Figure 3.

Reconstitution of Immune Cell Functions following LV Gene Therapy in Primary Recipients

Assays of immune cell function 16 weeks after BM transplantation. Bars show the mean ± SD. (A) The percentage of cells that underwent ≥1 cell division 72 hr after incubation with CD3/CD28 antibodies or P/I (as measured by CellTrace Violet dilution). Shown are cells within CD4 (left) or CD8 (right) staining gates by flow cytometry. Data are from one of the three independent experiments: n = 3 (KO and WT mock, MND.hWASp) and 5 (650.MND.hWASp). (B) Flow cytometry analysis showing CellTrace Violet labeled splenocytes 72 hr post-CD3/CD28 stimulation, gated on live and either CD4⁺ or CD8⁺ populations. Numbers indicate the percentages that have proliferated after CellTrace Violet labeling. (C) B cell (CD43⁻ splenocyte) migration in response to media only (−) or media supplemented with CXCL13 chemokine (+). Each dot indicates the percentage of B cells from a single mouse that migrated through the 5-μm-pore transwell. Data are from two independent experiments: n = 5 (KO mock, 650.MND.hWASp), 3 (MND.hWASp), and 8 (WT mock). (D) IgG and IgM and (E) anti-double-stranded DNA antibody levels in the serum of primary recipients as determined by ELISA 16 weeks post-transplant. Data are from three independent experiments: n = 5 (KO and WT mock), 4 (MND.hWASp), 2 (WT) (D only), and 12 (650.MND.hWASp). Serum from an autoimmune WAS chimera²¹ (E only) with high serum anti-DNA antibodies was used as a positive control. The p value was determined by unpaired two-tailed t test. *p < 0.024.