Journal: The EMBO Journal
Article Title: PTPN2 phosphatase deletion in T cells promotes anti‐tumour immunity and CAR T‐cell efficacy in solid tumours
Figure Lengend Snippet: PTPN2 deficiency enhances CAR T‐cell efficacy in vivo by promoting STAT‐5‐mediating homing to CXCL9/10‐expressing tumours HER‐2‐E0771 mammary tumours cells (2 × 10 5 ) were injected into the fourth inguinal mammary fat pads of female HER‐2 TG mice. Six days after tumour injection, HER‐2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 6 × 10 6 FACS‐purified CD8 + CD62L hi CD44 hi central memory HER‐2 CAR T cells generated from Ptpn2 fl / fl , Lck ‐Cre; Ptpn2 fl / fl , Lck ‐Cre; Ptpn2 fl / fl ; Stat5 fl /+ or Lck ‐Cre; Ptpn2 fl / fl ; Lck +/− splenocytes. Mice were injected with IL‐2 (50,000 IU/day) on days 0–4 after adoptive CAR T‐cell transfer and (B) monitored for tumour growth. (A, C) Lymphocytes were isolated from the tumours on (A) day 3 or (C) day 16 post‐adoptive transfer, and mCherry + CD45 + CD8 + CAR T‐cell numbers were determined by flow cytometry. In (C), TILs were stained for intracellular IFNγ and TNF after PMA/ionomycin treatment. HER‐2‐E0771 cells generated to inducibly overexpress PTPN2 in response to doxycycline (E0771‐HER‐2‐PTPN2 hi ) were pre‐incubated (24 h) with vehicle or doxycycline (DOX) subsequently stimulated with IFNγ for the indicated times. STAT‐1 Y701 phosphorylation (p‐STAT‐1) and PTPN2 levels were assessed by immunoblotting. Cxcl9 and Cxcl10 mRNA levels in vehicle versus DOX‐treated and IFNγ‐stimulated HER‐2‐E0771 cells were assessed by quantitative real‐time PCR. E0771‐HER‐2‐PTPN2 hi mammary tumour cells (2 × 10 5 ) were injected into the fourth inguinal mammary fat pads of female HER‐2 TG mice. Five days after tumour injection, mice were administered vehicle or DOX in drinking water followed by irradiation (4 Gy) on day 6 and the adoptive transfer of 6 × 10 6 FACS‐purified central memory Ptpn2 fl / fl versus Lck ‐Cre; Ptpn2 fl / fl HER‐2 CAR T cells. Mice were then injected with IL‐2 (50,000 IU/day) on days 0–4 post‐adoptive CAR T‐cell transfer, and (H) tumour growth was monitored. In (G), CD45 + CD8 + mCherry + TILs were quantified by flow cytometry at day 4 post‐adoptive transfer. Data information: Representative flow cytometry profiles and results (means ± SEM) from two independent experiments are shown. In (A, E), significance was determined using 1‐way ANOVA test. In (B, G, H), significance was determined using 2‐way ANOVA test. In (C), significance was determined using 2‐tailed Mann–Whitney U ‐test. * P
Article Snippet: Recombinant human IL‐2, murine IL‐7 and IL‐15 used for T‐cell stimulation or IFNγ used for stimulating tumour cells were purchased from the NIH or PeproTech, respectively.
Techniques: In Vivo, Expressing, Injection, Mouse Assay, Irradiation, Adoptive Transfer Assay, FACS, Purification, Generated, Isolation, Flow Cytometry, Cytometry, Staining, Incubation, Real-time Polymerase Chain Reaction, MANN-WHITNEY