Fig. 3. Ectopic expression of LAT containing the PIPRSP mutant motif, AIARSA, impairs thymocyte β- and positive-selection in vivo.

Flow cytometric analysis of thymocytes from lethally irradiated bone marrow BoyJ (CD45.1⁺) chimeras reconstituted with CD45.2⁺ LAT-deficient B6 hematopoietic stem cells transduced with lentiviruses expressing either WT LAT-P2A-mCherry (WT; n = 4 recipients) or mutant AIARSA LAT-P2A-mCherry (AIARSA; n = 4 recipients). Recipient mice were analyzed 6 – 8 weeks later. (a) Representative mCherry expression of thymocytes, gated on CD45.2⁺ cells. Numbers indicate percentages of mCherry⁺ cells in each gate. (b) Flow cytometric analysis of CD45.2⁺ mCherry⁺ thymocytes. Numbers in each quadrant indicate percent of cells in each. (c) Flow cytometry analysis of CD24 versus TCR expression of CD45.2⁺ mCherry⁺ CD8SP thymocytes. (d) Bar graphs representing the frequencies of DN, DP, CD4SP and CD24⁻TCR⁺ mature CD8SP thymocytes among CD45.2⁺ mCherry⁺ cells. Each symbol represents an individual mouse. Bars indicate the mean± s.d. n = 4 independent animals in two independent experiments. *P = 0.0286; ns, not significant; two-tailed Mann-Whitney test. (e) Flow cytometric analysis of DN1 to DN4 cell development based on CD44 and CD25 expression, gated on CD45.2⁺ mCherry⁺ DN thymocytes. (f) Frequency of DN1 (CD4⁺CD25⁺), DN2 (CD44⁺CD25⁺), DN3 (CD44⁻CD25⁺), DN4 (CD44⁻CD25⁻) of CD45.2⁺ mCherry⁺ DN T cells in each quadrant of (e). Bars indicate the mean± s.d. n = 4 independent animals in two independent experiments. *P = 0.0286; ns, not significant; two-tailed Mann-Whitney test. (g) Flow cytometric analysis of DP progression through thymic positive selection based on CD69 and TCR expression, gated on CD45.2⁺ mCherry⁺ DP thymocytes. (h) Frequency of post-selection DP3 (CD69⁺TCR^hi) DP cells. Bars indicate the mean± s.d. n = 4 independent animals in two independent experiments. *P = 0.0286; two-tailed Mann-Whitney test. All data are representative of two independent experiments with similar results.