SC11-LEU-0034RR- Naji et al. - 2
SUPPLEMENTAL DATA
Neoplastic B cell growth is impaired by HLA-G/ILT2 interaction
Abderrahim Naji1,2, 3, Catherine Menier1,2, 3, Guitta Maki1,2, Edgardo D. Carosella1,2,, and Nathalie Rouas-Freiss1,2
1CEA, DSV, I2BM, Service de Recherches en Hemato-Immunologie, Paris, France
2 Univ Paris Diderot, Sorbonne Paris Cité, IUH, Hopital Saint-Louis, UMR_E, Paris, France
3 A.N. and C.M. contributed equally to this work
Address Correspondence to: Nathalie Rouas-Freiss, CEA, DSV, I2BM, Service de Recherches en Hemato-Immunologie, Hopital Saint-Louis, Institut Universitaire d’Hematologie, 1, avenue Claude Vellefaux, 75010 Paris. Phone: +33 (0)1 57 27 68 01 Fax: +33 (0)1 57 27 67 80. Email:
Legend to Figures
Supplemental Figure 1 HLA-G inhibits Raji B lymphoma cells through ILT2 interaction leading to G0/G1 cell cycle arrest via PKC and AKT/mTOR pathways. (a) Expression of HLA-G receptors on Raji cells was evaluated by flow cytometry. (b) Raji cells were pre-treated with HLA-G or Ctrl for 18 h or medium (Ø). Cell proliferation was evaluated after 24 h. Results are expressed as means of thymidine incorporation corrected for background values (Dcpm) ± SEM (n=6). (c) siRNA specific for ILT2 was used to down-modulate ILT2 expression on Raji cells, as shown by flow cytometry. (d) Cell cycle distribution was determined at 24 h and 48 h by propidium iodide staining. One representative experiment is shown. (e) Raji cells were treated with HLA-G or Ctrl for 6, 16 and 24 h and analyzed for expression of cyclin D1 by intracellular flow cytometry using the same settings (dotted lines correspond to isotype controls, grey filled peaks to Ctrl and black lines to HLA-G). (f)
Raji cells were treated with Ctrl or HLA-G for 90 min. Phosphorylation of STAT-1, STAT-3, and STAT-5, PDK1, PTEN, AKTThr308 and Ser473, mTORSer2448 and Ser2481, GSK-3b, c-Raf, Foxo1, Foxo3a, and Foxo4, and PKCa/bII, PKCdSer643 and Thr505, PKCµSer744/748 and Ser916 was detected by immunoblot analysis with phosphospecific antibodies as indicated. Total protein level of AKT, mTOR, Raptor and Rictor was detected by immunoblot using specific antibodies. Means of relative quantification for each protein from 3 independent experiments are shown. Numbers correspond to arbitrary units of protein for Raji treated with HLA-G normalized against untreated Raji cells (set as 1).
Supplemental Figure 2 Model of regulatory cascades induced by HLA-G/ILT2 interaction in malignant B cells. A delicate balance between activating (cytokines and growth factors) and inhibitory signals regulates B cell fate decisions. Black arrows represent B cell signaling following activation with growth factors and cytokines. When HLA-G is present, it interacts with ILT2 inhibitory receptor on B cells whose downstream targets are both phosphatase(s) (green arrows) and kinase(s) (red arrows). The signaling events leading to inhibitory effects operate through increased phosphorylation of PKC along with decreased phosphorylation of STAT-3, STAT-5, AKT, mTOR, GSK-3b, c-Raf and Foxo. These effects converge to activate inhibitors or to inhibit activators of cell cycle. Focus on AKT/mTOR pathways shows that HLA-G directly blocks mTORC1 by decreasing both mTOR and Raptor, and mTORC2 by reducing mTOR protein level. All together, these molecular events lead to inhibition of B cell proliferation.