Efficient generation of B2m-null pigs via injection of zygote with TALENs

Abstract

Donor major histocompatibility complex class I (MHC I) molecules are the main targets of the host immune response after organ allotransplantation. Whether and how MHC I-deficiency of pig donor tissues affects rejection after xenotransplantation has not been assessed. Beta2-microglobulin (B2M) is indispensable for the assembly of MHC I receptors and therefore provides an effective target to disrupt cell surface MHC I expression. Here, we report the one-step generation of mutant pigs with targeted disruptions in B2m by injection of porcine zygotes with B2m exon 2-specific TALENs. After germline transmission of mutant B2m alleles, we obtained F1 pigs with biallelic B2m frameshift mutations. F1 pigs lacked detectable B2M expression in tissues derived from the three germ layers, and their lymphocytes were devoid of MHC I surface receptors. Skin grafts from B2M deficient pigs exhibited remarkably prolonged survival on xenogeneic wounds compared to tissues of non-mutant littermates. Mutant founder pigs with bi-allelic disruption in B2m and B2M deficient F1 offspring did not display visible abnormalities, suggesting that pigs are tolerant to B2M deficiency. In summary, we show the efficient generation of pigs with germline mutations in B2m, and demonstrate a beneficial effect of donor MHC I-deficiency on xenotransplantation.

Triple (GGTA1, CMAH, B2M) modified pigs expressing an SLA class Ilow phenotype—Effects on immune status and susceptibility to human immune responses

“…They are composed of a heavy α‐chain, noncovalently bound to the light chain, β 2 ‐microglobulin (B2M), and the presented peptide. Because B2M is required for expression of the MHC class I α‐chain, targeting of B2M is an effective approach to induce the absence of MHC class I …”

“…SLA class I–deficient pigs have already been generated, either by targeting directly the genes encoding for the heavy α‐chain or by knocking out B2M . Phenotypic studies on the consequences of B2M/SLA class I α‐chain targeting in pigs have already been described.…”

Role of human and porcine MHC DRB1 alleles in determining the intensity of individual human anti‐pig T‐cell responses

“…Furthermore, cells and tissues from pigs lacking SLA‐DRB1*06 alleles may be favored because of a lower stimulatory capacity for human T cells. Complete elimination of SLA class‐I and/or class‐II molecules in the pig would also reduce the immunogenicity of a xenograft . Because of the central significance of MHC molecules for the immune system and thus for survival, complete absence in the pig may not be desirable.…”

“…SLA molecules are not only activating ligands for human T cells, but also targets for cross‐reacting anti‐HLA antibodies which are present in sensitized individuals . Thus, genetically engineered deletion or modification of SLA molecules may be a strategy to decrease the antigenicity of a porcine xenograft . Because of the central role of MHC molecules for the immune system, complete absence of SLA molecules may be associated with major problems in pig breeding and maintenance.…”

Possible detrimental effects of beta‐2‐microglobulin knockout in pigs

“…Especially, the recognition of porcine MHC class I, also called swine leukocyte antigen (SLA) class I, by recipient T cells and NK cells leads to cell infiltration and cell lysis . Masking or removing SLA class I results in less lysis and prolonged graft survival . Patients sensitized against human MHC class I are thought to benefit from reduced SLA class I levels because of the potential cross‐reactivity with human anti‐MHC class I‐antibodies …”