Supplementary Information: Follicular regulatory T cells with Bcl6 expression suppress germinal center reactions by Yeonseok Chung, Shinya Tanaka, Fuliang Chu, Roza Nurieva, Gustavo J. Martinez, Seema Rawal, Yi-Hong Wang, Ho Yong Lim, Joseph M. Reynolds, Xiao-hui Zhou, Hui-min Fan, Zhong-ming Liu, Sattva S. Neelapu, and Chen Dong :contains 8 supplementary Figures
20 x 60 x Supplementary Figure 1. Foxp3+ cells in the germinal centers of human tonsils. Immunohistochemical staining for Foxp3 (dark brown) was performed on formalin-fixed paraffin-embedded human tonsil tissue. Original magnification 20 (left) and 60 (right) are shown for germinal centers. Data represent three different tonsil sections examined.
* * Supplementary Figure 2. Analysis of T reg population in Bcl6-deficient mice. Lymphoid cells from the thymus, spleen and mesenteric lymph node (MLN) of either Bcl6-deficient or littermate mice (6-8 weeks old, n=4 per group) were stained with antibodies against CD4, CD8, CD25, followed by intracellular staining with anti-foxp3. Data shown are mean ± SEM. *, p<0.05 in comparison with wild-type.
CXCR5 APC-A a 10 4 10 3 10 2 10 1 CD4 gate APC-A 10 4 10 3 10 2 10 1 10 0 10 4 0.84 76.1 0.48 22.6 10 0 10 1 10 2 10 3 10 4 FITC-A 0 1.88 b * * c 10 0 10 0 10 1 10 2 10 3 10 4 FITC-A GFP APC-A 10 3 10 2 10 1 10 0 0.16 98 10 0 10 1 10 2 10 3 10 4 FITC-A Supplementary Figure 3. Comparison of suppressive activity and gene expression profiles between CXCR5 + and CXCR5 - T reg cells. a, CXCR5 - GFP + or CXCR5 + GFP + CD4 T cells were FACS sorted from the lymphoid cells of Foxp3 gfp mice after staining with anti- CXCR5 and anti-cd4. b, Naïve CD4 + T cells from C57BL/6 mice were cultured with irradiated T cell-depleted splenocytes in the presence of soluble anti-cd3 (2 mg/ml). In some wells, T reg cells isolated as described in a were added at a ratio of 1:0.25 (Naïve T: Treg). Three days later, the proliferation of T cells was measured by [H-3]-thymidine incorporation for the last 8 hours. Data shown are mean ± SEM. *, p<0.05 in comparison with No T reg condition. c, The relative mrna expression of the FACS sorted CXCR5 + and CXCR5 - T reg cells was analyzed by using quantitative real-time PCR analysis. The values were obtained after normalized with Actb reference gene. Data are mean ± SD and represent two independent experiments.
Supplementary Figure 4. Effect of IL-6 and IL-21 stimulation on the gene expression of CXCR5 - T reg cells. CXCR5 - GFP + CD4 T cells were FACS sorted from the lymphoid cells of Foxp3 gfp mice after staining with anti-cxcr5 and anti-cd4 as described in supplementary Figure 3a. The sorted CXCR5-negative T reg cells were stimulated with plate-bound anti-cd3 in the presence or absence of IL-6 (10 ng/ml) and IL-21 (50 ng/ml) for 48 hours. The mrna levels of Foxp3, Cxcr5, Bcl6 were measured by quantitative RT-PCR. Data shown are mean + SD.
a CD95 b Treg donor Wild-type Bcl6 -/- GL7 * d Gated CXCR5 c on CD45.2 + CD4 + Treg donor Wild-type Bcl6 -/- BTLA e PNA Foxp3 Wild-type Bcl6 -/- Treg donor Supplementary Figure 5. Bcl6 -/- T reg cells were inefficient in controlling the germinal center reactions. CD25 - GITR - CD44 lo CD62L hi naïve CD4+ T cells (CD45.1 +, 3-4x10 6 cells) were mixed with CD25 hi CD4 + T cells (CD45.2 +, 0.3-0.4x10 6 cells) from either Bcl6 -/- or littermate mice before intravenously transferred into Tcrb -/- mice. The recipient mice were subcutaneously immunized with KLH in CFA. Nine days later, the lymphoid cells from the draining lymph nodes were analyzed for the GL7 + CD95 + cells among B220 + B cells (a, b), and CXCR5 + BTLA + cells among CD45.1 + population (c). d, The surface expression of BTLA and CXCR5 on donor Treg cells (CD45.2 + CD4 + ) were analyzed by flow cytometry. e, The draining LNs were isolated and stained with PE-labeled PNA to visualize germinal center together with FITC-labeled anti-foxp3. Scale bars shown are 50 mm *, p<0.05 in comparison with wild-type.
a Day5 post immunization b p=0.081 αnp26 IgM αnp26 IgG1 αnp26 IgG2a αnp26 IgG2b O.D. (Abs at 490 nm) p=0.997 p=0.0.035 p=0.936 p=0.046 αnp4 IgM αnp4 IgG1 αnp4 IgG2a αnp4 IgG2b p=0.982 p=0.0.419 p=0.778 p=0.849 c p=0.099 Serum dilution Supplementary Figure 6 Bcl6 -/- T reg cells were defective in limiting affinity maturation, plasma cell differentiation during active immunization. CD25 - GITR - CD44 lo CD62L hi naïve CD4+ T cells (CD45.1 + ) were mixed with CD25 hi CD4 + T cells (CD45.2 + ) from either Bcl6 -/- or littermate mice before intravenously transferred into Tcrb -/- mice. The recipient mice were subcutaneously immunized with KLH-NP15 in CFA. Sera from the recipients were collected on day 5 after immunization. Levels of high and global affinity immunoglobulin were determined by using NP4-BSA (for high affinity) and NP26-BSA (for global affinity)-specific ELISA (a) Data shown are mean ± SE (n=5). b, The KLH-NP26-specific IgG-secreting cells were measured by ELISPOT assay. c, Lymphoid cells from the draining LNs were stained with antibodies against B220, IgD, IgM. P-values shown in a were analyzed by two-way ANOVA test.
Supplementary Figure 7. Analysis of Th1, Th2 and Th17 responses in Bcl6 -/- T reg recipient mice. Tcrb -/- mice were given T reg cells from wild-type or Bcl6 -/- mice together with CD25 - GITR - CD44 lo CD62L hi naïve CD4 + T cells (CD45.1 + ). The recipient mice (n=3~4) were subcutaneously immunized with KLH in CFA. Nine days later, the lymphoid cells from spleen were restimulated with KLH for three days. Levels of IFNγ, IL-5, and IL-17 in the supernatant were measured by ELISA. Data shown are mean + SE.
T cell zone B cell zone ntreg Foxp3 Tfr Foxp3, Bcl6 Memory B cell Antigen + Inflammation (Adjuvants etc.) CXCR5 BTLA PD-1 ICOS B cell Germinal Center B cell Naïve CD4 T Tfh Bcl6 Plasma cell CXCR5 BTLA PD-1 ICOS Supplementary Figure 8. Analogous functional specialization of Tfh and Bcl6 + CXCR5 + follicular regulatory T (Tfr) cells. Upon antigenic stimulation in combination with inflammatory signals such as adjuvants, a subpopulation of naïve CD4 + T cells and natural Treg cells are differentiated to express Bcl6 and CXCR5, whose expression enables these cells to migrate into B cell zone. While Tfh cells help the differentiation of B cells into effector B cells, Bcl6 + CXCR5 + Tfr cells suppress early differentiation of B cells, therefore regulate germinal center reactions.