Page 102 - Haematologica June

S. Gon et al.
expressed the full transgenic OT-II TCR (usually TCRVα2 chain was expressed, but not associated with TCRVβ5 chain) (Table 1 and Figure 3). In line with the H-Y model above, this indicates that (pre)leukemic clones harboring OT-II fit TCR are counter-selected during oncogenesis. In striking contrast, most of TCRαβ+ T-ALL in the allogenic I-Ab/d background (9 of 10) expressed OT-II (Table 1 and Figure 3A). This indicates that in a context of sub-optimal positive selection, Ptendel OT-II+ blasts are not counter- selected, but rather bypass death-by-neglect during posi-
tive selection allowing further leukemia development. Altogether our data indicate that, in Pten-deficient T- ALL mouse models, fit TCR functions as a tumor suppres- sor impeding thymocytes to develop leukemia, while thy- mocytes expressing no or unfit TCR are prone to leuke-
mogenesis.
TCRαβ signaling is disabled in Pten-deficient T-ALL
We next asked whether endogenous TCRαβ+ from Ptendel mice, which presumably passed positive selection
F
AB
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of living cells. Representative data of Pten
del
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G
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Figure 4. TCRαβ signaling is disabled in Ptendel T-ALL blasts. (A) Indicated cells labeled with CFSE were either left unstimulated (US) or stimu- lated with anti-CD3/28 beads (CD3/28) for 24 or 72 hours (h), and then analyzed by flow cytome- try. SSC/FSC dot plots (left) and CFSE histograms (right) are shown. Numbers indicate percentage
T-cell acute lymphoblastic leukemias (T-ALL) (n=5) and Cdkn2a–/– T-ALL (n=5). (B) Analysis of early TCR signaling by immunoblots. Two representative cases of Ptendel T-ALL (n=5) and Cdkn2a–/– T-ALL (n=5), and wild-type (WT) thymocytes are shown. Cells were untreated (-) or stimulated (+) with anti-CD3/CD28 antibodies for 2 minutes and analyzed by immunoblotting with antibodies spe- cific for phosphorylated tyrosine (P-Tyr), phospho- rylated AKT S473 (P-AKT), AKT and Actin. (C) Levels of P-Tyr species normalized to Actin (top) and of P-Akt normalized to Akt (bottom) in unstimulated (US) and in CD3/CD28-stimulated (S) of indicated cells: WT thymus, Ptendel T-ALL (n=5) and Cdkn2a–/– (n=5) assayed in duplicate for P-Tyr. (D and E) Impact of TCR stimulation on human T-ALL cell survival. Cells were either left unstimulated or stimulated with beads coated with anti-CD3 and anti-CD28 antibodies (CD3/CD28) during 72 h and then stained with Annexin V and 7-AAD to monitor cell death by flow cytometry. (D) Typical dot plots for Xg9 and Xg35 are shown. Percentage of live cells (gated) is indicated. (E) Survival index as determined by ratio of live cells in treated (CD3/28) versus unstimulated conditions 72 h post induction. Each dot corresponds to the mean survival index (obtained from at least 2 assays) of one T-ALL xenograft. Xg35 sample is depicted as a white square. (F and G) Human thymus and T-ALL were analyzed as described in (B). Thymus NP and SP CD4+ correspond to total non-purified (NP) and purified CD4 SP cells, respectively, from healthy human thymus. (F) Two representative samples of human T-ALL: Xg13 (TCRneg) and Xg8 (TCRαβ+) are shown (see also Online Supplementary Figure S9). (G) P-Tyr activation index which corresponds to the ratio of P-Tyr species levels in stimulated (CD3/28) versus unstimulated samples (top); P-AKT normalized to AKT (bottom) in unstimulated (US) and in CD3/CD28-stimulated (S) (Bottom). Statistical significance of P-AKT levels between unstimulat- ed TCR+ PDX versus TCRneg PDX, NP or SP thymo- cytes are indicated with blue asterisks. TCRneg (n=5: Xg3, 13, 20, 23 & 40) and TCRαβ+ (n=5: Xg8, 9, 35, 38 & 47). P-Tyr species levels were normalized to ACTIN. (C, E and G) Error bars rep- resent means + Standard Deviation. Statistical significance was assessed using Mann-Whitney test; ns: non-significant P>0.05; *P<0.05;
**P<0.01.
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haematologica | 2018; 103(6)