Identification of Immunophenotyping Biomarkers and Characterization of Peripheral Blood Lymphocytes Populations Distribution in Minipigs


Zhong, M., Shoemake, C., White, D., Brocksmith, D., Liu, J., Bouchard, G.F., Stricker-Krongrad, A.


Background: Swine are one of the major animal species in which a number of human diseases
and surgical procedures have been modeled1,2. There are also a number of functional similarities
between immune systems of swine and human. The two species share many pathogens as well
as the genes and pathways that are involved in general immune responses3. The minipig breeds
most commonly used in research are Hanford, Yucatan, Sinclair, and Gottingen1,4,5. Minipigs
have been used as a species of increasing importance in preclinical safety testing4,5,6. Preclinical
toxicology studies include parameters to monitor the potential adverse effects of test articles on
the immune system, yet they provide limited information about the mechanisms involved in the
generation of these adverse effects. Lymphocyte immunophenotyping has been shown to be one
of the most predictive assays for detecting immunotoxicity of various products7.

Purpose: To develop a panel of flow cytometry biomarkers to characterize the major populations
of porcine peripheral blood mononuclear cells (PBMCs).

Methods: Whole blood samples were collected in sodium citrate tubes from 40 Hanford, Sinclair,
Yucatan, and 49 Gottingen minipigs (both genders for all four breeds). PBMCs were prepared
with gradient centrifugation with two volumes of Ficoll-Paque Plus (d=1.077, GE Healthcare).
Live cell staining were performed on ice in PBS (Gibco) supplemented with citrate-dextrose
solution (Sigma) and 2% of horse serum (Gibco). Dead cells were identified with propidium iodide
staining solution (BD biosciences). Stained PBMCs were analyzed with Accuri C6 flow cytometer
(BD biosciences).

Results: T lymphocyte marker (CD3ε) identified PBMCs other than myeloid cells (CD172a+) and
B lymphocytes (CD21+/B subset+). Natural killer (NK) cell marker CD8 separated CD8+ PBMCs
from CD172a+ cells and B lymphocytes. A number of PBMCs were CD172a+CD21+/B subset+,
which were distinguished by CD16. The CD16 antibody stained most of CD172a+ PBMCs but
not those positive for CD21/B subset. CD16 was expressed in a fraction of CD3ε+ PBMCs, and
was used to characterize NKT cells. Two populations of T lymphocytes express αβ or γδ T cell
receptors (TcRs), which were distinguished from each other with an antibody for TcR δ chain. The
current immunostaining procedure was shown to yield repeatable immunophenotyping data with
PBMC samples containing 0.3-1.0 million cells (CV% <15%). There were a number of common
features associated with distributions of the major PBMCs of four breeds of minipigs (Hanford,
Yucatan, Sinclair and Gottingen). On the other hand, a number of variations were also identified.
Taking everything in account, Hanford minipigs had fewer differences in different PBMCs from
three other breeds of minipigs.

Conclusion: A novel flow cytometry approach has been developed to characterize the major
porcine PBMCs. This assay will facilitate immune investigations during non-clinical pharmacology
and toxicology studies.

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