Supplementary Figure 6: ILT3-expressing T_reg cells represent a distinct T_reg cell subset that is elevated in T_H2 cell–mediated inflammation.

(a) Flow cytometric analysis of ILT3 expression on CD4⁺Foxp3⁺ T_reg cells from different tissues of Csnk2b^fl/fl and Csnk2b^fl/flFoxp3-Cre mice. Representative flow cytometry plots and percentages of ILT3⁺ among CD4⁺Foxp3⁺ T_reg cells in thymus, mesenteric lymph nodes (mLN), spleen and inguinal lymph nodes (iLN) are shown (n = 11 mice per group for thymus and n = 9 per group for mLN, spleen and iLN, combined from 2 independent experiments). Data show mean +/- SD. (b) Flow cytometric analysis of ILT3 expression in CD4⁺Foxp3⁺ T_reg cells, stimulated for 3 days with plate-bound anti-CD3 and anti-CD28 in the presence of 300ng/ml mrIL-2 and indicated concentration of CK2 inhibitors (DMAT and CX4945). T_reg cells were isolated at day 0 by CD25⁺ magnetic activated cell sorting from spleens of Csnk2b sufficient C57BL/6 mice. Data are representative for three independent experiments. (c) Flow cytometric analysis of ILT3 expression in CD4⁺Foxp3⁺ T_reg cells from lungs of non-immunized (PBS) or OVA/Alum immunized C57BL/6 mice upon OVA-challenge. Immunization was performed as described in online methods. Representative flow cytometry plots as well as the percentage of ILT3⁺ among CD4⁺Foxp3⁺ T_reg cells are shown. (n = 7 for PBS and n = 8 for OVA/alum treated mice pooled from 2 independent experiments). Data represent mean +/- SD. (d) Flow cytometric analysis of ILT3 expression in splenic CD4⁺Foxp3⁺ T_reg cells from Non-infected and L. sigmodontis infected BALB/c mice at day 76 after infection. Representative FACS plots as well as the percentage of ILT3⁺ among CD4⁺Foxp3⁺ T_reg cells are shown. (n = 10 for non-infected and n = 11 for L. sigmodontis infected group pooled from 2 independent experiments). Data represent mean +/- SD. (e) Total cell count in bronchoalveolar lavage fluid (BAL) of mixed bone marrow chimeras (Csnk2b^fl/fl + Csnk2b^fl/flFoxp3-Cre BMC) (n = 9) as well as from control Csnk2b^fl/fl and Csnk2b^fl/flFoxp3-Cre mice (n = 3). Csnk2b^fl/fl + Csnk2b^fl/flFoxp3-Cre BMC were generated as described in online methods. BMCs were analyzed 11 weeks post transfer. Data represent mean +/- SD from one single experiment. (f) Total cell count of eosinophils in bronchoalveolar lavage fluid (BAL) of Csnk2b^fl/fl + Csnk2b^fl/flFoxp3-Cre BMC as well as from control Csnk2b^fl/fl and Csnk2b^fl/flFoxp3-Cre mice. Data represent mean +/- SD from one single experiment. (g) Flow cytometric analysis of activated CD62L^-CD4⁺ T cells in the lungs of Csnk2b^fl/fl + Csnk2b^fl/flFoxp3-Cre BMC, control Csnk2b^fl/fl, and Csnk2b^fl/flFoxp3-Cre mice. Percentage of CD62L^- among CD4⁺Foxp3^- T cells is shown. Data represent mean +/- SD from one single experiment. (h) Flow cytometric analysis of the origin of ILT3⁺ T_reg cells in Csnk2b^fl/fl + Csnk2b^fl/flFoxp3-Cre BMC. Given is the percentage of ILT3⁺ among CD4⁺Foxp3⁺ T_reg cells derived either from CD90.1⁺ Csnk2b-competent C57BL/6 wildtype (Csnk2b^fl/fl BMC) or CD90.2⁺ Csnk2b-deficient Csnk2b^fl/flFoxp3-Cre bone marrow (Csnk2b^fl/flFoxp3-Cre BMC). Individual data points form Csnk2b^fl/fl BMC or Csnk2b^fl/flFoxp3-Cre BMC of respective BMC mice are shown. * = p<0.05; ** = p<0.01; *** = p<0.001. In case of non gausian distribution Mann-Whitney U test was used instead of students t-test.