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αβTCR signaling acts as a tumor suppressor
type or leukemic cells resuspended in RPMI medium (200 mL) and stimulated for 2 minutes (min) at 37°C with avidin (14 mg) and biotinylated anti-CD3 (10 mg; clone 2C11, BD Pharmingen, for mouse cells and clone UCHT1, eBiosciences for human cells) and biotinylated anti-CD28 (10 mg; clone 37.51, BD Pharmingen, for mouse cells and clone CD28.2, eBiosciences for human cells). Unstimulated (control) cells were incubated with avidin alone. After lysis in 2X TNE buffer (100 mM Tris, 2% Nonidet P-40, 40 mM EDTA) supplemented with protease and phosphatase inhibitors, protein extracts (approx. 60 mg) were analyzed by immunoblot (see Online Supplementary Methods and Online Supplementary Table S2). P-Tyr levels were quantified on the entire lane and normalized to ACTIN. P-AKT protein levels were nor- malized to AKT.
Proliferation and apoptosis assays following CD3/CD28 stimulation
For comparison of the TCR-signaling ability to mediate prolif- eration or apoptosis of normal or leukemic cells, 1.105 of non-puri- fied or CD4 SP purified T cells were mixed (ratio 1:1) with Dynabeads Mouse T-Activator CD3/CD28 (Life Technologies) or Dynabeads Human T-Activator CD3/CD28 (Life Technologies) in 96-well flat bottom plates and incubated for 24 or 72 hours (h) (37°C, 5% CO2). For unstimulated controls, thymocytes were incubated in the same conditions but without anti-CD3/CD28 coated beads. Proliferation was measured using CFSE labeling (CellTraceTM CFSE Cell Proliferation Kit, Life Technologies) and apoptosis was followed using AnnexinV labeling (BD Pharmingen) and 7-AAD (BD Pharmingen) according to the man- ufacturer’s instructions.
Statistical analysis
Kaplan-Meier survival curves and statistical analyses were per- formed using GraphPad Prism software. Survival curves were compared using log-rank (Mantle-Cox) test. Statistical significance
was evaluated by two-tailed Mann-Whitney U-test. P<0.05 was considered significant.
Further details are provided in the Online Supplementary Methods and antibodies used for flow cytometry are listed in Online Supplementary Tables S3 and S4.
Results
Ptendel thymocytes expressing transgenic TCR are counter-selected during leukemogenesis
Phosphatase and tensin homolog protein (PTEN) is a well-known tumor suppressor involved in numerous types of cancers, and represents the main negative regula- tor of PI3K/AKT signaling pathway.12 To investigate the role of the TCR in leukemogenesis, we used a Pten-defi- cient mouse model of T-ALL which has previously been shown to induce TCRαβ+ tumors.13,14 PtenFlox/Flox mice were crossed into a CD4-Cre background (hereafter referred to as Ptendel) in which Cre is fully active at the CD4+CD8+ double positive (DP) stage of thymocyte development.15 As previously described,16 Ptendel mice developed leukemia characterized by malignant proliferation of mono/oligo- clonal T cells (Online Supplementary Table S5), and enlarged thymus and spleen (Figure 1A). Peripheral leukemic blasts from Ptendel mice were typically CD4 SP and expressed αβTCR at their surface (Figure 1B and Online Supplementary Table S5), in line with previous reports.14,16,17 To analyze the impact of positive selection, we used the OT-II mouse model which expresses a transgenic Vα2/Vβ5.1 TCR recognizing the chicken ovalbumin anti- gen in the context of MHC-II molecules.18 Ptendel mice were crossed with OT-II Rag1-deficient mice, in which all developing T cells do express an OT-II fit TCR, designed to trigger positive selection and give rise to mature SP T
Table 1. Immuno-phenotypes of T-cell acute lymphoblastic leukemia developed by OT-II x Ptendel mice in I-Ab/b or I-Ab/d backgrounds.
Mouse #
25
26
28
70
83
114
140
141
350
2
11
12
17
19
278
281
291
321
354
I-A
I-Ab/b
I-Ab/b
I-Ab/b
I-Ab/b
I-Ab/b
I-Ab/b
I-Ab/b
I-Ab/b
I-Ab/b
I-Ab/d
I-Ab/d
I-Ab/d
I-Ab/d
I-Ab/d
I-Ab/d
I-Ab/d
I-Ab/d
I-Ab/d
I-Ab/d
CD4/CD8 TCRαβ CD4+ +
CD4+/DN + CD4+ + CD4+ + CD4+ + CD4+ + CD4+ +
CD4+/DN + CD4+ + CD4+ + CD4+ + CD4+ + CD4+ + CD4+ + CD4+ + CD4+ +
CD4+/DN + CD4+/DN +
TCR OT-II TCRVβ Neg Vβ14
TCRV α Vα2
Neg Vβ11 Neg Vβ14 Neg Vβ14 Neg Vβ14 Neg Vβ6 Neg Vβ8.1/8.2 Neg Vβ5 Neg Vβ14
Vα2 Vα2 Vα2 Vα2 Vα2 Vα2 Nd* Vα2 Vα2 Vα2 Vα2 Vα2 Vα2 Vα2 Vα2 Vα2 Vα2 Vα2
+ Vβ5 + Vβ5 + Vβ5
*Negativity for TCRVa2 and the TCRVα expressed was not determined.
CD4+/DP
+
Neg Vβ6 + Vβ5 + Vβ5 + Vβ5 + Vβ5 + Vβ5 + Vβ5
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