Fig 5. Following hematopoietic stem cell transplantation (HSCT) dominant characteristic T cells are regenerated.

(A) BALB/c mice were lethally irradiated and injected with 300 μg of a T cell- depleting CD4-specific monoclonal antibody. They were then rescued via intravenous transplantation of T cell-depleted syngeneic bone marrow cells (106 cells). Following lymphocyte recovery (6 weeks) mice were immunized subcutaneously with HEL emulsified in CFA. Lymph nodes were harvested 9 days later and primary cultures were incubated for 72h with HEL or serum-free medium alone. TCR CDR3 spectrotyping analysis reveald that the characteristic HEL-specific Vβ8.2Jβ1.5 T cell clonotype was not significantly exanded. Results are representative of four independent experiments. (B) ELISA was performed for IFN-γ detection on the 72-hour supernatants. Although the characterisitc expansion was not observed, mice were able to secrete significant amounts of IFN-γ (p = 0.007, Student’s t-test) and (C) were able to expand other HEL-specific T cell clones. (D) A second group of mice were treated exactly as described in “A” with exception that their immune system was allowed to recover for a longer period of time (100 days). Flow cytometry was done to confirmed the repletion of the CD4+ T cell compartment. Mice were immunized subcutaneously with HEL emulsified in CFA. Lymph nodes were harvested 9 days later and primary cultures were incubated for 72h with HEL or serum-free medium alone. (E) ELISA was performed for IFN-γ detection on the 72-hour supernatants. Results are representative of four independent experiments. Significant increase in IFN-γ secretion (p = 0.0014, Student’s t-test) was detected in culture supernantants when lymphocytes were incubated with HEL and (F) TCR CDR3 spectrotyping analysis demonstrated that the characteristic HEL-specific Vβ8.2Jβ1.5 T cell clonotype was strongly exanded in respone to culture with HEL. Results are representative of four independent experiments.