Supplementary Figure 1. Characterization of basophils after reconstitution of SCID mice

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1 Supplementary figure legends Supplementary Figure 1. Characterization of after reconstitution of SCID mice with CD4 + CD62L + T cells. (A-C) SCID mice (n = 6 / group) were reconstituted with 2 x 1 6 CD4 + CD62L + T cells from BALB/c mice (white bars) or received just PBS without T cells (grey bars). (A) On day 7, 14, 25 and 35 after T cell transfer the percentage of total and among CD11b + cells in the spleen was measured by flow cytometry. (B) On day 7 and 14 after reconstitution with T cells absolute numbers of total and as well as their percentage among CD11b + cells was measured in the bone marrow. (C) Representative FACS plots of Fc R1 + and in the bone marrow identified by expression of Fc R1 and. Data are represented as mean +/- SEM. Supplementary Figure 2. Percentage of among CD11b + cells in the blood after reconstitution of SCID mice with CD4 + CD62L + T cells. SCID mice (n=9 / group) were reconstituted with.5 x 1 6 CD4 + CD62L + T cells from BALB/c mice (white bars) or IL-3 deficient mice (black bars). The control group received just an injection of PBS without T cells (grey bars; 1 mice per group). Percentages of among CD11b + cells in the blood were measured by flow cytometry at various time points after reconstitution with T cells. Data are represented as mean +/- SEM. Supplementary Figure 3. Quantification of (absolute numbers and percentage of CD11b + cells) at day 37 after reconstitution with T cells. 1

2 SCID mice were reconstituted with 2 x 1 6 CD4 + CD62L + T cells from BALB/c mice (white bars) or received just an injection of PBS without T cells (grey bars; 1 mice per group) and were analyzed on day 37. (A) Flow cytometric quantification of among total cells in single cell suspensions of the spleen mesenteric lymph nodes and colon at day 37 after colitis induction. (B) Flow cytometric quantification of as percentage of CD11b + myeloid cells. (C) Representative dot plots showing flow cytometric identification of by expression of Fc RI and. Basophil subpopulations are characterized by expression of surface Fc R1 and high expression of. Data are represented as mean +/- SEM. Supplementary Figure 4. Ex vivo analysis of IL-6 release by after reconstitution of SCID mice with CD4 + CD62L + T cells. (A-B) SCID mice were reconstituted with 2 x 1 6 CD4 + CD62L + T cells from BALB/c mice (white bars) or IL-3 -/- mice (black bars) or received (grey bars; 4 mice per group). After 14 days splenocytes (A) or bone marrow cells (B) were obtained. Total cells or cells depleted of in vitro (Baso ) were cultured for 12 h in medium alone (Medium) or in medium with IL-3 (+ IL-3) (2 1 6 cells in 2 μl). The concentration of IL-6 was measured by ELISA. Data are represented as mean +/- SEM. Supplementary Figure 5. Characterization of FACS-sorted Fc R1 + and. (A) Basophils were expanded in RAG2 -/- mice for three days by twice daily i.p. injection of 2 ng IL-3 complexed with anti-il-3 (1 µg). Splenic Fc R1 + and were purified with magnetic beads and FACS-sorting as described in the methods. Representative FACS-plot of splenic Fc R1 + and after FACS-sort. (B) Various numbers of sorted Fc R1 + and splenic cells were cultured overnight with IL-3. Concentrations of IL- 2

3 4 and IL-6 in the supernatant were quantified by ELISA. (C) CFSE-stained B cells of C57BL/6 mice (5. cells/ well) were cultured with various numbers of sorted Fc R1 + and splenic together with CD4 + T cells of C57BL/6 mice (5. cells/ well), anti-cd3 antibody (.5 µg/ml) and IL-3 (1 ng/ml) for 3 days. Proliferation of B cells occurred in the presence of and was quantified by reduction of the CFSE signal. T cell derived cytokines IL-1 and IFN- were measured by ELISA in the supernatant. One out of three representative experiments is shown. Data are represented as mean +/- SEM. Supplementary Figure 6. Flowcytometric analysis of Fc R1 + cells in SCID mice. Spleen and bone marrow were prepared from naïve SCID mice and stained with antibodies against Fc R1 (MAR1), and CD11c. Fc R1 + cells express but not CD11c. Supplementary Figure 7. The antibody Ba13 depletes but not mast cells in SCID mice. SCID mice received a single i.v. injection (3µg/mouse) of the CD2R3 antibody Ba13 (black bars) or an isotype control antibody (white bars; 9 mice per group). 2 x 1 6 CD4 + CD62L + T cells from BALB/c mice were adoptively transferred 3 days later (day ). The control group received and was not treated with antibodies (grey bars; 4 SCID mice). Flow cytometric identification and quantification of and mast cells in the spleen by expression of Fc RI, and c-kit at day 8 after T cell transfer. Basophil subpopulations are characterized by expression of surface Fc R1 and high expression of but no expression of c-kit while mast cells express surface Fc R1 and c-kit but were negative for. Data are represented as mean +/- SEM. 3

