimal TFH cell responses, the identification of two novel cell surface markers should be useful in characterization and identification of TFH cells following vaccination and infection.

Keywords: B cells; CD4+ T cells; CD73; Folate R 4; Viral infection.

Figure 1

FR4 expression distinguishes T_FH and T_H1 subsets. Antigen-specific CD4 effectors in spleen were sorted based on Ly6C expression and mRNA levels of FR4 (A) and other folate transport genes (B) were examined. (C) Flow cytometric analysis confirmed high FR4 expression on Ly6C^lo antigen-specific CD4 T cells. (D) shows expression of CXCR5 and PD-1 on day 8 effectors and histograms show FR4 expression on T_FH and T_H1 subsets in. In (E) comparative phenotypic analysis of FR4^hi, CXCR5⁺ (red histogram) and FR4^lo, CXCR5⁻ subsets (blue histogram) is shown. FR4 is highly expressed by GL7⁺, CXCR5⁺ GC T_FH (F) and (G) shows localization of FR4 expressing CD4 T cells in the germinal centers in spleen at day 12 post-infection. (H) shows comparative expression profile of FR4 on antigen-specific CD4 T cells in the spleen, liver, and lung. FR4 expression on endogenous GP66 tetramer positive cells is shown in (I). Data are representative of three independent experiments.

Figure 2

FR4 expression on naive precursors does not determine T_FH/T_H1 commitment. Differentiation of FR4^hi and FR4^lo cells isolated from naive mouse spleen was determined as shown in (A). In (B), frequency and expression of FR4 on donor cells is shown in recipient spleen at day 8 post-infection. Bar graphs in (C) show number and frequency of donor cells per spleen. (D), shows % of T_FH identified by co-expression of CXCR5 and PD-1, and CXCR5 and FR4 and % of GL7⁺ GC T_FH in spleen at day 8 post-infection. Data are representative of three independent experiments. Bar graphs show mean ± SEM

Figure 3

Dynamic regulation of FR4 expression during T_FH differentiation. (A) Kinetic analysis of FR4 expression on naive and antigen-specific CD4 T cells. Red line indicates percentage of FR4^hi cells relative to isotype control staining. In (B), percentage of CXCR5⁺, PD-1^hi T_FH cells and CXCR5⁺, Bcl-6⁺ GC T_FH cells in FR4^lo versus FR4^hi subsets is shown. Data are representative of three independent experiments.

Figure 4

Re-expression of FR4 on T_FH cells requires interaction with cognate B cells. SMARTA CD4 T cells were transferred to WT or HEL Tg mice and responses were assessed 8 days after LCMV infection (A). Flow plots in (B) are gated on total B220⁺ B cells and show Fas⁺, PNA⁺, GC B cells in spleen. (C) shows % GC cells in spleen and total number of GC B cells in spleen is shown in (D). (E) LCMV enzyme linked immunosorbent assay (ELISA) was performed at day 8; LCMV-specific IgG endpoint titers in sera are shown. Flow plots in (F) are gated on CD4⁺ SMARTA cells in the spleen and show frequency of T_FH cells and GC T_FH cells in WT and HEL Tg mice. (G) shows frequency of FR4^hi, CXCR5⁺ cells and black histogram shows % FR4^hi antigen-specific CD4 T cells. Bar graph shows mean % T_FH and % FR4^hi antigen-specific CD4 T cells in peripheral blood (H) and spleen (I). (J), shows total number of T_FH and FR4^hi cells in spleen. Data are representative of two independent experiments. Bar graphs show mean ± SEM

Figure 5

Gene expression profiling of T_FH and T_H1 cells. (A) shows sorting strategy used to purify day 12 SMARTA CD4 T effectors based on FR4 and CXCR5 expression. Differentially regulated genes between T_FH and T_H1 subsets were identified using significance analysis of microarrays, which found 711 differentially regulated genes with a false discovery rate of 0.08 and a fold change of at least 2-fold. GSEA plots in B and C, show enrichment profile of T_FH and T_H1 genes in FR4^hi, CXCR5^hi and FR4^lo, CXCR5^lo subsets, respectively. Relative expression of select genes in T_FH/T_H1 subsets is shown in (D). Of note, CXCR5⁺, FR4^hi cells did not express Foxp3 transcript levels indicating that this subset did not comprise of T follicular regulatory cells. Data are representative of two independent replicates.

Figure 6

Gene expression profiling leads to identification of CD73 as a novel T_FH cell marker. Table A provides a list of select metabolic pathways enriched in T_FH. (B), shows genes related to purine metabolic pathway differentially expressed between T_FH and T_H1 subsets; P2rx7, purinergic receptor P2X ligand gated ion channel 7; ADA, adenosine deaminase; ADK, adenosine kinase; Pde2a, phosphodiesterase 2a; Dck, deoxycytidine kinase. (C) Phenotypic analysis of CD73 confirms high expression on FR4^hi and CXCR5⁺ antigen-specific T_FH cells. (D, E) Comparison of T_FH cells identified using CXCR5, PD-1 versus CD73 and FR4. Data are representative of three independent replicates.

Figure 7

Expression of FR4 is sustained on memory CXCR5⁺ antigen-specific cells. Kinetics of FR4 (A,B) and CD73 (C,D) expression on antigen-specific CD4 T cells from day 12 to day 280. Histograms show expression on CXCR5⁻ (grey) and CXCR5⁺ (black) subsets. Figure E shows expression of PD-1, ICOS, and Bcl-6 on antigen-specific CD4 T cells at effector and memory time-points. Data are representative of three independent experiments