Figure 5. Hoxb5-induced iT lymphocytes show normal immune functions in vivo.

(a) Kaplan-Meier survival curves of allogeneic skin grafts from Rag1−/− (NC group, n = 8 mice) and Hoxb5-Rag1^−/− mice (n = 8 mice). Median allogeneic graft survival: Primary allograft from Hoxb5-Rag1^−/− = 10.5 d (1^st group, n = 8 mice); Secondary allograft from Hoxb5-Rag1^−/− = 7.5 d (2^nd group, n = 8 mice) (P value < 0.001, Log-rank test). Time interval between primary and secondary allogeneic skin graft = 8 weeks. (b) Flow cytometry analysis of activation status of Hoxb5-induced iT lymphocytes in the allogeneic skin graft. Representative plots from rejected primary allogeneic skin grafts (1^st, day 10) and rejected secondary allogeneic skin grafts (2^nd, day 6) were shown. Single cells were harvested from rejected skin tissues by collagenase I digestion. Activated iT cells were defined as CD45.2⁺Ter119⁻Mac1⁻CD19⁻CD4⁺CD44^hiCD69⁺ or CD45.2⁺Ter119⁻Mac1⁻CD19⁻CD8⁺CD44^hiCD69⁺. (c) Intra-cellular staining of IFNμ and IL-17 in the activated CD45.2⁺ CD4⁺ or CD8⁺ iT lymphocytes from the allogeneic skin grafts. Representative plots from rejected primary allogeneic skin grafts (1^st, day 10) and rejected secondary allogeneic skin grafts (2^nd, day 6) were shown. (d) Immunofluorescence staining of GFP⁺ CD3⁺ iT lymphocytes in allogeneic skin grafts. Images from representative allogeneic skin tissue sections from Rag1^−/− and Hoxb5-Rag1^−/− mice were shown. Scale bars, 20 μm or 50 μm. Data are representative of three independent experiments (b-d).