Chronic graft-specific cell-mediated immune response toward candidate xenogeneic biomaterial

Katherine V. Gates, Leigh Griffiths

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)288-298
Number of pages11
JournalImmunologic Research
Volume66
Issue number2
DOIs
StatePublished - Apr 1 2018

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Biocompatible Materials
Pericardium
Rabbits
Transplants
T-Lymphocytes
Erythrocytes
Heterophile Antigens
Ammonium Chloride
Potassium Chloride
Heart Valves
Regulatory T-Lymphocytes
Humoral Immunity
Cell Extracts
Immune System
Immunization
Blood Cells
Flow Cytometry
Cell Death
Animal Models
Cell Culture Techniques

Keywords

  • Bovine pericardium
  • Proliferation
  • Rabbit
  • T lymphocytes
  • T regulatory cells

ASJC Scopus subject areas

  • Immunology

Cite this

Chronic graft-specific cell-mediated immune response toward candidate xenogeneic biomaterial. / Gates, Katherine V.; Griffiths, Leigh.

In: Immunologic Research, Vol. 66, No. 2, 01.04.2018, p. 288-298.

Research output: Contribution to journalArticle

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