Functional and epigenetic studies reveal multistep differentiation and plasticity of in vitro-generated and in vivo-derived follicular T helper cells - PubMed (original) (raw)

Functional and epigenetic studies reveal multistep differentiation and plasticity of in vitro-generated and in vivo-derived follicular T helper cells

Kristina T Lu et al. Immunity. 2011.

Abstract

Follicular T helper (Tfh) cells provide critical help to B cells for germinal center (GC) formation. Mutations affecting SLAM-associated protein (SAP) prevent GC formation because of defective T cell-B cell interactions, yet effects on Tfh cell differentiation remain unclear. We describe the in vitro differentiation of functionally competent "Tfh-like" cells that expressed interleukin-21, Tfh cell markers, and Bcl6 and rescued GC formation in SAP-deficient hosts better than other T helper (Th) cells. SAP-deficient Tfh-like cells appeared virtually indistinguishable from wild-type, yet failed to support GCs in vivo. Interestingly, both Tfh-like and in vivo-derived Tfh cells could produce effector cytokines in response to polarizing conditions. Moreover, Th1, Th2, and Th17 cells could be reprogrammed to obtain Tfh cell characteristics. ChIP-Seq analyses revealed positive epigenetic markings on Tbx21, Gata3, and Rorc in Tfh-like and ex vivo Tfh cells and on Bcl6 in non-Tfh cells, supporting the concept of plasticity between Tfh and other Th cell populations.

Copyright © 2011 Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. In vitro generated Tfh-like cells are distinct from other Th cells

(A, B) Sorted naïve CD4+ T cells from WT mice were assessed for IL-21, IFNγ, IL-4, IL-17, and IL-2 cytokine production after 3.5 days in Tfh-like or Th0 culture conditions in RPMI (A) or IMDM media [(A) and (B)]. (C) In vitro differentiated Th1, Th2, Th17, and Tfh-like cells were gated on CD4+CD19−CD44+ cells and evaluated for surface CXCR5 and PD-1. B cells and T-B cell doublets were excluded by CD19 staining. Mean ± SD of %CXCR5+PD-1+ cells are shown below each plot. Inserts: CXCR5 expression on Tfh-like cells and respective Th cells (grey). Summaries of IL-21+ and CXCR5+PD-1+ cells are shown in Figures S1A and C. (D) Relative expression of Blr1, compared in WT naïve and in vitro differentiated Th0, Th1, Th2, Th17, and Tfh-like cells. Expression was calculated in reference to 18s rRNA, and normalized to naïve CD4+ T cells. (E) Surface expression of ICOS, BTLA, and CD84. Flow cytometry is representative of 5 or more independent experiments using 2–3 mice per genotype. (F–G) Relative expression of genes encoding Th cell-specific transcription factors Bcl6 (F) and Tbx21, Gata3 and Rorc (G) in Tfh-like and other Th cell populations. Expression was calculated in reference to 18s rRNA and normalized to Th0 cells (Bcl6), naïve CD4+ T cells (Tbx21 and Gata3) or Th2 cells (Rorc). Data in (D, F–G) are representative of at least 3 samples per group, with 2–3 mice per sample. ** = P < 0.005, * = P < 0.05, nd = not detectable.

Figure 2

Figure 2. In vitro differentiated Sh2d1a−/− Tfh-like cells resemble WT Tfh-like cells

(A–C) WT and Sh2d1a−/− sorted naïve CD4+ T cells cultured under Tfh-like conditions and evaluated for IL-21 and IL-17 production (A, top and B), surface CXCR5+PD-1+ (A, bottom), and ICOS, BTLA, and CD84 (C). (D) Microarray pairwise comparison of gene expression in WT and Sh2d1a−/− Tfh-like cells (from independent biological triplicates, using 2–3 mice per experiment).

