DEV02510 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Control dot plots for endogenous H-2^k populations. (A) Control dot plots for H-2 cells. Ragγc^-/- mice were injected with H-2^b donor cells; C57BL/6 mice were used as a positive control (H-2^b cells) and Ragγc^-/- mice (H-2^k cells) as a negative control. (B) Dot plots describe the myeloid compartment of recipient mice (H-2^k) injected with either FL or FS. Plots are gated on live PI⁻ cells.

• Supplemental Figure 2 -

  Fig. S2. Loss of LTR potential is not due to molecules secreted by the FL. HSCs (cKit^hiSca1^hi) from E15 FL actin^GFP mice were sorted and used to reconstitute E15 irradiated (400 Rad) FL and FS explants in hanging drop for 2 weeks (1000 HSCs/explant). Then, FS and FL organ cultures (FSOC and FLOC) were harvested on filters for 4 days in either complete media or FL supernatant. The FL supernatant was obtained from E15 FL adherent cells cultured in complete media for 2 weeks. FLOC and FSOC cell suspensions were analyzed by cytometry to observe the maintenance of the HSC population. Only Lin⁻ GFP⁺ cells were analyzed for the co-expression of Kit and Sca1 markers. A clear LSK population is visible from the FLOC cell suspension (7 to 10%), whereas no real LSK population could be recovered from the FSOC cell suspension (1 to 4%).

• Supplemental Figure 3 -

  Fig. S3. Cytokine levels from various cell cultures. Cell culture supernatants were analyzed for Il10 and Il12p40 content by ELISA. For the assays, cytokine levels were determined from cultures of BM-DCs (cultured with GM-CSF), BM-macrophages (cultured with FSS), FSS1-macrophages (cultured with HSC-derived on FSS1), FSS1, FSS2-macrophages (cultured with HSC-derived on FSS2), and FSS2. FSS1 and FSS2 are different cell lines representative of the eight others. Il10 and Il12p40 levels were measured (pg/ml) by a standard sandwich ELISA with appropriate antibody pairs (BD PharMingen).

• Supplemental Figure 4 -

  Fig. S4. Tgfβ1 is mainly responsible for the anti-inflammatory capacity of the FS. (A) Tgfβ1 levels were measured after activation of cell line supernatants (OP9 S17 and FSS) to release the immunoreactive Tgfβ1 form. ELISA was performed using the Duoset mouse Tgfβ1 from R&D systems. (B) OT-II CD4⁺ T cells (3×10⁴) were stimulated by 10⁴ DCs pulsed with 1 μg/ml of OVA_323-336 in the presence of 3×10⁴ FSS1 or OP9 stromal cells, together with 5 μg/ml of anti-Tgfβ (α-Tgfβ) antibody or a control isotype (Ctrl Ig). After 3 days, supernatants were tested by ELISA to detect Ifnγ produced by activated T cells.