The coinhibitory receptor CTLA-4 controls B cell responses by modulating T follicular helper, T follicular regulatory, and T regulatory cells

Abstract

The receptor CTLA-4 has been implicated in controlling B cell responses, but the mechanisms by which CTLA-4 regulates antibody production are not known. Here we showed deletion of CTLA-4 in adult mice increased Tfh and Tfr cell numbers and augmented B cell responses. In the effector phase, loss of CTLA-4 on Tfh cells resulted in heightened B cell responses, whereas loss of CTLA-4 on Tfr cells resulted in defective suppression of antigen-specific antibody responses. We also found that non-Tfr Treg cells could suppress B cell responses through CTLA-4 and that Treg and/or Tfr cells might downregulate B7-2 on B cells outside germinal centers as a means of suppression. Within the germinal center, however, Tfr cells potently suppress B cells through CTLA-4, but with a mechanism independent of altering B7-1 or B7-2. Thus, we identify multifaceted regulatory roles for CTLA-4 in Tfh, Tfr, and Treg cells, which together control humoral immunity.

Figure 1. T Follicular Regulatory Cells Express High Amounts of CTLA-4

(A) Gating strategy to identify CD4+ICOS+CXCR5+FoxP3-CD19-Tfh and CD4⁺ICOS⁺CXCR5⁺FoxP3⁺CD19Tfr cells from dLN of immunized mice. (B) Histograms demonstrating intracellular CTLA-4 expression in Tfh and Tfr cells gated as in (A). DN= CD4⁺ICOS⁻CXCR⁻FoxP3⁻ cells and correspond to naïve CD4⁺ T cells. Inset numbers indicate MFI. (C) Quantification of intracellular CTLA-4 expression in FoxP3- and FoxP3+ cellular subsets. DN=CD4+ICOS−CXCR5−, ICOS+= CD4+ICOS+CXCR5−. (D) CTLA-4 expression correlates with ICOS expression. Staining of ICOS and CTLA-4 in Tfr cells (left). Quantification of CTLA-4 in Tfr cells with intermediate (int) or high (hi) ICOS expression (right). (E) CTLA-4 expression correlates with IRF4 expression. Staining of IRF4 and CTLA-4 in Tfr cells (left). Quantification of CTLA-4 in Tfr cells with intermediate (int) or high (hi) IRF4 expression (right). (F) CTLA-4 and Ki67 expression. Staining of Ki67 and CTLA-4 in Tfr cells (left). Quantification of CTLA-4 in Tfr cells positive (+) or negative (−) for Ki67 expression (right). (G) CTLA-4 and PD-1 expression. Staining of PD-1 and CTLA-4 in Tfr cells (left). Quantification of CTLA-4 in Tfr cells with intermediate (int) or high (hi) PD-1 expression (right). (H–I) CTLA-4 expression in Tfr cells from dLN, blood (BL.) or Peyer’s patches (PP). DN=CD4⁺ICOS⁻CXCR5FoxP3⁻ cells. Quantification is shown (I). (J) ICOS expression on Tfr cells from dLN, blood and PP in as in (H).

Figure 2. Inducible Global Deletion of CTLA-4 Results in Increased Tfr Cell Differentiation and Increased Germinal Center B cells

