Human dominant‐negative class II transactivator transgenic pigs – effect on the human anti‐pig T‐cell immune response and immune status

Abstract

SummarySwine leucocyte antigen (SLA) class II molecules on porcine (p) cells play a crucial role in xenotransplantation as activators of recipient human CD4 + T cells. A human dominant-negative mutant class II transactivator (CIITA-DN) transgene under a CAG promoter with an endotheliumspecific Tie2 enhancer was constructed. CIITA-DN transgenic pigs were produced by nuclear transfer/embryo transfer. CIITA-DN pig cells were evaluated for expression of SLA class II with/without activation, and the human CD4 + T-cell response to cells from CIITA-DN and wild-type (WT) pigs was compared. Lymphocyte subset numbers and T-cell function in CIITA-DN pigs were compared with those in WT pigs. The expression of SLA class II on antigen-presenting cells from CIITA-DN pigs was significantly reduced (40-50% reduction compared with WT; P < 0Á01), and was completely suppressed on aortic endothelial cells (AECs) even after activation (100% suppression; P < 0Á01). The human CD4 + T-cell response to CIITA-DN pAECs was significantly weaker than to WT pAECs (60-80% suppression; P < 0Á01). Although there was a significantly lower frequency of CD4 + cells in the PBMCs from CIITA-DN (20%) than from WT (30%) pigs (P < 0Á01), T-cell proliferation was similar, suggesting no significant immunological compromise. Organs and cells from CIITA-DN pigs should be partially protected from the human cellular immune response.

In vitro testing of an anti-CD40 monoclonal antibody, clone 2C10, in primates and pigs

“…The sub-confluent pAECs were activated for 72h by co-culture with recombinant pIFN-γ (50ng/mL, R&D Systems, Minneapolis, MN). Activation of the cells was evaluated by staining with swine leukocyte antigen (SLA) class I (mouse anti-pig SLA class I, clone JM1E3, Serotec, Raleigh, NC) and SLA class II (DR) (mouse anti-pig SLA class II, clone 2E9/13, BD Biosciences, San Jose, CA) using flow cytometry [38]. …”

“…The binding of anti-CD40mAb to CD21 + B cells in PBMCs was measured by LSR II flow cytometry (BD), and analyzed by FlowJo software (Treestar. Ashland, OR) [38]. …”

“…To investigate the potency of 2C10R4 and belatacept by suppression of the proliferation of PBMCs, primate and pig PBMCs were stimulated with phytohemagglutinin (PHA, Roche, Basel, Switzerland) as previously described [38]. Responder PBMCs (1×10 5 /well) from primates and pig were cultured with 10µg/ml PHA with/without 2C10R4 and/or belatacept in AIM V culture media, and incubated at 37°C in 5% CO 2 for 3 days.…”

Bringing Home The Bacon: Update on The State of Kidney Xenotransplantation

“…The T cell response may also be reduced by genetic engineering of the pig, for example, by the introduction of a mutant gene that reduces MHC class II (swine leukocyte antigen [SLA] class II) expression [14], or by knockout of MHC class I (SLA class I) [15]. The transgenic expression of a co-stimulation blockade agent, for example, CTLA4-Ig, to provide local suppression of the T cell response, has also proved possible [6].…”

Synthetic Biology in Cell and Organ Transplantation

“…However, this may have the unintended effect of enhancing the display of the immunogenic donor peptides by the modified donor cells, actually increasing host T-cell activity against the donor tissue. A complementary approach uses genome engineering to eliminate or reduce the expression of donor HLA and preventing antigen presentation (Hara et al 2013). Although this approach reduces immunogenicity of donor cells, it increases the risk of infection and leads to "missing self " recognition by NK cells (van Bergen et al 2009).…”