Chronic graft-specific cell-mediated immune response toward candidate xenogeneic biomaterial
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Despite rabbits becoming an increasingly popular animal model, a flow cytometry panel that combines T cell markers (CD3, CD4, CD8, CD25, FOXP3) with a method for monitoring proliferation is lacking in this species. It has been shown that the rabbit model can be used to identify xenoantigens within bovine pericardium (BP), a common biological heart valve replacement material; however, these methods rely on monitoring the humoral immune response. The development of a rabbit T cell proliferation assay has utility in monitoring graft-specific cell-mediated immune responses toward bovine pericardium. Isolation and culture conditions were optimized to avoid cell death, red blood cell contamination, and non-specific proliferation. Effect of cell culture and stimulation on distribution and intensity of T cell markers was analyzed and compared between cells isolated from naïve and BP-immunized rabbits. Submaximal levels (0.25 μg/mL) of concavalin A were used to stimulate proliferation toward BP extract, with resultant proliferation compared between naïve and BP-immunized rabbits. Density stratification followed by ammonium potassium chloride (ACK) lysis yielded the greatest number of viable peripheral blood mononuclear cells with the least amount of erythrocyte contamination. Flat-bottomed plates were necessary to reduce non-specific proliferation in culture. T cells responded appropriately to maximal mitogenic stimulation (5 μg/mL concavalin A). Interestingly, immunization increased the intensity of FOXP3 in T regulatory cells compared to cells from naïve animals. With addition of submaximal levels of concavalin A, T cells from immunized rabbits proliferated in response to BP protein extract, while cells from naïve rabbits did not. In immunized rabbits, not only did more CD4+ T cells proliferate in response to BP re-stimulation, but the intensity of CD25 was increased indicating cell activation. This research provides a functional cell-mediated screening assay for assessment of BP-based biomaterials in rabbits, overcoming the limitations of previous humoral immune system-based assessments of biomaterial antigenicity in this important experimental animal species.
KeywordsRabbit T lymphocytes T regulatory cells Proliferation Bovine pericardium
Research supported by grant number R01HL115205 from the National Heart Lung and Blood Institute (NHLBI) at the National Institutes of Health (NIH) and grant number 5T32OD010931-10 from the NIH.
KYG and LGG were equally involved in the design and conception of the research, although KYG predominantly performed the collection and analysis of data. KYG contributed to the drafting and refinement of the manuscript with the assistance and oversight of LGG, who gave final approval of the version to be published.
Compliance with ethical standards
All animal procedures were conducted in accordance with the guidelines established by University of California, Davis IACUC, Mayo Clinic IACUC and the Guide for the Care and Use of Laboratory Animals.
Conflict of interest
The authors declare that they have no conflict of interest.
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