CD3/TCR stimulation and surface detection Determination of specificity of intracellular detection of IL-7Rα by flow cytometry

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1 CD3/TCR stimulation and surface detection Stimulation of HPB-ALL cells with the anti-cd3 monoclonal antibody OKT3 was performed as described 3. In brief, antibody-coated plates were prepared by incubating overnight with OKT3 (10μg/ml) at 37 C in PBS. Subsequently, plates were gently washed twice with PBS. Cells (10 6 /ml), pre-treated or not with Filipin (5μg/ml) for 1h, were added to the coated plates and stimulated for 1h. Subsequently, cells were washed twice in PBS and surface TCR/CD3 complex expression was determined by evaluating CD3 levels by flow cytometry using OKT3 primary monoclonal antibody (1:200 dilution) followed by secondary detection with anti-mouse PEconjugated antibody. Determination of specificity of intracellular detection of IL-7Rα by flow cytometry To ensure that the detection of total (intracellular and surface) detection of IL-7Rα was not affected by a high unspecific background which can sometimes affect the staining and subsequent flow cytometry analysis of intracellular proteins, an IL-7Rα negative cell line (HEK- 293) was used as a negative control, whereas HEK-293 ectopically expressing IL-7Rα were used as positive control. The human HEK-293 IL-7Rα cell line was generated by transfecting wild type HEK-293 cells with an IL-7Rα expression plasmid verified by sequencing. Transfection was performed using Effectene (Qiagen) and pools of stable transfectants were selected in 0.8mg/ml Geneticin (Gibco). HEK-293 IL-7Rα and HEK-293 wild type cells were cultured in DMEM supplemented with 10% serum, 4mM L-glutamine, and penicillin-streptomycin solution (Gibco). Intracellular flow cytometry staining for IL-7Rα in HEK-293 cell lines was performed as described in the manuscript ( Materials and Methods ).

2 Figure S1. IL-7 induces rapid IL-7Rα downregulation in primary thymocytes Primary thymocytes were isolated and cultured in the presence or absence of IL-7 (50ng/ml) in culture medium for the indicated time and subsequently analyzed by flow cytometry for surface IL-7Rα expression, as described in Materials and Methods. Thymocytes were incubated with anti-cd4 FITC-conjugated and CD8 APC-conjugated antibodies to determine the double positive (DP), CD4 single positive (SP4) and CD8 single positive (SP8) sub-populations. Relative IL-7Rα expression was calculated as the geometric mean intensity of fluorescence normalized to the zero (unstimulated) time point. Results are representative of two independent thymuses.

3 Figure S2. IL-7 does not interfere with anti IL-7Rα antibody staining Representative flow cytometry histogram overlay of IL-7Rα surface expression in HPB-ALL cells either unstimulated (black line), or stimulated for 30 minutes with IL-7 at 4 C in PBS / 1%BSA / 1mM Sodium Azide (red line) to prevent receptor internalization or with IL-7 at 37 C (green line). IL-7Rα was detected as described in Materials and Methods, using a commercially available PE-conjugated antibody (clone 40131; R&D) reported to be noncompetitive. 1,2 Values indicate geometric mean intensity of fluorescence for each condition. The levels of IL-7Rα detection when IL-7 was added at 4 C in the presence of Sodium Azide (red line histogram) were similar to those in cells without IL-7 (black line histogram). Since the former condition prevents receptor internalization, while still allowing for IL-7 binding to the receptor, these data indicate that decreased staining in IL-7 stimulated cells under normal conditions (37 C; green line histogram) was indeed due to internalization of the receptor and not to IL-7 blockade of anti IL-7Rα antibody binding.

4 Figure S3. IL-7 induces surface IL-7Rα downregulation in a dose-dependent manner (A) HPB-ALL cells were cultured in the presence of increasing doses of IL-7 (0 to 100ng/ml) in culture medium for 30 minutes and subsequently analyzed by flow cytometry for surface IL-7Rα expression, as described in Materials and Methods. Relative IL-7Rα expression was calculated as the geometric mean intensity of fluorescence normalized to the zero (unstimulated) time point. Data are mean±sem from two independent experiments. (B) Representative flow cytometry histograms of IL- 7Rα surface expression in HPB-ALL cells stimulated for 30 minutes with IL-7 at the indicated concentrations. The vertical line in each histogram was arbitrarily set at the same value in all histograms to facilitate their visual comparison.

