Pigs with deletion of 3 carbohydrate xenoantigens (triple knock-out, TKO) are expected to be optimal donors for human xenotransplantation. We hypothesized that concomitantly inserted human transgenes (hTGs) are important to attenuate anti-xenograft immune responses.  In the current study, kidney xenotransplants from four TKO pig lines with different hTGs as well as TKO without hTGs were evaluated.

Nineteen cynomolgus monkeys received kidneys from four different TKO pig lines (TKO-A to D) with various expression of human immune regulatory proteins (ImmuRPs), complement regulatory proteins (CompRPs) and coagulation regulatory proteins (CoagRPs) or no hTGs. Recipients were treated with anti-thymocyte globulin (ATG) and rituximab induction followed by anti-CD154 antibody (every 1-2 weeks) and daily mycophenolate mofetil (MMF). Prednisone and either rapamycin or tacrolimus were also administered for the first two months. 

Two recipients of TKO-A, which expressed higher ImmuRP with lower CompRPs, survived for 2 and 61 days, while recipients of TKO-B with high CompRPs and lower ImmuRPs survived for 15, 20, 71, 135, 265 and 316 days (Table 2). 15 NHPs received xenografts from TKO-C with CompRP, ImmuRP, CoagRPs TM/EPCR, and with or without endogenous retrovirus inactivation (RI). Ongoing recipients (7) show no signs of rejection or thrombotic microangiopathy (TMA), currently at days >489, >482, >292, >160, >104, and >48, treated with only anti-CD154 mAb and MMF after 2 months. Rejection and TMA appeared to contribute to graft loss in the remaining 8 recipients between 8 and 240 days following transplant. Both recipients of TKO-D, in which CoagRPs TM and TFPI were present, survived for 243 and 267 days without rejection or TMA but were euthanized due to infectious complications. Finally, both recipients of TKO without hTG lost their xenografts early on day 4 and 50, due to severe tubular injury and significant proteinuria (final pathologic diagnosis pending but AMR is suspected) respectively.

Prolonged (>1 year) rejection and TMA-free survival of kidney xenografts with TKO and multiple hTGs have been achieved.  Whether the hTGs are essential for long-term xenograft survival remains to be determined with more control animals without hTGs. Our preliminary results that two recipients of TKO without hTG lost their xenografts early, suggest an essential role of hTGs for long-term xenograft survival.