Determination of Absolute Counts of Circulating Regulatory T Cells in Cynomolgus Macaques Using an Optimized Flow Cytometric Method

Abstract

Regulatory T cells (Tregs) are a rare subset of lymphocytes that inhibit the activation and effector functions of T cells and are important regulators of immune responses. Although Tregs are well characterized in humans and rodents, little is known about their immunophenotyping (IP) profile in cynomolgus macaques (Macaca fascicularis), which is an important species for pharmacological and toxicological evaluation of potential immune modulators because of their similar physiologic, genetic, and metabolic response patterns to humans. The authors have developed an immunophenotyping panel using a high-throughput 96-well microtiter plate-based assay to detect circulating Tregs (and have determined the normal range for the number of Tregs in naive healthy cynomolgus macaques to be 56.4 to 179.7 cells/mL (mean + SEM ¼ 113.6 + 5.1 cells/mL; n ¼ 25). Furthermore, the authors compared the resulting FoxP3 þ Treg profiles with a CD127 lo cell-surface panel (CD3) and found a close correlation between the absolute numbers of CD3hi CD127 lo cells (mean + SD ¼ 120 + 8.0 cells/mL). Quantification of circulating Tregs in cynomolgus macaques in this high-throughput assay may help to identify drug candidates that affect this rare, but critical, immunoregulatory cell population.

Immunophenotyping: Application to Safety Assessment

“…in human and cynomolgus macaques (Liu et al 2006;Seddiki et al 2006;Clark, Narayanan, and Fort 2012). CD4 þ and CD8 þ T cells are separated into naive and various memory subsets with additional cellular markers.…”

“…Treg is highly dependent on cytokine IL-2 for development, maintenance, and function and Foxp3 for function (Plitas and Rudensky 2016). In addition, Treg cells express low level of CD127 (IL-7R α) and are defined as CD3 + /CD4 + /CD25 + /CD127 low /Foxp3 + in human and cynomolgus macaques (Liu et al 2006; Seddiki et al 2006; Clark, Narayanan, and Fort 2012). CD4 + and CD8 + T cells are separated into naive and various memory subsets with additional cellular markers.…”