ICOS receptor instructs T follicular helper cell versus effector cell differentiation via induction of the transcriptional repressor Bcl6

Abstract

The nature of follicular helper CD4(+) T (Tfh) cell differentiation remains controversial, including the minimal signals required for Tfh cell differentiation and the time at which Tfh cell differentiation occurs. Here we determine that Tfh cell development initiates immediately during dendritic cell (DC) priming in vivo. We demonstrate that inducible costimulator (ICOS) provides a critical early signal to induce the transcription factor Bcl6, and Bcl6 then induces CXCR5, the canonical feature of Tfh cells. Strikingly, a bifurcation between Tfh and effector Th cells was measurable by the second cell division of CD4(+) T cells, at day 2 after an acute viral infection: IL2Rα(int) cells expressed Bcl6 and CXCR5 (Tfh cell program), whereas IL2Rα(hi) cells exhibited strong Blimp1 expression that repressed Bcl6 (effector Th cell program). Virtually complete polarization between Bcl6(+) Tfh cells and Blimp1(+) effector Th cell populations developed by 72 hr, even without B cells. Tfh cells were subsequently lost in the absence of B cells, demonstrating a B cell requirement for maintenance of Bcl6 and Tfh cell commitment via sequential ICOS signals.

Figure 1. Bcl6⁺CXCR5⁺ Tfh cell differentiation occurs within 72 hours in vivo

(A–D) LCMV-specific SM CD4⁺ T cells were transferred into B6 mice that were subsequently infected with LCMV. (A) Bcl6⁺CXCR5⁺ Tfh cells analyzed 7 days post-infection (p.i.) (“d7”) vs. naïve (“N”, CD44^loCD62^hi) CD4⁺ T cells of uninfected control mice. (B) Bcl6 protein expression in d7 SM as Δ MFI (e.g. Bcl6 MFI_Tfh - Bcl6 MFI_naiveCD4). (C) Bcl6⁺CXCR5⁺ Tfh cells, 3 and 4 days p.i. (“d3”, “d4”). “N”, uninfected mice. FACS plot shows SM CD4⁺ T cells. (D) Increase in SM CXCR5 MFI compared to that of naive (CD44^loCD62^hi) CD4⁺ T cells of uninfected mice. (E–H) Day 3 and 4 after LCMV infection, PD-1 (E) and CD69 (F) co-expression with CXCR5 on SM CD4⁺ T cells. (G) PD-1 and (H) CD69 MFIs of CXCR5⁺ SM vs. CXCR5⁻ SM. Data are representative of three or more independent experiments; n = 4 per time point for all panels. *** P<0.001.

Figure 2. Early Bcl6⁺CXCR5⁺ Tfh cell differentiation is B cell-independent and does not require SAP- or CD40L–mediated signals

(A) Naïve Sh2d1a^−/− or WT SM CD4⁺ T cells were transferred into B6 mice subsequently infected with LCMV. Bcl6⁺CXCR5⁺ Tfh cell differentiation analyzed at 3 and 4 days p.i. FACS plots show Sh2d1a^−/− SM and gates identify Bcl6⁺CXCR5⁺ Tfh cells. (B) WT SM CD4⁺ T cells were transferred into either B6 or Cd40^−/− mice subsequently infected with LCMV. Tfh cell differentiation was analyzed as shown in (A). (C, D) SM CD4⁺ T cells were transferred into B6 or B cell-deficient (µMT) mice, infected with LCMV, and the CD4⁺ T cell response was analyzed 7 days later. (C) SM CD4⁺ T cells are shown. Gate indicates SLAM^loCXCR5^hi Tfh cells. (D) SM cells are shown and gates identify Bcl6⁺CXCR5⁺ Tfh cells. Bcl6 protein calculated Δ MFI (e.g., Bcl6 MFI_Tfh - Bcl6 MFI_naiveCD4). (E, H) SM CD4⁺ T cells were transferred into B6 and µMT (or MD4-µMT. B def.) mice. Mice were then infected with LCMV and analyzed 3 (E) and 4 (H) days p.i. FACS plots show total SM cells, and gates identify Bcl6⁺CXCR5⁺ Tfh cells. Data are a composite of 3 or more independent experiments (n=15–16 per group for day 3, n=17–20 per group for day 4). (F, G) LCMV Gp61 peptide pulsed DCs were activated in vitro with LPS and transferred into B6 mice that had received naïve SM CD4⁺ T cells. SM were analyzed 4 days after DC immunization. (F)Left, SM CD4⁺ T cells in DC immunized (red line) vs. unimmunized mice (gray filled) compared to that of endogenous naïve (CD44^lo) CD4⁺ T cells in DC immunized mice (black line). Right, CXCR5 MFIs calculated. Day 4 SM (“day4”) vs. SM in unimmunized mice (“N”). (G) Bcl6⁺CXCR5⁺ Tfh cells. (A-D, F and G) Data are representative of two independent experiments. n = 4 per group. n.s., no statistically significant difference (P>>0.05). *P<0.05, ***P<0.001.