(A) Schematic diagram of inducible deletion strategy to study the role of CTLA-4 in regulating B cell responses. UBC-iCre⁻Ctla4^F/F or UBC-iCre⁺Ctla4^F/F mice were given tamoxifen daily for 3 days and on the third day, immunized with NP-OVA s.c. 9 or 21 days later dLN or blood was harvested for analysis. (B) Histogram demonstrating deletion of CTLA-4 in Tfh and Tfr cells. Tfh and Tfr cells were gated as in Figure 1A. Inset numbers indicate MFI. (C) Tfh cell numbers are increased after total deletion of CTLA-4. Representative histograms of Tfh cell gating (left, at d9) and quantification of Tfh cells (right, at d9 and d21). (D) Increased ICOS expression on Tfh cells after CTLA-4 deletion. ICOS expression on Tfh cells gated as in (C) at d9 post immunization. (E) Increased Tfr cell percentages after deletion of CTLA-4. Representative histograms of Tfr cell gating (left, at d9) and quantification of Tfr cells (right, at d9 and d21 post immunization). Tfr cell percentages reported as a percentage of all CD4⁺ T cells. (F) Tfr cell percentages reported as a percentage of all FoxP3⁺ T cells 9 days after immunization. (G) ICOS expression on Tfr cells 9 days after immunization. (H) Tfr cell percentage of total CD4⁺CXCR5⁺ T cells 9 or 21 days after immunization. (I) Enhanced germinal center B cells after CTLA-4 deletion. Gating of CD19⁺GL7⁺FAS⁺ germinal center (GC) B cells (left, at d9). Quantification of GC B cells (middle at d9, and right at d21 after immunization). (J) Quantification of B7-1 and B7-2 on GC and total B cells from (I) 9 days after immunization. (K) Quantification of serum antibody levels 14 and 21 days after immunization for total IgG1 (left), NP specific IgG1 (middle), or IgE (right). (L) Quantification of serum antibody levels 240 days after CTLA-4 deletion in unimmunized mice. See also Figure S1.

Figure 3. CTLA-4 Inhibits Tfh stimulation of B cells

(A) Schematic diagram of experimental approach. 10–20 UBC-iCre− or + Ctla4^F/FFoxp3^IRES-GFP mice were immunized with NP-OVA s.c. and 4 days later tamoxifen was administered i.p.; 3 days later CD4⁺ICOS⁺CXCR5⁺FoxP3CD19 (Tfh) cells were sorted and plated with CD19⁺ cells (from dLN of immunized iCre- control mice) along with anti-CD3, anti-IgM and 4OHT for 6 days. (B) Histogram demonstrating expression of CTLA-4 on Tfh cells in stimulation assay after deletion. Numbers indicate percent positive based on gate. (C) Enhanced stimulatory capacity of Tfh cells after deletion of CTLA-4. B cells from cocultures as in (A) were stained for GL7 and intracellularly for IgG1 to identify class switched B cells. Gating strategy (left) and quantification (right). (D) Enhanced stimulatory capacity of Tfh cells after deletion of CTLA-4 using NP-OVA. Cocultures were performed as in (A) but NP-OVA was added to wells instead of anti-CD3/IgM. 6 days later supernatants were analyzed for IgG. (E) Expression of B7-1 (left) and B7-2 (right) on B cells from stimulation assays as in (A). (F) Intracellular expression of Ki67 in Tfh cells from stimulation assays as in (A) (G) CTLA-4 expression in Tfh cells from mice defined in (A) and also with Cre+ mice injected with tamoxifen in vivo but not cultured with 4OHT to achieve the 50% Tfh deletion of CTLA-4. (H) CTLA-4 inhibits Tfh stimulatory function in a cell intrinsic manner. Expression of Ki67 in CTLA-4 non-expressing (−) or CTLA-4 expressing (+) Tfh cells from stimulation assays. See also Figure S2.

Figure 4. Inducible deletion of CTLA-4 in Treg cells Results in Increased Tfr and Tfh cells

