Follicular regulatory helper T cells control the response of regulatory B cells to a high-cholesterol diet

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Reference

Follicular regulatory helper T cells control the response of regulatory B cells to a high-cholesterol diet

BURGER, Fabienne, et al.

Abstract

B cell functions in the process of atherogenesis have been investigated but several aspects remain to be clarified.

BURGER, Fabienne, et al. Follicular regulatory helper T cells control the response of regulatory B cells to a high-cholesterol diet. Cardiovascular Research, 2021, vol. 117, no. 3, p. 743-755

DOI : 10.1093/cvr/cvaa069 PMID : 32219371

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http://archive-ouverte.unige.ch/unige:152604

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Follicular regulatory helper T cells control the response of regulatory B cells to a high-cholesterol diet.

Fabienne Burger¹, Daniela Baptista¹, Aline Roth¹, Kapka Miteva¹, Rodrigo A. Fraga-Silva², Catherine Martel³, Nikolaos Stergiopulos², François Mach¹, Karim J. Brandt^1*

Supplemental Methods Immunofluorescence staining

Mouse aortic sinus was serially cut in 5 μm transversal sections, as previously described^{15, 16}. Sections from mouse specimens were fixed in acetone and immunostained with specific antibodies anti-CD68 (BioRad), anti-iNOS (Abcam), and anti-Arginase 1 (GeneTex) antibodies and SYTO 13 (Thermofisher) for nuclear detection. Intracellular immunostainings were performed in PBS with 0.2%BSA, 0.1% Tween20 and 5% mouse or rabbit serum respectively.

For intracellular staining, slides were permeabilized with Triton 0.5% PBS, then treated with 10mM citric acid buffer pH6 and blocked with PBS 5% BSA. Images were captured with Zeiss Axioscan.Z1 and quantifications were performed using Definiens software.

Figure S2: T_FR cells influence Th1, Th2 and Th17 populations. a) Flow cytometry gating strategy for characterization of Th1, Th2 and Th17 cell populations and Effect of Bcl-6 inhibitor on secondary lymphoid organs Th1, Th2 and Th17 cell populations on a normal cholesterol diet (NCD) and high cholesterol diet (HCD) fed mice. (n = 6 to 8 mice per group) b) Effect of TFR cell transfer on secondary lymphoid organs Th1, Th2 and Th17 cell populations on a normal choles- terol diet (NCD) and high cholesterol diet (HCD) fed mice. (n = 5 to 8 mice per group). The nonparametric Mann-Whit- ney U test was used for statistical analysis: *: p ≤ 0.05; **: p ≤ 0.005; ***: p ≤ 0.0005. All data were represented as mean ± s.e.m.

Figure S3: T_FR cells control FOB cells but not MZB cells differentiation in vitro. a) Flow cy- tometry gating strategy for characterization of B cell population. b) Quantification of immu- noglobulin isotypes in serum of mice treated with vehicle (PBS) or Bcl-6 inhibitor under NCD and HCD (n = 6 to 7 mice per group) c) Effect of TFR cell transfer on immunoglobu- lin isotypes in serum of mice on normal cho- lesterol diet (NCD) and high cholesterol diet (HCD) fed mice (n = 6 to 8 mice per group).

Figure S1: Localization of T_FR cells. a) Flow cytometry-gating strategy for T_FH and T_FR characterization. b) T_FR cells sorting strategies and control of CXCR5, foxp3 and Bcl-6 expression by sorted TFR cell population c) Localization of Yellow trace-stained T_FR cells after 3 days post adoptive transfer. d) Localization of Yellow trace-stained T_FR cells after 3 weeks post adoptive transfer (n = 3). For c) and d): Numbers correspond to geometrical mean for Bcl-6. *: p ≤ 0.05;

Figure S4: a) Quantification of FOB and MZB cell differentia- tion relative to initial FOB and MZB cell population, respec- tively, and expressed in fold increased (n = 5).

Figure S5: T_FR cell transfer has not significant effects on M1 and M2 subset of macrophages present in aortic roots. a) Representative microphotographs of aortic root plaques with Arginase, iNos and CD68 expression in Apoe^-/- mice treated with vehicle (PBS) or T_FR cell transfer under NCD and HCD. (n=8 mice per group) b) Percentage of CD68⁺ cells expressing iNos only (M1 Macrophages), or Arginase (Arg) only (M2 Macrophages), or expressing both markers on total CD68⁺ cells (n = 6 to 8 mice per group). c) Intensity of iNos and Arginase expression in each CD68⁺ cell of the roots in Apoe^-/- mice treated with vehicle (PBS) or T_FR cell transfer under NCD and HCD with black lines defining the thresholds of arginase and iNos positive cells. (n=8 mice per group) The nonparametric Mann-Whitney U test was used for statistical analysis: *: p ≤ 0.05; **: p ≤ 0.005; ***: p ≤ 0.0005. All data were represented as mean ± s.e.m.

Figure S6: Th2 cells-dependent IL-4 production is not involved in lymphangiogenesis a) Effect of Bcl-6 in- hibitor on secondary lymphoid organs CD4⁺GATA3⁺IL-4⁺-producing cells in normal cholesterol diet (NCD) and high cholesterol diet (HCD) fed mice (n = 10 mice per group). b) Effect of T_FR cell transfer to secondary lymphoid organs CD4⁺GATA3⁺IL-4⁺-producing cells in normal cholesterol diet (NCD) and high cholesterol diet (HCD) feed mice (n = 5 to 8 mice per group). c) Effect of Bcl-6 inhibitor on mRNA expression of Li- ver X receptor alpha (lxra) on the spleen of normal cholesterol diet (NCD) and high cholesterol diet (HCD) fed mice (n = 5 to 10 mice per group). d) Effect of T_FR cell transfer on mRNA expression of Liver X receptor alpha (lxra) in the spleen of normal cholesterol diet (NCD) and high cholesterol diet (HCD) fed mice (n = 6 mice per group). e) Effect of T_FR cell transfer on immunoglobulin isotypes in serum on established atheros- clerosis mice (n = 6 to 8 mice per group). *: p ≤ 0.05; **: p ≤ 0.005.

Supplementary Table 1

Antibodies Fluorophore Compagnies Clone

B220 AF488 Bioscience RA-3-6B2

Bcl6 PerCP-Cy5.5 Biolegend 7D1

CD1d PE Bioscience 1B1

CD4 AF488 Bioscience RM 4-5

CD5 PerCP Bioscience 53-7.3

CD21 PECy7 eBioscience eBio8D9

CD23 BV421 Bioscience B3B4

CD25 BV605 Bioscience PC61

CD43 APC Bioscience S7

CXCR5 BV421 Bioscience 2G8

Foxp3 PE CF594 Bioscience MF23

IgM PE-Cy7 Bioscience R6-60.2

IL-4 APC Bioscience 554436

PD-1 PE-Cy7 Biolegend 29F.1A12