Figure 3

Figure 3. WT Tfh-like cells promote GC response in Sh2d1a−/− mice

GC B cells (B220+IgDloGL7hiFashi) in draining lymph nodes six days post-immunization with NP-OVA in Alum. (A) Left: WT or Sh2d1a−/− mice without cell transfers; middle and right: transfer of WT or Sh2d1a−/− in vitro generated Th2 or Tfh-like cells into Sh2d1a−/− hosts one day prior to immunization. (B) Summary of % GC B cells in Sh2d1a−/− mice after cell transfer and immunization. Immunized WT mice had an average of 14.35 +/− 0.65 SEM % GC B cells. Transferred cells: WT, filled circles; Sh2d1a−/, open circles; no cells transferred (nt), open triangles. P<0.005 for WT Tfh-like cell transfers compared to all other cell transfers. (C) Tfh cells (CXCR5+PD-1+) in Sh2d1a−/− mice after transfer of Tfh-like cells and immunization. Left: total Tfh cells (gated on CD4+CD44hi); right: transferred donor cells (gated on CD4+CD44hiGFP+). (D) Summary of % GFP+ cells, 6 days post-immunization in draining LNs of Sh2d1a−/− mice that received WT or Sh2d1a−/− Tfh-like cells. Data points represent one mouse, from three or more independent experiments. (E) Immunofluorescence of draining lymph node sections. Top Panel: GFP+ OT-II cells (GFP+, green); GC (PNA, red); follicle (IgD, blue), 6 days after immunization. Middle panel: IgD and GFP+ cells only. Lower panel: GFP staining alone for better visualization. (F) NP-specific antibody titers (total IgG, IgG1, IgG2b) measured by ELISA. Data in (A), (C) and (F) are representative of three or more independent experiments.

Figure 4

Figure 4. Epigenetic state and cytokine plasticity of Tfh-like cells

Evaluation of cytokine production (A) and CXCR5+PD-1+ expression (B) after 2 rounds of Tfh-like polarization. Percentage of CXCR5+PD-1+ cells after one round of Tfh-like polarization is listed below (B). Data are representative of 3 or more independent experiments. (C–F) Genome browser view of ChIP-Seq profile maps. (C, D) Distributions of H3K4me3 (blue) and H3K27me3 (red) on genes encoding Th cell-determining transcription factors, Tbx21 (chr11:96,942,182–96,993,793), Gata3 (chr2:9,764,070–9,826,790), and Rorc (chr3:94,143,165–94,220,573) shown for (C) double-polarized in vitro generated Th cells and Tfh-like cells, and (D) sorted CD4+CD19−CD44hiCXCR5+PD-1+ in vitro differentiated Tfh-like cells and Tfh cells isolated ex vivo from SRBC-immunized mice. Biological triplicates of Th cell cultures with comparison to data from (Wei et al., 2009) are shown in Figure S3A. (E,F) Distributions of H3K4me3 (blue) and H3K27me3 (red) on Th cell-specific cytokine genes Ifng (chr10:117,864,373–117,896,679), Il4 (chr11:53,418,049–53,445,596), and Il17a (chr1:20,710,659-20,734,904) shown for (E) double-polarized in vitro generated Th cells and Tfh-like cells and (F) sorted CD4+CD19−CD44hiCXCR5+PD-1+ in vitro Tfh-like cells and ex vivo Tfh cells. Gene structure is from the UCSC Genome Browser. Where no signals were detected, corresponding columns are blank. Vertical scales are noted. (G) Cytokine production examined after repolarization of Tfh-like cells under Th1, Th17, and Th2 conditions. (H) Cytokine production of sorted Tfh cells after isolation ex vivo and rested one day in the presence of IL-2 (top) and after repolarization under Th1, Th2, and Th17 conditions (bottom). Data in (G) and (H) are representative of three or more independent experiments.

Figure 5

Figure 5. Histone H3K4me3 and H3K27me3 modifications of Bcl6 and Prdm1.

H3K4me3 (blue) and H3K27me3 (red) modifications on (A, B) Bcl6 (chr16:23,941,577-24,012,259) and (C, D) Prdm1 (chr10:44,133,792-44,279,402) loci in (A, C) naïve CD4+ T cells, and double-polarized Th1, Th2, Th17, Tfh-like, and (B, D) CD4+CD19−CD44hiCXCR5+PD-1+ sorted Tfh-like cells and Tfh cells isolated ex vivo from SRBC-immunized mice. Biological triplicates for Th cell cultures are shown in Figure S4.

Figure 6

Figure 6. Repolarization of Th1, Th2, and Th17 cells in Tfh-like conditions

(A) Cytokine production from polarized Th1, Th2, and Th17 cells. (B) Cells were rested, repolarized under Tfh-like conditions, and evaluated for cytokine production (B) and surface markers CXCR5 and PD-1 (C). The percentages of CXCR5+PD-1+ cells of the original polarized Th1, Th2, and Th17 cell populations are listed in parentheses. Data are representative of three or more independent experiments.

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