(A–D) Expression of K^b on liver leukocytes and splenocytes was equivalent between Tap1KOHep and Tap1^fl/fl mice, while much lower levels were detected on hepatocytes from Tap1KOHep. Representative flow plots from n = 6. (C) Tap1KOHep and Tap1^fl/fl livers are morphologically normal. IHC staining demonstrates expression of K^b and D^b in nonparenchymal cells from Tap1KOHep livers, while K^b and D^b are absent from hepatocytes in these mice (representative images, n = 3). (D) Thick sections (150 μm) from Tap1KOHep or Tap1^fl/fl livers were stained with antibodies against H-2K^b, CD31, CK19, and CD45. Confocal micrographs were obtained. K^b is ubiquitously expressed in Tap1^fl/fl, but absent from hepatocytes in Tap1KOHep (representative images, n = 3). (E and F) While H-2K^d was clearly present on hepatocytes from Tap1KOHep transduced with AAV-HC-K^d, expression did not reach that in TAP-sufficient mice transduced with the same vector. To achieve robust cell surface expression of K^d, we designed a construct where point mutations (Y84C and A139C; YCAC) stabilize expression of K^d when empty or loaded with low-affinity peptides. Inoculation of Tap1KOHep mice with AAV-HC-K^d-YCAC yielded comparable expression to that of TAP-sufficient mice transduced with AAV-HC-K^d. Representative flow plots from n = 3. (G) Thick sections of Tap1KO/YCAC and Tap1^fl/fl/YCAC livers were stained with antibodies against H-2K^d, CD31, CK19, and CD45. Equivalent strong expression of K^d in hepatocytes, but not other cell types, was observed for both treatment groups (representative of n = 3). Scale bars: 100 μm (C and E), 40 μm (D and G). In B and F, mean ± SEM are shown. Statistical analysis comprised unpaired Student’s t test: ***P < 0.001, ****P < 0.0001.