title: Granzyme B secretion by human memory CD4 T cells is less strictly regulated compared to memory CD8 T cells.
abstract: 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 1x10(5) 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 (~170/1x10(5)) and memory CD8 (~200/1x10(5)) 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.
authors.forename: Lin
authors.initials: L
authors.lastname: Lin
authors.forename: Jacob
authors.initials: J
authors.lastname: Couturier
authors.forename: Xiaoying
authors.initials: X
authors.lastname: Yu
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Medina
authors.forename: Claudia A
authors.initials: CA
authors.lastname: Kozinetz
authors.affiliation: Division of Infectious Diseases, Department of Internal Medicine, University of Texas Health Science Center at Houston, 6431 Fannin St,, MSB 2,112, Houston 77030, TX, USA. Dorothy.E.Lewis@uth.tmc.edu.
authors.forename: Dorothy E
authors.initials: DE
authors.lastname: Lewis
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D001782:Blood Donors; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D016207:Cytokines / Q000378:metabolism; D057075:Enzyme Assays; D053804:Granzymes / Q000378:metabolism*; D006801:Humans; D007156:Immunologic Memory; D007694:Killer Cells, Natural / Q000276:immunology; D052118:Lysosomal-Associated Membrane Protein 1 / Q000378:metabolism; D054353:Perforin / Q000378:metabolism; D050378:T-Lymphocytes, Regulatory / Q000276:immunology; D018417:Th1 Cells / Q000276:immunology
chemical_list: D015415:Biomarkers; D016207:Cytokines; D052118:Lysosomal-Associated Membrane Protein 1; D054353:Perforin; D053804:Granzymes