Extended Data Fig. 4: CITE-Seq with 5’ TCR profiling reveals the presence of double-negative gamma-delta CAR T-cells in patient 2 at month 3 and year 3.

a, UMAP showing expression of key marker genes and TCRαβ clonotype for patient 2 at month 3, and (b) patient 2 at year 3. Aliquots of peripheral blood were sorted for CD3⁺CD14⁻CAR⁺ cells, and 5’ CITE-Seq with TCRαβ clonotyping was performed. High-quality cells were computationally identified by retaining cells with 200–5000 genes detected and less than 5% mitochondrial RNA, and shown are the 552 (month 3) and 242 (year 3) cells that were verified as CAR T-cells with at least one read aligned to the 5’ CAR construct. UMAP plots showing normalized RNA expression are colored in shades of blue; plots showing protein expression via CITE-Seq antibody-derived tags are colored in shades of green; TCRαβ clonotype plots (bottom-right of panels a and b) are colored using a spectral color scheme, with cells with no detected TCRαβ clonotype colored light grey. Red arrows indicate the double-negative CAR T-cell population in both time points, with gamma-delta identity demonstrated by protein expression of the γδ TCR, lack of protein expression of the αβ TCR, specific RNA expression of TRDV1 and TRGV4, and non-detection of the TCRαβ clonotype.