4 Supplementary Figure 8. Depletion of with the antibody MAR1 upregulates the Th-1 phenotype of adoptively transferred T cells at day 25. Basophils were depleted in SCID mice by twice daily i.p. injections of the antibody MAR1 for 3 days (black bars) or remained undepleted (injection of the isotype control antibody; white bars; 1 mice per group). 2 x 1 6 CD4 + CD62L + T cells from BALB/c mice were adoptively transferred 2 days later (=day ) to induce colitis and mice were monitored until day 25. A control group received (grey bars; 4 SCID mice). (A) The weight of the mice was measured until day 25. (B) Histological analysis of colon tissue to quantify epithelial damage and inflammation at day 25. (C) On day 25 intracellular cytokine expression of CD4 + T cells from spleen was quantified by flow cytometry. Data are represented as mean +/- SEM. 4

5 A % splenic (total) / CD11b with T cells d 7 d 14 d 25 d 37 % splenic / CD11b d 7 d 14 d 25 d 37 B Total x 1, / femur d 7 d 14 % femur Basophils (total) / CD11b d 7 d 14 C.2%.2% BM.2%.8% BM with T cells d7 x 1, / femur d 7 d 14 % femur / CD11b d 7 d 14.2%.4% Fc R1 BM with T cells d14 Supplementary Figure 1

6 % blood (total) / CD11b n.s. n.s. d 8 d 16 d 29 d 84 WT T cells IL-3 -/- T cells Supplementary Figure 2

7 A Cells x 1, / spleen with T cells Total Day 37 SCID after T cell transfer Cells x 1, / 1 cm 3 colon 1,5 1,,5 Total Cells x 1, / mes. LNs 1,5 1,,5 Total B % splenic cells / CD11b Total % colonic cells / CD11b Total % mes.ln cells / CD11b Total C Day 37 SCID after T cell transfer Spleen 1 5 Fc R1 + Colon Mes. LN Fc RI with T cells 1 5 Fc RI no T cells Supplementary Figure

8 Spleen IL-6 release (pg/ml) A Total cells Baso- Total cells Baso- Reconstitution: WT T cells IL-3 -/- T cells BM IL-6 release (pg/ml) B Medium + IL-3 Total cells Baso- Total cells Baso- Medium + IL-3 Supplementary Figure 4

9 A Sorted Fc R1 + Sorted Fc R1 B IL-4 (pg/ml) Basophils IL-6 (pg/ml) Fc R1 + +IL-3 +IL Basophils C CFSE (median) IFN- (pg/ml) Basophils Basophils IL-1 (pg/ml) B cells B cells + + Fc R CFSE Fc R Basophils +IL-3 +IL-3 Supplementary Figure 5

10 Spleen (SCID mice) Fc R1 Bone marrow (SCID mice) CD11c Supplementary Figure 6

11 A Basophils x 1, / spleen Mast cells x 1, / spleen Day 8 Day 8 Day 8 Isotype Ba13 B Day 8 Fc R1 Fc R1 Fc R Spleen (Ba13) Spleen (Ba13) Spleen () Spleen () Spleen (Isotype) Spleen (Isotype) c-kit Supplementary Figure 7

12 A 11 B % of initial weight Isotype MAR Days after T cell transfer Histological score 2 1 Epithelium Inflammation C Cytokine + CD4 + T cells (%) Day 25 after T cell transfer p=,6 IFN- TNF IL-17a IL-2 Supplementary Figure 8