Figure 6. Early transgene expression in engineered preTs is required for their in vivo survival and directs final differentiation towards CD8 T cells.

(A–E) Lethally irradiated B6 mice received 3 × 10⁶ syngeneic TCDBM cells together with either 8 × 10⁶ non-transduced or model TCR gene-transduced preTs. For one group doxycycline was added early starting the day of transplantation while the second group did not receive any doxycycline. One month after transplantation, splenocytes were harvested and cultured ex vivo for four more days in the presence of doxycycline. (B and C) Expression of CD4 and CD8 T cell markers were assessed on T cell progenies of precursor preTs. (D) The ability for intracellular IFNγ generation was assessed upon stimulation with SIINFEKL. (E) Expression levels of a broad range of TCR Vβ families (other than Vβ5) on eGFP⁺ progenies of preTs were quantified by flow cytometry. (F) One month after transplantation, splenocytes were harvested and cultured ex vivo in the presence of doxycycline. After four days of culture 10⁵ GFP⁺CD3⁺ cells were sorted and analyzed by complete TCR Vβ spectratyping as described previously¹⁷. Data were further analyzed by GeneMapper software (Life Technologies) comparing the area under the curve (AUC) representing various CDR3-size lengths. Peak Scanner software (Life Technologies) was used for calculations. Statistical analysis was done applying the Student’s t-test. A representative example is shown.