Differential survival of thymic and peripheral T cells in the mutant mice and the underlying mechanism. (A) Bone marrow cells from Lck-Cre+-Crryflox/flox mice (CD45.2) and WT mice (CD45.1) were mixed at 1:1 ratio and transplanted into lethally irradiated WT or C3−/− recipients (n = 4 per group). After 2 months, the ratios of CD45.1/CD45.2 thymocytes (DN, DP, CD4 single positive, CD8 single positive), and peripheral (lymph node and blood) CD4+ or CD8+ T cells were determined by flow cytometry. CD45.1 T cells were enriched in the lymph nodes (LNs) and blood (BL) but not in the thymus of WT recipients. As expected, no enrichment was observed at any sites in C3−/− recipients. (B) Lck-Cre+-Crryflox/flox and Lck-Cre−-Crryflox/flox mice (n = 4 per group) were treated twice weekly with clodronate liposome (+) or control liposome (−) for 4 weeks, and blood CD4+ and CD8+ T-cell counts were monitored by flow cytometry. Clodronate liposome but not control liposome treatment time dependently increased blood CD4+ and CD8+ T-cell counts (NS, not statistically significant; *P < .05, Student t test). Clodronate-liposome treatment had no significant effect on blood T-cell counts in Lck-Cre−-Crryflox/flox mice. (C) Lck-Cre+-Crryflox/flox and Lck-Cre−-Crryflox/flox mouse thymocytes were labeled with CFSE, mixed at 1:1 ratio, and injected into different recipient mice (n = 2 per group). The ratios of Cre+/Cre− thymocytes (determined with the use of Crry as a marker among CFSE-positive cells) in the spleens of the recipient mice were determined 1 hour after cell injection. Some WT mice received clodronate-liposome or clodronate-PBS (control) treatment 24 hours before thymocyte transfer. (D) Same experimental procedure as in panel C except that the thymocytes were first incubated in 5% WT mouse serum at 37°C for 45 minutes before adoptive transfer. The top FACS histograms in panels C and D show C3 deposition levels on the thymocytes with (D) or without (C) ex vivo incubation in WT mouse serum. All values shown are mean plus or minus SEM.