Figure 3. Early Tfh cell commitment requires ICOS signals for Bcl6 expression

WT or Icos^−/− SM CD4⁺ T cells were transferred into B6 mice subsequently infected with LCMV. (A, B) Bcl6⁺CXCR5⁺ Tfh cell development was analyzed 3 and 4 days p.i. by FACS (A) and quantified (B). (C) Bcl6 protein expression was quantified in WT CXCR5⁺ SM and WT CXCR5 SM vs. Icos^−/− SM CD4⁺ T cells. (D) PD-1 expression on WT and Icos^−/− SM CD4⁺ T cells. (E)Left, CXCR5 expression on WT (black) and Icos^−/− (red) SM CD4⁺ T cells, compared to isotype control (gray filled). Right, CXCR5 MFIs. Data are representative of three independent experiments; n = 4 per group. ***P<0.001.

Figure 4. ICOS-dependent Tfh cell maintenance and germinal center formation

B6 and Icos^−/− mice were infected with vaccinia virus (VACV). Mice were analyzed 8 days p.i. (A–D) and 12 days p.i. (E–G). (A) Representative FACS plots of germinal center B cells (Fas⁺PNA⁺). Total B220⁺ B cells are shown. Right, GC B cells were quantified as % of total B cells. (B) Representative FACS plots of WT and Icos^−/− polyclonal Tfh cells. Activated CD4⁺ T cells (CD44^hiCD62L^lo) are shown and gates identify SLAM^loCXCR5⁺ Tfh cells. Tfh cell frequencies (% of activated CD4⁺ T cells (CD44^hiCD62L^lo)) and CXCR5 MFIs were quantified. (C) Representative FACS plots of WT and Icos^−/− polyclonal GC Tfh cells. Activated CD4⁺ T cells (CD44^hiCD62L^lo) are shown and gates identify GL7⁺CXCR5^hi GC Tfh cells. GC Tfh cell frequencies were quantified (% of activated CD4⁺ T cells (CD44^hiCD62L^lo)). (D) Representative FACS plots of Bcl6 expression by CD4⁺ T cells in B6 and Icos^−/− mice. Bcl6⁺CXCR5⁺ Tfh cells were quantified (% of activated CD4⁺ T cells). Tfh cells (E), GC Tfh cells (F), and germinal center B cells (G) were analyzed 12 days p.i., as for day 8 (A–C). Data are representative of two independent experiments for day 8 and day 12; n = 4 per group. (H–K) B6 mice were infected with VACV and treated with anti-ICOSL (αICOSL) or isotype control mAb at day 3, 5, and 7 as schematically shown in Figure S4E. Tfh cells (H), GC Tfh cells (I), Bcl6 expression (J), and GC B cells (K) were analyzed 8 days p.i. “C” = isotype control mAb treated mice. “αICOSL” = anti-ICOSL treated mice. “N” = naïve uninfected mice. n = 5–6 per group. Data are representative of two independent experiments. *P<0.05, **P<0.01, ***P<0.001.

Figure 5. ICOS → Bcl6 → CXCR5

(A–E) Blimp1 expressing (Prdm1-RV⁺, GFP⁺) and non-transduced (GFP) SM CD4⁺ T cells were transferred into the same (A) or separate (B–E) B6 recipient mice. (A) SM CD4⁺ T cells at day 4 after LCMV infection. (B) WT SM vs. Prdm1-RV⁺ SM CD4⁺ T cell expansion (% of total CD4⁺ T cells). (C, D) CXCR5 expression by Prdm1-RV⁺ SM and WT SM, day 4 p.i. (C) Representative FACS plots showing WT (GFP) and Prdm1-RV⁺ (GFP⁺). Gates identify CXCR5⁺Bcl6⁺ SM. (D) Quantitation of CXCR5⁺ and CXCR5⁺Bcl6⁺ SM. (E) CXCR5⁺ SM and Bcl6⁺CXCR5⁺ Tfh cell frequencies day 3 p.i. (F, G) Bcl6 shRNA-RV⁺ (GFP⁺Ametrine⁺) and non-transduced (GFP⁻Ametrine) SM CD4⁺ T cells were transferred into separate B6 recipient mice subsequently infected with LCMV. (F) SM CD4⁺ T cell expansion, day 4 p.i. (% of total CD4⁺ T cells). (G) Representative FACS plots of CXCR5 and Bcl6 expression by WT SM and Bcl6 shRNA⁺ SM CD4⁺ T cells. Total SM cells are shown and gates identify Bcl6⁺CXCR5⁺ SM. Data are representative of three (A–E) and two (F–G) independent experiments; n = 4 per group per time point. **P<0.01, ***P<0.001.

