Figure 3.
Figure 3. F5 TreIL-7ROFF T cells activate, expand, and develop effector function similarly to F5 control T cells. (A) Splenocytes from F5 TreIL-7R donors taken off dox food 3 days previously were CFSE labeled and stimulated with different concentrations of NP68 peptide. After 72 hours, CFSE profiles were analyzed. Graphs show percent cells divided (triggering) and division index of dividing cells calculated (burst size) for cultures of control (□) and F5 TreIL-7ROFF T cells. (B) In similar cultures, T cells were stimulated with 10 nM NP68 for 72 hours. Viable cells were isolated by centrifugation over Ficoll gradient and then cultured for a further 4 days in IL-2. Cell recoveries were enumerated both after 3 days of peptide stimulation and after 4 days of further IL-2 culture. Bar charts show fold expansion over input cell number in NP68-stimulated and IL-2 expansion cultures. (C) Effector function of cultured T cells from panel B was tested in vivo. C57BL/6J Ly5.1 targets were pulsed in vitro with increasing concentrations of NP68 and then labeled with different levels of CFSE. The antigen dose for each individual CFSE peak is indicated above the histograms. Targets were mixed in equal numbers and 2 × 107 total cells cotransferred with 2 × 107 cultured F5 effectors to Rag1–/– recipients. Targets alone were injected into Rag1–/– or naive F5 Rag1–/– recipients as control for no killing. Twenty-four hours later, spleen from recipient mice was analyzed by FACS for the presence of Ly5.1-positive target cells. Histograms show CFSE labeling of Ly5.1-positive targets cotransferred with effectors (solid line) and targets transferred alone as control (gray fill) for the F5 effector donor indicated. Target cell recoveries from naive F5 Rag1–/– were identical to those recovered from Rag1–/– hosts (data not shown). (D) Bar chart indicates the percent killing of targets pulsed with the indicated concentrations of NP68 by F5 control (open) and F5 TreIL-7ROFF effectors (filled). Data are representative of at least 2 independent experiments.

F5 TreIL-7ROFF T cells activate, expand, and develop effector function similarly to F5 control T cells. (A) Splenocytes from F5 TreIL-7R donors taken off dox food 3 days previously were CFSE labeled and stimulated with different concentrations of NP68 peptide. After 72 hours, CFSE profiles were analyzed. Graphs show percent cells divided (triggering) and division index of dividing cells calculated (burst size) for cultures of control (□) and F5 TreIL-7ROFF T cells. (B) In similar cultures, T cells were stimulated with 10 nM NP68 for 72 hours. Viable cells were isolated by centrifugation over Ficoll gradient and then cultured for a further 4 days in IL-2. Cell recoveries were enumerated both after 3 days of peptide stimulation and after 4 days of further IL-2 culture. Bar charts show fold expansion over input cell number in NP68-stimulated and IL-2 expansion cultures. (C) Effector function of cultured T cells from panel B was tested in vivo. C57BL/6J Ly5.1 targets were pulsed in vitro with increasing concentrations of NP68 and then labeled with different levels of CFSE. The antigen dose for each individual CFSE peak is indicated above the histograms. Targets were mixed in equal numbers and 2 × 107 total cells cotransferred with 2 × 107 cultured F5 effectors to Rag1–/– recipients. Targets alone were injected into Rag1–/– or naive F5 Rag1–/– recipients as control for no killing. Twenty-four hours later, spleen from recipient mice was analyzed by FACS for the presence of Ly5.1-positive target cells. Histograms show CFSE labeling of Ly5.1-positive targets cotransferred with effectors (solid line) and targets transferred alone as control (gray fill) for the F5 effector donor indicated. Target cell recoveries from naive F5 Rag1–/– were identical to those recovered from Rag1–/– hosts (data not shown). (D) Bar chart indicates the percent killing of targets pulsed with the indicated concentrations of NP68 by F5 control (open) and F5 TreIL-7ROFF effectors (filled). Data are representative of at least 2 independent experiments.

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