PHENOTYPIC CHARACTERISATION OF PERIPHERAL BLOOD CYTOTOXIC T LYMPHOCYTES: REGULATORY AND EFFECTOR MOLECULES

Abstract

Cytotoxic T lymphocytes (CD3+CD8+, Tcyt) play a major role in protective immunity against intracellular pathogens and can eradicate malignant cells. As based on CD45RA and CD62L expression, the peripheral CD3+CD8+ blood lymphocytes were divided into "na ve" (N) cells, central memory (CM) and effector memory (EM), as well as "terminally-differentiated" CD45RA-positive effector cells (TEMRA). Using multicolor flow cytometry, a co-expression of effector (perforin, granzyme B and CD57) and regulatory (CD27, CD28, CD244 (2B4), CD279 (PD-1) and KLRG1) molecules was studied on all these subsets. CD57 was expressed in 2.39±0.31% “na ve” and 5.45±0.91% of central memory Tcyt. Meanwhile, within EM and TEMRA Tcyt subset, its expression was identified on the cell membranes of 26.53±2.20% and 51.43±2.55% of cells, respectively. Cytolytic effector molecules (granzyme B and perforin) were detected in cytoplasmic granules of 4.22±0.36% and 5.30±0.43% of na ve Tcyt, respectively. For CM cells, these values were 10.09±1.17% and 24.90±3.10%, respectively. Dramatic increases of granzyme B and perforin expression were observed in the “EM → TEMRA” cell lineage, when the relative number of granzyme B-positive cells increased to 41.05±2.63% and 66.73±3.29%, respectively, while perforin was detected in 59.33±4.26% and 75.08±3.12% of cells, respectively. For regulatory molecules, CD244 and KLRG1, the similar dynamics were observed, their expression increased from “na ve” to late maturation stages, while the expression of two main costimulatory molecules – CD27 and CD28, decreased in the lineage N → CM → EM → TEMRA cells. The highest level of CD279 was observed in EM cells. It was shown that CD57-positive cells contain perforin and granzyme B in their cytoplasmic granules and lack CD28 expression. Furthermore, CD57 can be used as a surrogate marker for multicolor immunophenotyping to identify most mature effector cells containing cytolytic enzymes. Our results on the co-expression of all the beforementioned molecules suggest that the most mature CD45RA+CD62L– effector peripheral blood cytotoxic T cells express CD244 and CD57, lack costimulation molecules CD27 and of CD28, as well as inhibitory receptors KLRG1 and CD279.

Keywords

• flow cytometry
• multicolor immunophenotyping
• cytotoxic t cell subsets
• cd3+cd8+ maturation
• effector molecules
• inhibitory receptors