Phenotypic characterization of bovine memory cells responding to mycobacteria in IFNγ enzyme linked immunospot assays

Bovine tuberculosis (bTB) remains a globally significant veterinary health problem. Defining correlates of protection can accelerate the development of novel vaccines against TB. As the cultured IFNγ ELISPOT (cELISPOT) assay has been shown to predict protection and duration of immunity in vaccinated cattle, we sought to characterize the phenotype of the responding T-cells. Using expression of CD45RO and CD62L we purified by cytometric cell sorting four distinct CD4⁺ populations: CD45RO⁺CD62L^hi, CD45RO⁺CD62L^lo, CD45RO^-CD62L^hi and CD45RO^-CD62L^lo (although due to low and inconsistent cell recovery, this population was not considered further in this study), in BCG vaccinated and Mycobacterium bovis infected cattle. These populations were then tested in the cELISPOT assay. The main populations contributing to production of IFNγ in the cELISPOT were of the CD45RO⁺CD62L^hi and CD45RO⁺CD62L^lo phenotypes. These cell populations have been described in other species as central and effector memory cells, respectively. Following in vitro culture and flow cytometry we observed plasticity within the bovine CD4⁺ T-cell phenotype. Populations switched phenotype, increasing or decreasing expression of CD45RO and CD62L within 24h of in vitro stimulation. After 14 days all IFNγ producing CD4⁺ T cells expressed CD45RO regardless of the original phenotype of the sorted population. No differences were detected in behavior of cells derived from BCG-vaccinated animals compared to cells derived from naturally infected animals. In conclusion, although multiple populations of CD4⁺ T memory cells from both BCG vaccinated and M. bovis infected animals contributed to cELISPOT responses, the dominant contributing population consists of central-memory-like T cells (CD45RO⁺CD62L^hi).