Granzyme B secretion by human memory CD4 T cells is less strictly regulated compared to memory CD8 T cells

Background: Granzyme B (GrzB) is a serine proteinase expressed by memory T cells and NK cells. Methods to measure GrzB protein usually involve intracellular (flow cytometry) and extracellular (ELISA and ELISpot) assays. CD8 T cells are the main source of GrzB during immunological reactions, but activated CD4 T cells deploy GrzB as well. Because GrzB is an important mediator of cell death, tissue pathology and disease, clarification of differences of GrzB expression and secretion between CD4 and CD8 T cells is important for understanding effector functions of these cells. Results: Memory CD4 and memory CD8 T cells were purified from human peripheral blood of healthy donors, and production of GrzB was directly compared between memory CD4 and memory CD8 T cells from the same donors using parallel measurements of flow cytometry (intracellular GrzB), ELISpot (single cell secretion of GrzB), and ELISA (bulk extracellular GrzB). Memory CD8 T cells constitutively stored significantly more GrzB protein (~25%) compared to memory CD4 T cells as determined by flow cytometry (~3%), and this difference remained stable after 24 hrs of activation. However, measurement of extracellular GrzB by ELISA revealed that activated memory CD4 T cells secrete similar amounts of GrzB (~1,000 pg/ml by 1x10⁵ cells/200 μl medium) compared to memory CD8 T cells (~600 pg/ml). Measurement of individual GrzB-secreting cells by ELISpot also indicated that similar numbers of activated memory CD4 (~170/1x10⁵) and memory CD8 (~200/1x10⁵) T cells secreted GrzB. Expression of CD107a further indicated that Grzb is secreted similarly by activated CD4 and CD8 T cells, consistent with the ELISA and ELISpot results. However, memory CD8 T cells expressed and secreted more perforin compared to memory CD4 T cells, suggesting that perforin may be less associated with GrzB function for memory CD4 T cells. Conclusions: Although measurement of intracellular GrzB by flow cytometry suggests that a larger proportion of CD8 T cells have higher capacity for GrzB production compared to CD4 T cells, ELISpot and ELISA show that similar numbers of activated CD4 and CD8 T cells secrete similar amounts of GrzB. Secretion of GrzB by activated CD8 T cells may be more tightly controlled compared to CD4 T cells.