(A) A schematic diagram illustrating the immunoaffinity purification workflow. (B and C) Unique peptides were identified from transduced hepatocytes, skin, and spleen. For H-2K^d, the peptide repertoires of C57BL/6 hepatocytes transduced with AAV-HC-K^d, B6.Kd spleen, and B6.Kd skin grafts collected 7 days after transplantation were determined, while for H-2K^b, the corresponding tissues were B10.BR hepatocytes transduced with AAV-HC-K^b, 178.3 spleen, and 178.3 skin grafts sampled at 7 days after transplant. Data from 2 independent experiments are shown. Within each replicate experiment, samples from 3 to 4 mice were pooled per condition. For K^d, 880 9-mer peptides were found to be shared across all 3 tissue types, while 1083 K^b peptides (8- to 11-mer, IC₅₀<500 nM) were common to the 3 tissues. (D) Length distribution of filtered H-2K^d peptides from hepatocytes, spleen, and skin graft tissue samples. The number of peptides of each length identified with a 5% FDR cut-off are shown. (E) Peptide-binding motifs for H2-K^d peptides generated from a nonredundant list of 9-mer peptides using GibbsCluster 2.0. (F) Length distribution of H-2K^b peptides from hepatocytes, spleen, and skin graft tissue samples (as for D). Most eluted peptides are 8-mers, with 9-mers also relatively frequent. (G) Peptide-binding motifs for H2-K^b peptides generated from a list of 8- to 11-mer peptides using the GibbsCluster 2.0 algorithm as above. The canonical binding motifs were observed for all 3 tissues for both H2-K^d and H2-K^b. (H) The extent of peptide sharing between the H2-K^d repertoires of hepatocytes, spleen, and skin was substantially reduced when Tap1KOHep, Tap1^fl/fl, or C57BL6 mice inoculated with AAV-HC-K^d-YCAC were substituted for C57BL/6 transduced with AAV-HC-K^d. This reduction was particularly striking for the combination of Tap1KOHep with AAV-HC-K^d-YCAC.