Molecular immunology profiles of monkeys following xenografting with the islets and heart of α‐1,3‐galactosyltransferase knockout pigs

Abstract

Effective immunosuppression strategies and genetically modified animals have been used to prevent hyperacute and acute xenograft rejection; however, the underlying mechanisms remain unknown. In this study, we evaluated the expression of a comprehensive set of immune system-related genes (89 genes, including five housekeeping genes) in the blood of cynomolgus monkeys (~5 yr old) used as graft recipients, before and after the xenografting of the islets and heart from single and double α-1,3-galactosyltransferase (GalT) knockout (KO) pigs (<6 weeks old). The immunosuppressive regimen included administration of cobra venom factor, anti-thymocyte globulin, rituximab, and anti-CD154 monoclonal antibodies to recipients before and after grafting. Islets were xenografted into the portal vein in type 1 diabetic monkeys, and the heart was xenografted by heterotopic abdominal heart transplantation. Genes from recipient blood were analyzed using RT(2) profiler PCR arrays and the web-based RT(2) profiler PCR array software v.3.5. Recipients treated with immunosuppressive agents without grafting showed significant downregulation of CCL5, CCR4, CCR6, CD4, CD40LG, CXCR3, FASLG, CXCR3, FOXP3, GATA3, IGNG, L10, IL23A, TRAF6, MAPK8, MIF, STAT4, TBX21, TLR3, TLR7, and TYK2 and upregulation of IFNGR1; thus, genes involved in protection against viral and bacterial infection were downregulated, confirming the risk of infection. Notably, C3-level control resulted in xenograft failure within 2 days because of a 7- to 11-fold increase in all xenotransplanted models. Islet grafting using single GalT-KO pigs resulted in upregulation of CXCL10 and MX1, early inflammation, and acute rejection-associated signals at 2 days after xenografting. We observed at least 5-fold upregulation in recipients transplanted with islets grafts from single (MX1) or double (C3, CCR8, IL6, IL13, IRF6, CXCL10, and MX1) GalT-KO pigs after 77 days; single GalT-KO incurred early losses owing to immune attacks. Our results suggest that this novel, simple, non-invasive, and time-efficient procedure (requiring only 1.5 ml blood) for evaluating graft success, minimizing immune rejection, and blocking infection.

Immune molecular profiling of whole blood drawn from a non‐human primate cardiac xenograft model treated with anti‐CD154 monoclonal antibodies

“…This may be because of the relatively low dosage of C3 used. 19 At 2 days after heterotopic cardiac xenograft, ATG did not achieve complete reduction of helper T cells; however, additional treatment with anti-CD154 accelerated the downregulation of helper T cells. Therefore, we observed effective reduction of T cells, resulting in regulation of the costimulation blockade pathway (CD40 and CD40L) using anti-CD154, similar to a previous study.…”

“…CVF functions in complement depletion and is used for inhibition of HAR; however, C3 levels were not appropriately regulated after heterotopic cardiac xenograft transplantation, although no significant increase was observed owing to the limited number of recipients in this study. This may be because of the relatively low dosage of C3 used . At 2 days after heterotopic cardiac xenograft, ATG did not achieve complete reduction of helper T cells; however, additional treatment with anti‐CD154 accelerated the downregulation of helper T cells.…”

“…Activation of T cells and natural killer (NK) cells was also expected owing to upregulation of IL15 and IL18 . In our previous study, we tested the effects of immunosuppressants, including anti‐CD154, in recipients without grafts and found downregulation of the same genes . In particular, recipients treated with anti‐CD154 showed downregulation of IL4 , which regulates the pathway from Th cells to Th2 cells.…”

“…For analysis, a 25‐μL solution containing cDNA, 2× SABiosciences RT 2 qPCR Mastermix, diluted first‐strand cDNA synthesis solution, and RNase‐free water was transferred to each well of an RT 2 Profiler PCR Array Plate (Rhesus Macaque Innate & Adaptive Immune Responses; Qiagen). This plate was used to evaluate genes involved in the host response to bacterial infection and sepsis, including 89 genes and 5 housekeeping genes, ACTB, B2M, GAPDH, LOC709186, and RPL13A . These data were analyzed based on 2 stable genes ( B2M and LOC709186 ) of the 5 included housekeeping genes (Table S1).…”

“…Previously, we reported a new method using circulating mRNAs in peri-operative blood samples from xenograft recipients in an NHP 19 to elucidate the immune molecular profiles. Accordingly, in this study, we analyzed the effects of anti-CD154 mAb treatment on CD40/CD154 signaling by immune molecular profiling to elucidate changes in inflammation or coagulation pathways in heterotopic cardiac xenografts using organs from GalT-KO pigs in an NHP model.…”