In vivo development and functions of murine T cells after various period of culture. (A, B) T-cell development in CD3-KO mice after injection of 10⁷ fresh (day 0) or cultured mT cells (days 2-6). Each point represents percentages (○) and numbers (•) of CD4 (A) and CD8 (B) T cells in the blood and spleen from individual mice (n = 4 per group) 3.5 and 5 months after adoptive transfer, respectively. Of the 4 mice injected with day 6 cultured cells, 3 died 4 months after transfer. ^*Indicates blood and ^**spleen: significant values as compared to those of day 0 (P < .05 Fisher PLSD test). (C) Allogeneic MLR of mT cells from reconstituted CD3-KO mice: the allogeneic proliferative response of splenocytes from CD3-KO mice against BALB/c-irradiated splenocytes was measured 5 months after injection of cultured T cells. Cell proliferation was determined by ³H-Tdr incorporation after 4 days of coculture. □ indicates an individual mouse. ^*Significant values as compared to those of day 0 (P < .05 Fisher PLSD test). (D) 10⁷ fresh day 0 (▵, n = 8), day 2 (▪, n = 5), day 4 (•, n = 7), or day 6 (▴, n = 6) cultured mT cells were injected into B6 nude mice that were grafted with allogeneic skin tail fragments from BALB/c mice 2 weeks later. Skin graft survival was monitored over time. Results are presented as Kaplan-Meier skin survival graph. (E-F) Ability of cultured donor mT cells to induce GVHD after injection into 6-Gy-irradiated B6D2F1: noninjected mice (X, n = 11), mice receiving 10⁷ fresh donor T cells (day 0, ▵, n = 17) were used as controls. Results are presented as Kaplan-Meier survival graph after T-cell injection. (E) Effect of the length of T-cell culture: 10⁷ cells were injected after day 2 (▪,n = 8), day 4 (•, n = 4), or day 6 (▴, n = 13) of culture. ▵ indicates day 0 (noncultured cells). (F) Effect of injecting increased amounts of 6-day cultured T cells: 10⁷ (▴, n = 13), 2 × 10⁷ (•, n = 4), 2.5 to 3 × 10⁷ (○, n = 8), 4 × 10⁷ (▪, n = 7), and 8 × 10⁷ (▾, n = 4). ▵ indicates day 0 (noncultured cells).