Extended Data Fig. 3: Comparative analysis of Vβ and Vα frequencies and Vβ14+Vα2+ CD8 expansion captured by CyTOF versus TCR sequencing during LADD-OVA infection.

(a) Schematic of experimental approach to compare CyTOF to mRNA-based TCR-seq (iRepertoire) from spleens of LADD-OVA-infected mice. (b) Two-tailed spearman correlation analysis of Vβ and Vα frequencies in CD8+ T cells (left) and CD4+ T cells (right). (c-d) Comparative quantification of the TRBV/Vβ and TRAV/Vα chains captured by CyTOF of all CD8+ T (c) and CD4+ T cells (d). Note: Anti-Vα3.2 RR3- 16 clone binds specifically to a Vα3.2 allele preferentially expressed in CD8+ T cells over CD4+ T cells59. (e) Quantification of the TRBV/Vβ chain (top) and TRAV/Vα chain (bottom) frequencies in SIINFEKL-loaded H-2Kbtetramer+ CD8+ T cells sorted from spleens. (f) Fold change calculation of TRBV/Vβ chain and TRAV/Vα chain frequencies of SIINFEKL-loaded H-2Kb tetramer+ CD8+ T cells over frequencies of bulk CD8+ T cells. (g) Schematic comparison of approach used in this study compared to Straub et al.³⁷. (h) Re-annotated clusters identified by Straub et al. plotted in UMAP space. (i) Scaled gene expression within the scRNA-seq dataset of genes encoding the proteins measured in Fig. 1, stratified by cluster. (j) UMAP visualization of all clustered cells colored by Ki67 or PD-1 gene expression. (k) UMAP visualization of all clustered cells colored by TRBV31 assignment or TRAV14D-2 assignment (left), and UMAP visualization of TRBV31 assigned cells colored by TRAV14D-2 assignment (right). (l) UMAP visualization of all clustered cells colored by identified specificity. (m) Quantification of TRBV gene usage among SIINFEKL-reactive cells (right) or of cells of unidentified reactivity (left). (n) Quantification of TRBV gene usage among SIINFEKLreactive cells expressing TRAV14D-2. (o) Quantification of TRBV/Vβ chain assignment of all CD8 T cells as measured by CyTOF or scRNA-seq. (p) Quantification of the TRBV/Vβ chains measured in the CyTOF assay of all CD8 T cells that were assigned a TRBV/Vβ chain by either CyTOF or scRNA-seq. (q) Two-tailed spearman correlation analysis of data shown in (j). Results from n=3 LADD-OVA infected mice for bulk TCRseq (a-f), data are presented as mean values ± s.e.m.