5 Figure S4. IL-7 induced IL-7Rα internalization is lipid-raft independent (A) HPB-ALL cells were cultured for 30 min. in the presence or absence of IL-7 (50ng/ml), with or without 1h pre-treatment with the lipid-raft inhibitor Filipin (5μg/ml). Samples were analyzed by flow cytometry for surface IL-7Rα expression, as described in Materials and Methods. Relative IL-7Rα expression was calculated as the geometric mean intensity of fluorescence normalized to the zero (unstimulated) time point. Data are mean±sem from two independent experiments. (B) Representative flow cytometry histogram overlay of IL-7Rα surface expression in HPB-ALL cells stimulated for 30 minutes with IL-7 (50ng/ml), with or without 1h of pre-treatment with Filipin (5μg/ml). The indicated values correspond to the geometric mean intensity of fluorescence for each condition. (C) Effectiveness of Filipin used in (A,B) was confirmed by blocking lipid raftdependent internalization of CD3-stimulated TCR complex 3. Representative flow cytometry histogram overlay of TCR/CD3 surface expression in HPB-ALL cells stimulated for 1h with platebound immobilized anti-cd3 antibody OKT3 (10μg/ml), with or without 1h of pre-treatment with Filipin (5μg/ml). Expression of surface TCR/CD3 complex expression was assessed, after collecting and washing the cells with PBS, using OKT3 antibody and secondary detection with antimouse PE-conjugated antibody. The indicated values correspond to the geometric mean intensity of fluorescence for each condition.

6 Figure S5. Inhibition of clathrin-coated pit-mediated receptor endocytosis, using either hyperosmotic sucrose or monodansylcadaverine, affects significantly IL-7 mediated, but not TCR-dependent, signaling HPB-ALL cells were pretreated with 0.5M sucrose (hypertonic media) or 100 μm monodansylcadaverine (MDC) for 1h, to prevent the formation of clathrin-coated pits, and then stimulated with 50ng/ml IL-7 for 30 min. (A), with 10μg/ml of immobilized OKT3 for 1h (B) or left untreated (Unst.). TCR/CD3-mediated signaling is largely lipid raft-dependent 4 and was analyzed to ensure that the effects of hyperosmotic sucrose and MDC on IL-7 mediated signaling were specific. Assessment of phosphorylated JAK1/3, STAT5a/b and AKT was performed by immunoblotting. Actin was used as loading control. Data are representative of two independent experiments.

7 Figure S6. Intracellular detection of IL-7Rα is not affected by unspecific background To ensure that the protocol used to detect IL-7Rα total (surface plus intracellular) expression (see Materials and Methods ) was not significantly affected by unspecific binding of the antibody leading to high background, the endogenous expression of IL-7Rα in HPB-ALL cells was compared with that in a negative control (wild type HEK-293 cells, which lack IL-7Rα) or a positive control (HEK-293 cells ectopically expressing IL-7Rα). Representative flow cytometry histogram overlays of IL-7Rα total (surface plus intracellular) expression in (A) HPB-ALL and (B) HEK-293 cells transfected (293-IL7Rα; black line) or not (293-WT; blue line) with an IL- 7Rα expression plasmid. IgG indicates irrelevant isotype matched negative control. In panel B, the 293-IL7Rα IgG PE control histogram overlapped with the 293-WT IgG PE control and therefore it is not displayed.

8 REFERENCES 1. Blom-Potar MC, Bugault F, Lambotte O, Delfraissy JF, Theze J. Soluble IL-7Ralpha (scd127) and measurement of IL-7 in the plasma of HIV patients. J Acquir Immune Defic Syndr. 2009;51: Janot-Sardet C, Assouline B, Cheynier R, Morre M, Beq S. A validated assay to measure soluble IL-7 receptor shows minimal impact of IL-7 treatment. J Immunol Methods Monjas A, Alcover A, Alarcon B. Engaged and bystander T cell receptors are downmodulated by different endocytotic pathways. J Biol Chem. 2004;279: Barr VA, Balagopalan L, Barda-Saad M, et al. T-cell antigen receptor-induced signaling complexes: internalization via a cholesterol-dependent endocytic pathway. Traffic. 2006;7:

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