Figure 6. Rapid Bcl6 versus Blimp-1 bifurcation of CD4⁺ T cell differentiation in vivo

(A) Blimp1 and CXCR5 expression by Blimp1-YFP BAC tg SM CD4⁺ T cells was analyzed at 8 days p.i. in B6 recipient mice. Gates identify Blimp1⁺CXCR5⁻ effector Th cells and Blimp1⁻CXCR5⁺ Tfh cells. (B)Left, Bcl6 expression in Blimp1⁺CXCR5⁻ (gray filled) vs. Blimp1CXCR5⁺ (black line) SM CD4⁺ T cells. Right, Bcl6 MFIs. (C) Day 3 p.i., analysis of Blimp1, Bcl6, and CXCR5 expression by Blimp1-YFP BAC Tg SM CD4⁺ T cells in B6 recipients. Representative FACS plots of SM cells from LCMV infected (“Day3”) and uninfected (“Naïve”) recipients are shown. CXCR5 and Bcl6 expression by Blimp1⁺ vs. Blimp1⁻ SM CD4⁺ T cells were quantified. “Ctrl” = isotype control stain. (D) MD4-µMT recipient mice of Blimp1-YFP BAC tg SM CD4⁺ T cells, day 3 p.i. Representative FACS plot of SM cells and CXCR5 quantitation. (E–G) Blimp1-YFP SM CD4⁺ T cells were transferred into B6 recipients and analyzed for IL2Rα expression 3 days after LCMV infection. IL2Rα^hi vs. IL2Rα^lo SM CD4⁺ T cells are identified in co-stains with CXCR5 (E), Bcl6 (F), and Blimp1 (G). Data in all panels are representative of three or more independent experiments; n = 3–4 per group. **P<0.01, ***P<0.001.

Figure 7. Tfh versus effector Th cells are distinguishable by the second cell division, in association with differential early IL-2Rα expression levels

Early virus-specific Tfh vs. effector Th cell differentiation was analyzed at 48 hrs after acute LCMV infection. (A) Representative FACS plots of SM CD4⁺ T cells from LCMV infected (“Day 2”) and uninfected (“Naïve”) mice. Gates identify IL2Rα^hiCXCR5^lo vs. IL2Rα^intCXCR5^hi SM CD4⁺ T cells. (B) CXCR5 expression by IL2Rα^hi (black) vs. IL2Rα^int (red) SM CD4⁺ T cells. Isotype control = gray filled histogram. (C) Bcl6 expression by IL2Rα^hi vs. IL2Rα^int SM CD4⁺ T cells. (D) Expression of IL2Rα and CXCR5 by SM CD4⁺ T cells in MD4-µMT recipients, day 2 p.i. (E) Bcl6 expression by SM CD4⁺ T cells in MD4-µMT recipients, day 2 p.i. (F) IL2Rα and Blimp1 expression by Blimp1-YFP SM CD4⁺ T cells in B6 recipients, day 2 p.i. Representative SM CD4⁺ T cell FACS plot and quantitation are shown. (G) IL2R α expression (MFI) by SM CD4⁺ T cell populations gated in (F). (H) Bcl6 expression by SM CD4⁺ T cells in B6 recipients at day 2 p.i. Gates identify IL2Rα^hi vs. IL2Rα^int SM CD4⁺ T cells. Bcl6 protein expression (MFI) by each population was calculated. (I, J) CFSE-labeled SM CD4⁺ T cells were transferred into B6 recipient mice subsequently infected with LCMV. Cell divisions and differentiation were analyzed at day 2 p.i. (I) Cell divisions. Gray filled histogram = SM CD4⁺ T cells in uninfected mice. (J) Representative FACS plots are shown of SM CD4⁺ T cells that have undergone 2 (left) and 3 (right) cell divisions. Gates identify IL2Rα^hiBcl6⁻ and IL2Rα^intBcl6⁺ SM CD4⁺ T cells. Data in all panels are representative of three or more independent experiments; n = 3–4 per group. **P<0.01, ***P<0.001.