Human tonsil B-cell lymphoma 6 (BCL6)-expressing CD4+ T-cell subset specialized for B-cell help outside germinal centers

Abstract

T follicular helper (Tfh) cells represent a Th subset engaged in the help of B-cell responses in germinal centers (GCs). Tfh cells abundantly express the transcription repressor B-cell lymphoma 6 (Bcl6), a factor that is necessary and sufficient for their development in vivo. Whether Tfh or Tfh-committed cells are involved in the help of B cells outside GCs remains unclear, particularly in humans. In this study, we identified a previously undefined BCL6-expressing CD4(+) T-cell subset in human tonsils. This subset expressed IL-7 receptor and chemokine receptor 5 (CXCR5) and inducible costimulator (ICOS) at low levels (CXCR5(lo)ICOS(lo)), and it was found exclusively outside GCs. CXCR5(lo)ICOS(lo) CD4(+) T cells secreted larger amounts of IL-21 and IL-10 than CXCR5(hi)ICOS(hi) GC-Tfh cells upon activation, and they induced proliferation and differentiation of naïve B cells into Ig-producing cells more efficiently than GC-Tfh cells. However, this subset lacked the capacity to help GC-B cells because of the induction of apoptosis of GC-B cells through the FAS/FAS-ligand interaction. CXCR5(lo)ICOS(lo) CD4(+) T cells expressed equivalent amounts of BCL6 transcript with CXCR5(hi)ICOS(hi) GC-Tfh cells, but they expressed less Bcl6 protein. Collectively, our study indicates that CXCR5(lo)ICOS(lo) CD4(+) T cells in human tonsils represent Tfh lineage-committed cells that provide help to naïve and memory B cells outside GCs.

Fig. 1.

Four Th populations in pediatric tonsil samples. (A) Four tonsillar Th populations were defined according to the expression of ICOS and CXCR5. (B) The frequency of each Th population (n = 20). One-way ANOVA Bonferonni multiple comparison test. ***P < 0.001. (C) Expression of cell surface molecules (a representative from three experiments).

Fig. 2.

CXCR5^loICOS^lo cells localize exclusively outside GCs. (A) Localization of IL-7R⁺ cells in tonsils was analyzed by immunohistochemistry using a frozen tonsil section. FM, follicular mantle. (Magnification: 4×.) (B and C) Localization of CXCR5^loICOS^lo CD4⁺ T cells was analyzed by the coexpression of CD4 (red), CD45RO (green), and IL-7R (blue) by immunofluorescence microscopy (10×). Triple positive cells illustrated by the white color include CXCR5^loICOS^lo CD4⁺ T cells. A higher magnification view (40×) is shown in C (analyzed by confocal microscopy).

Fig. 3.

Tonsillar Th populations differentially help B-cell subsets. (A–C) Ig production by B-cell subsets cocultured with tonsillar Th populations. (A) GC-B cells (n = 11 except CXCR5^loICOS^hi, which is n = 3), (B) naïve B cells (n = 4), and (C) memory B cells (n = 6 except CXCR5^loICOS^hi, which is n = 3) were cocultured with each Th population in the presence of SEB for 8 d. One-way ANOVA Bonferonni multiple comparison test. ***P < 0.001, **P < 0.01, and *P < 0.05. (D and E) Recovery of GC-B cells cocultured with Th populations. GC-B cells cultured for 8 d with each Th population were stained with CD3 and CD4 mAbs. Surface IgG and IgA expression by B cells cultured with CXCR5^hiICOS^hi GC-Tfh cells is shown in Right (a representative from four experiments). Absolute number of viable GC-B cells per well (day 8) are shown in E (n = 5). One-way ANOVA Bonferonni multiple comparison test. (F) Recovery of T cells cocultured with GC-B cells. Absolute number of viable T cells per well (day 8; n = 5). One-way ANOVA Bonferonni multiple comparison test. (G) Proliferation and CD38 expression of CFSE-labeled naïve B cells cultured for 8 d with each Th population (a representative from three experiments). (H) Recovery of naïve B cells cocultured with Th populations. Absolute number of viable CD38⁺ plasmablasts per well (day 8; n = 3). One-way ANOVA Bonferonni multiple comparison test. (I) Recovery of T cells cocultured with naïve B cells. Absolute number of viable T cells per well (day 8; n = 3). One-way ANOVA Bonferonni multiple comparison test.

Fig. 4.

Cytokine secretion profiles of tonsillar Th populations. (A and B) Cytokine secretion on interaction with B-cell subsets. The four Th populations were cultured with either naïve B cells (A; n = 6) or GC-B cells (B; n = 5), and the secretions of IL-10, IL-21, IL-17A, and IL-4 were analyzed on day 2. One-way ANOVA Bonferonni multiple comparison test. ***P < 0.001, **P < 0.01, and *P < 0.05. (C) CXCL13 secretion on interaction with naïve B cells (Upper; n = 4) or GC-B cells (Lower; n = 3). One-way ANOVA Bonferonni multiple comparison test. (D) Kinetics of IL21 and CXCL13 expression. The four Th populations were analyzed for the expression of IL21 and CXCL13 by real-time RT-PCR before and after stimulation with CD3/CD28 mAb-coated beads (n = 2–3). One-way ANOVA Bonferonni multiple comparison test for n = 3 and paired t test for n = 2.