(A) Schematic diagram of experiment. Foxp3^iCre/iCreCtla4^F/+ or Foxp3^iCre/iCreCtla4^F/F mice were injected with tamoxifen daily for three days before immunization with NP-OVA s.c. 9 or 21 days later dLN and blood were collected for analysis. (B) Histogram showing deletion of CTLA-4 in Tfr cells. Cre- ICOS-CXCR5-FoxP3-cells (Cre- DN FoxP3⁻) are included as controls. Inset numbers indicate MFI. (C) Quantification of CTLA-4 staining in Tfr cells. (D) Increased Tfr cell percentages after Treg cell-specific deletion of CTLA-4. Representative Tfr staining, gated on FoxP3+ cells (left, at d9), and Tfr quantification, as a percentage of total CD4⁺ T cells (right, at d9 and d21). (E) Quantification of FoxP3⁺ cells after Treg cell-specific CTLA-4 deletion 9 and 21 days after immunization. (F) Increased ICOS expression on Tfr cells after CTLA-4 deletion. ICOS expression was quantified on Tfr cells 9 and 21 days after immunization. (G) Cell death of Tfr cells after deletion of CTLA-4 in vivo. Cell death in Tfr cells and total Treg cells was measured by staining with the activated caspase staining reagent VAD-FMK 9 days after immunization. (H) Increased Tfh cell percentages after Treg cell-specific CTLA-4 deletion. Representative gating (left, at d9) and quantification (right, at d9 and d21) is shown. (I) ICOS expression on Tfh cells after Treg-specific CTLA-4 deletion 9 or 21 days after immunization. (J) Tfr cells are more abundant compared to Tfh cells in the germinal center. Percentage of Tfr cells of all CD4⁺CXCR5⁺ cells is shown 9 or 21 days after immunization. See also Figure S3.

Figure 5. Inducible deletion of CTLA-4 in Treg cells Results in Increased Germinal Center B cells in vivo

(A) Enhanced GC B cell responses after Treg cell-specific deletion of CTLA-4. Foxp3^iCre/iCreCtla4^F/+ or Foxp3^iCre/iCreCtla4^F/F mice were injected with tamoxifen daily for three days before immunization with NP-OVA s.c. 9 or 21days later dLN was collected for analysis. (B–C) Expression of B7-1 and B7-2 on total CD19+ B cells or on CD19⁺GL7⁺FAS⁺ GC B cells 9 days after immunization. Representative histograms (B) and quantification (C) are shown. (D) Serum IgG1 levels. Serum IgG1 was analyzed in mice 14 or 21 days after immunization as in (A). See also Figure S4.

Figure 6. Inducible deletion of CTLA-4 on Tfr Cells During Suppression Results in Decreased Suppressive Function in vitro and in vivo

(A) Schematic representation of experiment. 20 UBC-iCre⁻ Ctla4^F/FFoxp3^IRES-GFP (WT) or UBC-iCre⁺Ctla4^F/FFoxp3^IRES-GFP (iCre) mice were immunized with NP-OVA s.c. and 4 days later mice were given tamoxifen i.p.; 3 days later CD4⁺ICOS⁺CXCR5⁺FoxP3CD19- (Tfh) and CD19+ B cells from iCre- WT mice were cultured with anti-CD3, anti-IgM and 4OHT. CD4+ICOS+CXCR5+FoxP3+CD19- Tfr cells from Cre- or Cre+ mice were added. Cells were analyzed 6 days later. (B) Deletion of CTLA-4 in Tfr cells after suppression assay as in (A). Tfr cells from cultures were identified as CD4⁺FoxP3⁺CD19 cells. (C) Deletion of CTLA-4 in Tfr cells results in decreased suppression of class switch recombination. Representative plots of B cells showing GL7⁺IgG1⁺ class switched B cells from suppression assays detailed in (A)(left). Quantification of class switched B cells (right). (D) Effect of deletion of CTLA-4 in conventional Treg cells in suppression assays. Experiments were performed as in (A) except CD4+ICOS-CXCR5-FoxP3+ conventional Treg cells, or Tfr cells, were cultured with B cells. “Control” indicates B cells alone. (E) GL7 expression on B cells from suppression assays as in (A). (F) B7-1 expression on B cells from suppression assays as in (A). (G) Deletion of CTLA-4 in Tfr cells results in decreased suppression of Tfh cells. Tfh cells from suppression assays in (A) were intracellularly stained for Ki67 and Bcl6. Tfh cells were identified as CD4⁺FoxP3⁻CD19⁻ cells from cultures. Representative plots are shown. (H) Deletion of CTLA-4 in Tfr cells after differentiation results in diminished suppressive capacity in vivo. Schematic representation of assay. Foxp3^iCre/iCreCtla4^F/F or Foxp3^iCre/iCreCtla4^F/+ mice were immunized and tamoxifen was administered one day before sorting total CD4⁺ICOS⁺CXCR5⁺CD19 (Tfh and Tfr) cells on day 6. Sorted cells were adoptively transferred to Cd28^−/− mice that were immunized with NP-OVA and tamoxifen was administered on days 0 and 1 after immunization. Organs were harvested 9 days later for analysis. (I) Quantification of Tfh cells in recipient mice from transfers in (H). (J) NP-specific IgG serum levels from recipient mice in transfers as in (H). (K) B7-1 and (L) B7-2 expression on total CD19⁺ (B) or CD19⁺GL7⁺FAS⁺ (GC B) B cells from transfers in (H). See also Figure S5.

