Extended Data Fig. 4: In vitro and in vivo abrogation of pre-TCR function by silencing of CD2AP gene encoding the CMS functional adaptor of pTα.
From: Pre-TCR-targeted immunotherapy for T cell acute lymphoblastic leukemia
(A) Immunoblot analysis of CMS expression in SupT1 cells that were either transfected with several shRNAs (shCMS1-5) specific for CD2AP, the gene encoding CMS, or left untransfected (NT) (left panel). Relative CMS expression normalized to α-tubulin expression used as loading control is shown in the right panel. (B) Immunoblot analysis of CMS expression (upper) and PLCγ tyrosine (Tyr) phosphorylation (bottom) in JR.pTα pre-TCR+ cells transduced either with shCMS4 or with shSC as control, prior to activation with an anti-CD3ε monoclonal antibody for the indicated times. PLCγ Tyr-phosphorylation was analysed after immunoprecipitation with anti-PLCγ1 monoclonal antibody by probing with an anti-Y783-PLCγ1 antibody. Tubulin expression was analysed as loading control. (C) Relative NFAT activity of JR.pTα cells transduced with either shCMS4 or shSC as control, analysed upon stimulation with an anti-CD3ε monoclonal antibody by a luciferase assay. Data are shown as mean percentages ± SEM of NFAT activity relative to activated shSC cells analysed by two-tailed unpaired t test (n = 3). ****P < 0.0001. (D) Immunoblot analysis of CMS in human CD34+ early thymic progenitors (ETPs) transduced with a lentivirus encoding either shCMS4 and GFP or shSC and GFP as control. (E) Absolute numbers of shCMS- or shSC-transduced ETP-derived human cells reconstituting the thymus of RAG-2−/−γc−/− mice at the indicated weeks post-transplant (n = 6 week 3, n = 4 week 5; ***P = 0.0002). ETP transduction efficiencies with shCMS and shSC were 24,5% ± 4,5% and 21,5% ± 3,5%, respectively. (F) Absolute numbers of shCMS- or shSC-transduced human thymocytes in (E) expressing the double positive CD3lo TCRαβ− phenotype at 3- and 5-weeks post-transplant (left panel; *P = 0.0222) or the post-β selected DP CD3+ TCRαβ+ phenotype at 5-weeks post-transplant (right panel; *P = 0.0146). Data in (E, F) are shown as mean numbers ± s.e.m. of transduced (GFP+) cells normalized to 105 transduced input cells from two independent experiments, analysed by two-tailed unpaired t test.
