Figure 6. In Vitro Culture with High Dose IL-7 Promotes Proliferation and Down regulation of CD62L on CD8 T-cells.

(A-C). Splenic CD62L^lo or CD62L^hi T-cells were isolated from Bim^-/- mice and sorted using anti-CD62L antibody and magnetic beads as described in Materials and Methods. Cells were placed in culture without IL-7 or with 10 or 150 ng/mL of IL-7 and assayed after 7 or 14 days. (A). T-cell numbers from populations of cells that were initially sorted for CD62L^lo or CD62L^hi expression was determined using the Accuri flow cytometer and confirmed by microscopic evaluation as described in Materials and Methods. (B). Dot plots display ratio of CD8 and CD4 T-cells and surface levels of CD62L on CD62L^lo or CD62L^hi T-cells after 14 days of culture in 150 ng/mL IL-7. Phenotype was determined by staining with a FITC-conjugated anti-CD4 antibody or a PerCP-conjugated anti-CD8 antibody and a PE-conjugated CD62L antibody and analyzed by flow cytometry as described in Materials and Methods. Results shown were acquired from the viable cell gate. (C). Cell cycling of CD62L^lo or CD62L^hi T-cells was assessed after 7 days of culture in 150 ng/mL IL-7 by measuring the incorporation of BrdU into replicating DNA with a FITC-conjugated BrdU antibody and CD4 or CD8 expression determined as described using flow cytometry (A). Quadrants were established using control antibodies. Percentages in bold indicate CD4 or CD8 T-cells that had incorporated BrdU. (D). Unsorted lymph node T-cells were isolated from Bim^-/- mice and placed in culture without IL-7 or with high dose IL-7 (150 ng/mL) for 7 or 14 days. At the indicated time points, cells were stained with antibodies for CD4, CD8, CD44 and CD62L as described in Materials and Methods. Surface expression was analyzed by flow cytometry. Table 2 data is gated on CD8 T-cells and is presented as a percentage of total viable cells expressing the indicated phenotype. Results from a representative experiment of two experiments performed are shown.