Figure 7. Deletion of CTLA-4 in Tfr cells Results in Enhanced B cell Responses in Peyer’s Patches

(A) Representative gating of Tfh and Tfr cells from Peyer’s patches from UBC-iCre⁺Ctla4^F/F or UBC-iCre⁻ Ctla4^F/F mice 9 days after the last dose of tamoxifen administration; tamoxifen was administered on d −2, d −1, d0. (B) Quantification of Tfh cells (gated as in A) from UBC-iCre⁺Ctla4^F/F and UBC-iCre⁻ Ctla4^F/F mice (left 3 panels) or Foxp3^iCre/iCreCtla4^F/F and Foxp3^iCre/iCreCtla4^F/+ mice (far right panel) 9, 21 or 240 days after final tamoxifen treatment. (C) Quantification of Tfr cells (gated as in A) from UBC-iCre⁺Ctla4^F/F and UBC-iCreCtla4^F/F mice (left 3 panels) or Foxp3^iCre/iCreCtla4^F/F and Foxp3^iCre/iCreCtla4^F/+ mice (far right panel) reported as a percentage of CD4+ cells 9, 21 or 240 days after final tamoxifen treatment. (D) Quantification of Tfr cells (gated as in A) from UBC-iCre⁺Ctla4^F/F and UBC-iCreCtla4^F/F mice (left) or Foxp3^iCre/iCreCtla4^F/F and Foxp3^iCre/iCreCtla4^F/+ mice (right) reported as a percentage of total CD4⁺CXCR5⁺ cells 9 days after final tamoxifen treatment. (E) Increased germinal center B cells after Tfr deletion of CTLA-4 in Peyer’s patches. Identification of CD19⁺FAS⁺GL7⁺ B cells in UBC-iCre⁺Ctla4^F/F and UBC-iCre⁻ Ctla4^F/F mice (representative gating, far left and quantification middle 3 panels) 9, 21 or 240 days or Foxp3^iCre/iCreCtla4^F/F and Foxp3^iCre/iCreCtla4^F/+ mice (far right panel) 9 days after final tamoxifen treatment. (F–G) B7-1 and B7-2 expression on CD19⁺ (Total B) or CD19⁺GL7⁺FAS⁺ (GC B) cells from Peyer’s patches as in (E) from UBC-iCre⁺Ctla4^F/F and UBC-iCre⁻ Ctla4^F/F mice (F) or Foxp3^iCre/iCreCtla4^F/F and Foxp3^iCre/iCreCtla4^F/+ mice (G) 9 days after final tamoxifen treatment. (H) Serum IgA levels in UBC-iCre⁺Ctla4^F/F and UBC-iCre⁻ Ctla4^F/F mice 21 or 240 days after last tamoxifen treatment. See also Figure S6.