Transcription factor IRF4 determines germinal center formation through follicular T-helper cell differentiation

Abstract

Follicular T-helper (T(FH)) cells cooperate with GL7(+)CD95(+) germinal center (GC) B cells to induce antibody maturation. Herein, we identify the transcription factor IRF4 as a T-cell intrinsic precondition for T(FH) cell differentiation and GC formation. After immunization with protein or infection with the protozoon Leishmania major, draining lymph nodes (LNs) of IFN-regulatory factor-4 (Irf4(-/-)) mice lacked GCs and GC B cells despite developing normal initial hyperplasia. GCs were also absent in Peyer's patches of naive Irf4(-/-) mice. Accordingly, CD4(+) T cells within the LNs and Peyer's patches failed to express the T(FH) key transcription factor B-cell lymphoma-6 and other T(FH)-related molecules. During chronic leishmaniasis, the draining Irf4(-/-) LNs disappeared because of massive cell death. Adoptive transfer of WT CD4(+) T cells or few L. major primed WT T(FH) cells reconstituted GC formation, GC B-cell differentiation, and LN cell survival. In support of a T-cell intrinsic IRF4 activity, Irf4(-/-) T(FH) cell differentiation was not rescued by close neighborhood to transferred WT T(FH) cells. Together with its known B lineage-specific roles during plasma cell maturation and class switch, our study places IRF4 in the center of antibody production toward T-cell-dependent antigens.

Fig. 1.

Lack of GC formation in Irf4^−/− mice. Mice of the indicated genotypes were infected with L. major. Two weeks later, popliteal LNs were prepared. (A) Tissue sections were stained for CD4 (red), GL7 (green, Left), or B220 (green, Right), and analyzed by fluorescence-microscopy (20× magnification). (Scale bars, 50 μm.) (B and C) LN cell suspensions were stained for the indicated surface molecules and analyzed by flow cytometry. Numbers indicate percentages of positive cells in the respective circles or rectangles. Panels are from one representative mouse per group. Three different experiments, each with two or three mice per group. (B) B220⁺ B-cell gate. Percentages (mean ± SD) of GL7⁺CD95⁺ B cells compiled from all mice tested.

Fig. 2.

Lack of T_FH cells in L. major infected Irf4^−/− mice. Irf4^−/−, WT mice, and Irf4^+/− mice (three per group) were infected with L. major and their popliteal LN cells analyzed 2 wk later for expression of extracellular ICOS, CXCR5, and PD-1 (A) and intracellular BCL-6 (B) and IL-21 (C). (A; B; C, Right; D; and E) Direct ex vivo analysis. (C) Analysis after restimulation for 4 h with PMA and ionomycin (Left). (Right) CD4⁺ cells from pooled LN cells of all mice per group sorted according to ICOS expression (see A), and analyzed for IL-21 compared with HPRT (hypoxanthine-guanine phosphoribosyl transferase) expression by RT qPCR. Bars denote the SD of duplicate qPCR determinations of each sample. The asterisk signifies that this value was set to one. n.t., not tested. (D and E) Single CD4⁺ cells were gated similar to gate B of Fig. S2B. Numbers in A–E indicate percentages of cells in the respective rectangles. Data are from one representative mouse per group. Three experiments with similar outcomes.

Fig. 3.

Lack of T_FH cell differentiation in PP of naive Irf4^−/− mice. (A–C) PP of naive mice were analyzed as described for Figs. 1 and 2.

Fig. 4.

Disappearance of lesion-draining LNs in L. major infected Irf4^−/− mice. Mice of the indicated genotypes were infected with L. major. At the indicated time points, popliteal LNs were prepared and (A) cell numbers in single-cell suspensions counted (mean ± SD) or (B) tissue sections processed for HE staining and microscopy (40× magnification). (Scale bars, 10 μm.) (C) To control for proliferative capacity 2 wk after infection, cells were labeled with 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), stimulated for 72 h with PMA/ionomycin, and stained for CD4. Data are representative of five (A) or three (B and C) different experiments, each with three mice per group and (B and C) show the results of one representative mouse per group.

Fig. 5.

Rescue of GC formation by WT CD4⁺ T cells. Irf4^−/− mice (A–C) or Irf4^+/− mice (B and C) were infected with L. major. Where indicated, Irf4^−/− mice received 8 × 10⁶ Ly5.1 congenic WT CD4⁺ cells by intraperitoneal adoptive transfer on the day of infection. Two weeks later, popliteal LNs were prepared. (A) Tissue sections were stained with antibodies to CD4 (blue), Ly5.1 (red), and GL7 (green) followed by fluorescence-microscopy (20× magnification). (Scale bars, 50 μm.) (B and C) Cells in suspension were stained for B220, GL7, ICOSL, and CD95 and analyzed (B220⁺ gate) by flow cytometry. Numbers refer to percentages in the quadrant below the number (C) or within the indicated gate (B). SSC, side scatter. Data are from one representative mouse per group. Three (A) or two (B and C) different experiments, each with three mice per group, were performed with similar outcome.

Fig. 6.

Rescue of LN cell survival by WT CD4⁺ cells. (A–C) Where indicated, Irf4^−/− mice received 8 × 10⁶ Ly5.1 congenic WT B or CD4⁺ cells or both by intraperitoneal transfer, were infected, and analyzed 6 wk later in comparison with infected WT or Irf4^+/− mice. (A) Popliteal LN cell numbers. (B) H&E staining (40× magnification) (Scale bars, 10 μm.) (C, Upper) total life cell gate. Numbers refer to the percentage of CD4⁺Ly5.1⁻ or CD4⁺Ly5.1⁺ among total cells. (Lower) Relative cell percentages among CD4⁺Ly5.1⁺ cells (Upper numbers) or CD4⁺Ly5.1⁻ cells (Lower) within the respective rectangles. (D and E) T_FH cells (CD4⁺ICOS^hiCXCR5⁺) or control CD4⁺/ICOS⁻/CXCR5⁻ cells sorted (Fig. S6) from 2 wk-infected Ly5.1⁺ WT mice were transferred intraperitoneally (2 × 10⁵ per mouse) into Irf4^−/− mice which, together with control mice, were infected with L. major. After 6 wk, cell numbers of the draining LNs were counted (D) and the proliferative capacity of the cells determined by CFSE dilution (E). Data are representative of three (A–C) or two (D and E) different experiments, each with three (A–C) or two (D and E) mice per group. (D) Accumulated data ± SD of all mice per group in the two experiments or data from one representative mouse (E).

Fig. 7.

Role of IL-21. Irf4^−/− mice were infected with L. major. Where indicated, they also received 8 × 10⁶ purified Il21^−/− or WT CD4⁺ cells by intraperitoneal adoptive transfer. Two weeks (A and B) or 6 wk (C) after infection, draining LNs were prepared (B and C) as cell suspensions. (A) Tissue sections stained with antibodies to CD4 (red) and GL7 (green) and analyzed by fluorescence-microscopy (20× magnification). (Scale bars, 50 μm.) (B) FACS analysis of gated B220⁺ B cells stained for GL7 and CD95. Numbers indicate frequencies of B cells coexpressing GL7 and CD95. (C) Cell numbers within the draining LNs of individual mice with or without transfer of the indicated cells. Data are from one mouse representative for all experiments (A and B) or were compiled from the results of three individual experiments (C).