Background.CD56+human natural killer (NK) cells are the principal anti-pig cytotoxic effectors in vitro. Expression of certain human leukocyte antigen (HLA) class I molecules in porcine cells can inhibit NK cell-mediated natural cytotoxicity in serum-free medium, but had not been shown to inhibit antibody-dependent cellular cytotoxicity (ADCC) by CD16+NK cells in the presence of human xenoreactive immunoglobulin G. Moreover, expression of HLA molecules might amplify the previously weak CD8+cytotoxic T-lymphocyte (CTL) response against porcine cells.Methods.A novel porcine B-lymphoblastoid cell line (13271) was stably transfected withHLA-Cw*0304gene constructs encoding wild-type (wt) Cw3 or genetically modified Cw3 unable to interact with CD8 (Cw3-D227K). The Cw3 transfectants were used in limiting dilution assays to estimate the CTL precursor frequency in CD56-depleted human peripheral blood mononuclear cells (PBMC) obtained from eight unrelated donors. The 13271 transfectants were also used as targets for clonal and polyclonal NK cells in the presence and absence of human serum, to measure inhibition of ADCC.Results.Expression of Cw3-wt in 13271 cells significantly increased the human CTL response compared with the empty-vector control transfectant, whereas no significant increase resulted from expression of CD8-interaction mutant Cw3-D227K molecules. The Cw3-D227K mutant was indistinguishable from Cw3-wt in its ability to inhibit both natural cytotoxicity and ADCC mediated by human NK clones that have the appropriate CD158b inhibitory receptor.Conclusions.Transgenic expression of HLA molecules in pig cells will likely amplify the CD8+CTL response against the xenograft. Disruption of HLA-CD8 interaction could minimize this amplification without compromising NK-cell inhibition.