Fig 2: Efficient gene ablation with the one-shot CRISPR system(A) Measurement of specific gene disruption with the one-shot CRISPR. gRNAs were designed targeting either TRAC, TRBC, B2m, Fas or PD1, an empty gRNA was used as a control. PD1 ablation was confirmed and measured on day 3 after the re-stimulation of CD3/CD28 beads. Gene disruption was confirmed by flow cytometry. gEmp:Empty-gRNA. (B) Enrichment of CD3 or Fas genetically edited CAR T cells by negative selection. Targeted gene and CAR expression before and after selection was measured by flow cytometry.

Fig 3: The Breast Cancer Immune Landscape(A) Frequencies of selected immune cell types in juxta-tumoral and tumor samples.(B) t-SNE plots of the normalized marker expression of 40,000 T cells from all samples.(C) t-SNE of T cells colored by PhenoGraph cluster.(D) Heatmap of normalized T cell marker expression for 20 T cell clusters. CM, central memory; Eff/Mem, effector and memory; Reg, regulatory; PD-1, PD-1+.(E) Boxplots showing the frequencies of the CD4+ (left) and CD8+ T cell clusters (right) in juxta-tumoral and tumor samples.(F) PD-1+ T cell frequency (top) and mean PD-1 expression (bottom) among tumor-derived CD4+ and CD8+ T cells.(G) Comparison of the PD-1+ T cell frequency and mean PD-1 expression for CD8+ (top) and CD4+ T cells (bottom).(H and I) Frequencies of selected T cell clusters in (H) ER+ and ER- tumors and (I) luminal A and B tumors.(J) t-SNE plots of normalized marker expression of 40,000 myeloid cells from all samples.(K) t-SNE of myeloid cells colored by PhenoGraph cluster.(L) Heatmap of normalized myeloid marker expression for 19 myeloid clusters. Mono, monocyte; T.-res, tissue-resident; E. im., early immigrant; TAM, tumor-associated macrophage; MDSC, myeloid-derived suppressor cell.(M) Frequencies of the myeloid clusters in juxta-tumoral and tumor samples.(N and O) Frequencies of the indicated myeloid clusters in (N) ER+ and ER- tumors and (O) luminal A and B tumors.Wilcoxon rank-sum test was used for statistical analysis. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S2.

Fig 4: PD1/PDL1 Blocking Leads to Exhaustion Reversal in Patient PBMCsPBMCs were isolated 5 years post-injection. (A) PBMCs were stimulated in vitro with overlapping peptides (15-mers overlapping by 10 aa) spanning the VP1 capsid protein sequence or AAV1 immunodominant peptides. (B) PBMCs were stimulated in vitro with AAV1 immunodominant peptides for 48 h in the presence or absence of PD1/PDL1 antibodies or isotype controls. For each experiment in (A) and (B), the negative control consisted of unstimulated cells (medium), whereas CD3/CD28 stimulation was used as a positive control for cytokine secretion. Responses were considered positive when the number of spot-forming units (SFUs) per 1e6 cells was >50 and at least 3-fold higher than in the control condition (dotted line); asterisks denote positive response. (C) IFN? secretion fold change expressed as percent reactivity relative to the AAV1 peptide stimulation condition. Each condition was performed in triplicate. Error bars represent SEM.

Fig 5: Tregs in Human Fetal Skin Demonstrate an Effector Memory PhenotypeTwenty-three weeks g.a. fetal torso skin and healthy adult torso skin samples were analyzed for 22 markers using mass cytometry.(A) PCA plot of Tregs (cluster C, Figure 2) and CD4+ Tconv (clusters A & B, Figure 2) from 23 week fetal skin versus adult skin based on relative expression of key markers as assessed by mass cytometry.(B–E) Graphs showing median expression intensity (m.e.i.) of (B) Foxp3, (C) CD25, (D) PD-1, and (E) CTLA4 by mass cytometry on fetal versus adult skin Tregs.(F and G) Cells were isolated from 17–23 weeks g.a. fetal skin (scalp and/or torso) as well as adult (torso) skin and analyzed by flow cytometry. (F) Percentage of CD45RO+ Tregs in skin by age and (G) accompanying representative flow plots.(H) Representative histogram and (i) quantification of Nur77 MFI on fetal skin CD45RO+ versus CD45RA+ Tregs.(J) Nur77 in fetal skin CD45RA+ CD8+, CD4+ Tconv, and Tregs.Each point in (A)–(E) represents data from an individual donor. Points in (F), (I), and (J) point represent data from an individual tissue sample; for some fetal samples data from scalp and torso skin from the same fetal donor are included as separate points. ns, not significant (p > 0.05); *p < 0.05; **p < 0.01; ***p < 0.001.