Purpose: Miniature swine is a pre-clinical large animal model well suited to validate protocols of tolerance induction to vascularized composite allotransplantation (VCA). However, tools to assess the modulation of alloreactivity are limited. We present an optimized assay to assess to quantitate the function of porcine alloreactive lymphocytes.

Methods: Heterotopic hind limb transplantation was performed across full swine leukocyte antigen mismatch (KK to GG) in two MGH miniature swine. All animals received non-myeloablative conditioning (50 cGy total body and 300 cGy thymic irradiation) and transient immunosuppression. The recipients were diagnosed as clinically tolerant upon assessment of both graft appearance and histological analysis of skin biopsies. Mixed lymphocyte reactions were performed using responder T cells positively selected from PBMCs (recipients, control GG) and stained with CFSE. The negative fraction was irradiated and used as stimulators (donors, KK, DD). Following six days of responder/stimulator co-culture, cells were stained and analyzed via flow cytometry for proliferation of T cell subsets (with live/dead discrimination).

Results: Third party stimulation (DD) induced equivalent proliferation of responder T cells from either recipients (CD4: 27.4 ± 1; CD8: 30 ± 3.9) or controls (CD4: 30 ± 3.4; CD8: 39.3 ± 0.9; p = n.s). Donor specific stimulators induced the proliferation of controls (CD4: 19.2 ± 2.4; CD8: 15.4 ± 1.9) but not the recipients (CD4: 1.7 ± 1.3; CD8: 0.4 ± 0.1; p < .01). Interestingly, the recipients also had a reduced proliferative response to donor strain-matched (KK) stimulators (CD4: 9.1 ± 1.8; CD8: 11.4 ± 3.3) compared to the response observed in controls (CD4: 41.7 ± 2.5; CD8: 26.7 ± 2.5; p < .05).

Conclusions: Taken together these results confirm induction of donor specific tolerance with preservation of an allo-response to third party. Interestingly, the tolerant state extends to other donor SLA-matched swine. Therefore, this optimized assay provides an objective measure of swine alloreactivity that correlates with clinical findings and aids in assessing protocols for induction of VCA tolerance.

CITATION INFORMATION: Walch J, Alonso-Escalante J, Wang H, Oh B, Iglesias M, Lee A, Brandacher G, Raimondi G. Assessing the Induction of Tolerance to Vascularized Composite Allotransplantation in Miniature Swine. Am J Transplant. 2016;16 (suppl 3).

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