Introduction: The use of pigs to overcome human organ shortage proved to be a plausible alternative in 2022. However, the modulation of the acquired immune response by engineered pigs needs further research. Our goal was to generate human xenospecific CD8⁺ T cells (CTL) in vitro to analyze their response to porcine aortic endothelial cells (PAEC) from transgenic animals in functional assays.

Methods: Human CD8⁺ T cells were purified from healthy donors’ blood and stimulated weekly with lethally irradiated wild-type (WT) PAEC for 21 days in the presence of autologous dendritic cells (DC). CTL were tested for specificity in non-radioactive DELFIA cytotoxicity assays using primary WT PAEC (from German Landrace pig) and PEDSV.15 cells (from minipigs of dd-haplotype) as targets. Specific cell lysis was analysed in static (2D) co-cultures at 1:1 effector to target (E:T) ratio using live-imaging microscopy by capturing frames during 3.5h. Adhesion of CTL and lysis of pig target cells, distinguishing apoptosis from necrosis, were quantified by manual counting of apoptotic bodies and necrotic Draq7⁺ cells. To further improve in vitro assays and mimicking physiological conditions, WT PAEC were cultured in 3D microfluidic channel and perfused with CTL under pulsatile flow during 2h. Using video time-lapse acquisition, adhesion of CTL and dead WT PAEC were quantified.

Results: The purity of DC as verified by HLA-DR⁺CD11c⁺ phenotyping accounted for 78.52 ± 11.49% of the cells. The purity of CD8⁺ T cells (CD3⁺CD8⁺) was 94.38 ± 3.49%; contaminating cells of < 6% comprised NK, B and myeloid cells. CTL were highly specific for WT PAEC, displaying an effector number-dependent cytotoxicity in DELFIA assay. However, the effect was evident only at high E:T ratios (10:1 to 5:1), whereas there was no difference in killing of WT PAEC and PEDSV.15 at 3:1 ratio. In 2D live imaging, we found that CTL lysed WT PAEC predominantly via apoptotic pathways (11.26 ± 0.55%) compared to necrosis (6.02 ± 1.73%). The opposite was detected for PEDSV.15 targets: 4.95 ± 0.74% died by apoptosis while 8.46 ± 2.11% underwent necrosis. In 3D live imaging CTL adhesion to WT PAEC layers and a gradual increase of Draq7 signal in WT PAEC nuclei (necrosis) was observed over time.

Conclusion: We standardized the protocol to generate human xenospecific CTL in vitro. In addition to conventional assays, interactions between human CTL and porcine endothelial cells were assessed using a more sensitive live imaging method under static and flow conditions. This method allows to quantify and to differentiate the pathways of T cell cytotoxicity against PAEC. Although CTL showed specificity for WT PAEC in conventional assays, at lower and more physiological E:T ratio we were able to detect differences in the pathway of cytotoxicity against the two targets. The observed non-specific killing of PEDSV.15 needs to be further explored as well as the candidate transgenes to attenuate human anti-pig CTL responses.