Fig. 5.

Molecules associated with helper activity of tonsillar Th populations. (A) IL-21R/Fc chimera protein was added to the cultures of Th populations and the indicated B-cell subsets. Ig concentrations at day 8. A representative from four experiments. Paired t test. ***P < 0.001, **P < 0.01, and *P < 0.05. (B) ICOS mAb was added to the cultures of Th populations and the indicated B cells. Ig concentrations at day 8 (a representative from four experiments). Paired t test. (C) Cytokine secretion. CD40L mAb, ICOS mAb, or an isotype control was added to the cultures of Th populations and the indicated B cells. Cytokine concentrations at day 2 (a representative from three experiments).

Fig. 6.

CXCR5^loICOS^lo CD4⁺ T cells induce apoptosis of GC-B cells through the FAS/FAS-L interaction. (A) Expression of cell surface FAS by GC-B cells. (B) Soluble FAS-L production by Th populations cocultured with GC-B cells. FAS-L concentrations at day 2 (n = 4). One-way ANOVA Bonferonni multiple comparison test. ***P < 0.001, **P < 0.01, and *P < 0.05. (C) FASLG mRNA expression by tonsillar Th populations analyzed by real-time RT-PCR. Expression of FASLG transcript was normalized to that of HPRT1 transcript (n = 3). (D) Ig secretion by GC-B cells cocultured with tonsillar Th populations in the presence of FAS mAb. Ig concentrations were measured at day 8 (a representative from four experiments). (E) Ig secretion by naïve B cells cocultured with CXCR5⁻ICOS⁻ CD4⁺ T cells, CXCR5^loICOS^hi CD4⁺ T cells, or CXCR5^hiICOS^hi GC-Tfh cells supplemented with titrated doses of recombinant IL-21. Ig secretion measured at day 8 (a representative from two experiments).

Fig. 7.

CXCR5^loICOS^lo CD4⁺ T cells express large amounts of BCL6. (A) Expression of BCL6 and PRDM1 transcripts by tonsillar Th populations was analyzed by real-time RT-PCR. Expression of each transcript was normalized to that of HPRT1 transcript (n = 3). One-way ANOVA Bonferonni multiple comparison test. ***P < 0.001, **P < 0.01, and *P < 0.05. (B) Expression of Bcl6 and Blimp-1 proteins in tonsillar Th populations. Sorted Th populations were lysed, and equal amounts of protein were loaded per well to analyze the expression of Bcl6 and Blimp-1 by Western blotting. For the positive controls, the lysate of GC-B cells was used for Bcl6 detection, and BJAB (a B-cell line) cell lysate was used for Blimp-1 detection. Expected Blimp-1 band is indicated with an arrow. Densitometry score ratio for Bcl6 and Blimp-1 against control actin protein is indicated in number (a representative of four experiments). (C) Analysis of Bcl6 expression in tonsillar CD4+ Th populations by flow cytometry (a representative of four experiments). (D) Up-regulation of ICOS and PD-1. The four Th populations were cultured with naïve B cells, and the expression of ICOS and PD-1 on T cells was analyzed at day 5 (a representative from two experiments).

Fig. 8.

CXCR5^loICOS^lo CD4⁺ T cells in adult tonsils. The four Th populations in pediatric tonsil samples, adult tonsil samples, and human spleen samples. The same number of T-enriched cells (after removing B cells with magnetic beads) was stained and analyzed in parallel in identical conditions. The four Th populations were determined based on the analysis of pediatric tonsil samples, and the same gates were applied for the adult and spleen samples. (A) A representative of at least two experiments gated to CD3⁺CD4⁺ T cells. (B) The frequency of each Th population in adult tonsils (n = 3) and spleen (n = 2). One-way ANOVA Bonferonni multiple comparison test for adult tonsil samples. *P < 0.05.

Fig. P1.

Two distinct Tfh-committed cells in human tonsils differentially help B-cell subsets. CXCR5^loICOS^lo CD4⁺ T cells localize exclusively outside germinal centers (GC) and induce naïve and memory B cells to become antibody-secreting cells. CXCR5^hiICOS^hi GC-Tfh cells are specialized to help B cells in GCs. Whereas CXCR5^loICOS^lo CD4⁺ T cells produce higher levels of IL-21 upon interaction with B cells, GC-Tfh cells produce higher levels of CXCL13. CXCR5^loICOS^lo CD4⁺ T cells and GC-Tfh cells show similar expression profiles of BCL6 and PRDM1 transcripts. Our study suggests that tonsillar CXCR5^loICOS^lo CD4⁺ T cells represent Tfh-committed extrafollicular helper cells and/or precursors of GC-Tfh cells.