Figure 4. Bi-allelic disruption of PTPN22 in CD4⁺ cells using rAAV6 donor delivery leads to increased T cell activation in response to TCR engagement.

(A) PTPN22 disrupting AAVs within the PTPN22 locus after homology directed repair. Promoter – (GFP or BFP) reporter – WPRE/polyA constructs disrupt exon 14 of PTPN22. Identical constructs with homology arms that align to the CCR5 cut site used to make CCR5 control populations. (B) Representative flow plots of PTPN22 and CCR5 AAV edited CD4⁺ T cells from the same human donor at Day 7 post-editing. Cells were expanded 7 days post-editing and rested 24 hours in cytokine free media, as in Fig. 1A, prior to lysis. (C) Editing outcomes for PTPN22 or CCR5 AAV-edited T cells (bars represent mean +/− SEM, n=4 independent human donors, percentages reflect summary data). (D) Quantified PTPN22 protein expression relative to HSP90 and normalized to mock edited values from the same T cell donor (bars represent mean +/− SEM, n=4 independent human donors). (E) Representative western blot of PTPN22 expression in sorted PTPN22 AAV-edited CD4⁺ T cells from the same human donor. (F-G) Human CD4⁺ T cells were edited as in (A) and stimulated using plate bound anti-CD3 for 48 hours. Upper panels: Representative flow overlays of CD25 (F) CD71 (G) and PD-1 (H) in GFP+/BFP+ PTPN22 and CCR5 AAV-edited cells from the same donor. Lower panels: Summary data below represent median fluorescence of GFP+/BFP+ cells normalized to the median fluorescence of stimulated, mock edited CD4⁺ T cells from the same donor (n=4, paired t test). Lines illustrate data derived from each individual donor. RNP- ribonucleoprotein. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.