Improvements in immune function of patient P2 following gene therapy. (A) Increases in peripheral-blood CD4⁺, CD8⁺, and total CD3⁺ T-cell counts at 6 months after gene therapy. (B) TRECs, initially absent from peripheral-blood T cells, appearing at 9 months after gene therapy (normal adult range 1000-2200 TRECs/μg DNA, shading). (C) Shift in T-cell chimerism after gene therapy (area under PCR-amplified polymorphic allele peaks; Cofiler; Applied Biosystems, Foster City, CA), from before gene therapy (top set of peaks) where there is 51% maternal bone marrow donor (left peak) and 49% P2 host (stippled middle peak) to 6 months after gene therapy (bottom set of peaks) where the ratio is 13% donor versus 87% host. (D) CD4⁺ T-cell proliferation measured by decrease in fluorescence in response to PHA mitogen stimulation, absent before gene therapy (empty top left quadrant of the bottom left panel) but normal at 12 months after gene therapy (top left quadrant of the bottom right panel). P2 blood mononuclear leukocytes were labeled with the permanent cell-membrane–binding dye CFSE and cultured for 5 days in medium alone (control; top panels) or PHA (stimulus; bottom panels), then labeled with phycoerythrin-labeled anti-CD4 antibody. CD4⁺ T cells appear in the top half of each panel, and proliferation of the CD4⁺ T cells (dilution of CSFE fluorescence) is seen to occur only after gene therapy and only in response to PHA stimulation (bottom right panel). Not shown is that CD8⁺ T-cell response to PHA and both CD4⁺ and CD8⁺ responses to ConA, PWM, and Candida antigen increased similarly as measured by the CSFE assay. (E) Spectratyping of the Vβ TCR repertoire of CD3⁺ T cells²³  from P2, demonstrating very restricted diversity before gene therapy (middle panel; total absence of representation of 3 Vβ families and almost monoclonal single-peak representation within 6 or 7 families) but increased diversity at 12 months after gene therapy (right panel; some representation in all the Vβ families, almost monoclonal single-peak representation only within 3 or 4 families, and clear improvement in multipeak polyclonality within 12 of the 23 families represented). A typical healthy control is shown in the left panel.