TY - JOUR
T1 - Chronic graft-specific cell-mediated immune response toward candidate xenogeneic biomaterial
AU - Gates, Katherine V.
AU - Griffiths, Leigh G.
N1 - Publisher Copyright:
© 2018, Springer Science+Business Media, LLC, part of Springer Nature.
PY - 2018/4/1
Y1 - 2018/4/1
N2 - 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.
AB - 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.
KW - Bovine pericardium
KW - Proliferation
KW - Rabbit
KW - T lymphocytes
KW - T regulatory cells
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U2 - 10.1007/s12026-018-8985-8
DO - 10.1007/s12026-018-8985-8
M3 - Article
C2 - 29446013
AN - SCOPUS:85042131443
SN - 0257-277X
VL - 66
SP - 288
EP - 298
JO - Immunologic Research
JF - Immunologic Research
IS - 2
ER -