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.

Figure 1

Comparison of intracellular and extracellular GrzB protein levels by memory CD4 and memory CD8 T cells. (A) Representative flow cytometry dotplots and mean ± sem intracellular GrzB protein expression by 1×10⁵ untreated (UT) or activated (CD3/CD28 costimulation) memory T cells after 24 hrs culture (n = 10-13). (B) Representative ELISpot wells and mean ± sem GrzB spots (per 1×10⁵ cells) of untreated or activated (CD3/CD28 costimulation) memory T cells after 24 hrs culture (n = 10-13). (C) Extracellular GrzB production (ELISA) by 1×10⁵ untreated or activated (CD3/CD28 costimulation) memory T cells after 24 hrs culture (mean ± sem, n = 10-13).

Figure 2

Inter-assay correlations between intracellular and extracellular GrzB assays of memory T cells, and intra-donor correlations of GrzB production between memory T cells. (A-F) Spearman correlations between intracellular (flow cytometry) and extracellular (ELISA and ELISpot) GrzB measurements of 1×10⁵ untreated (UT) or activated (CD3/CD28 costimulation) memory CD4 (A-C) or memory CD8 (D-F) T cells (n = 20-26). (G-I) Spearman correlations comparing GrzB production (costimulation values) of memory CD4 vs. memory CD8 T cells within single donors as measured by flow cytometry, ELISpot, or ELISA (n = 9-10).

Figure 3

Association of memory T cell GrzB secretion with activation and degranulation markers. (A-D) Memory T cell flow cytometry dotplots and mean ± sem (n = 16-17) total surface CD69 expression or CD69+/GrzB+ coexpression (gated on total memory CD4 or memory CD8 T cells), or CD69 expression gated on GrzB+ memory CD4 or memory CD8 T cells. (E-H) Flow cytometry dotplots and mean ± sem (n = 6) total surface CD107a expression or CD107a+/GrzB+ coexpression (gated on total memory CD4 or memory CD8 T cells), or CD107a expression gated on GrzB+ memory CD4 or memory CD8 T cells. (I) GrzB ELISA of memory T cells conducted in parallel with CD107a experiments. (J-K) Spearman correlations between CD107a and intracellular (flow cytometry) or extracellular (ELISA) GrzB of memory T cells (n = 8-12).

Figure 4

Comparison of GrzB and perforin expression between memory CD4, memory CD8, and natural killer cells. (A) Representative GrzB/perforin dotplots of memory CD4, memory CD8, and NK cells. Cells were purified from peripheral blood, and 5×10⁵ cells (in 1 ml medium) were untreated (UT) or stimulated with IL2 (100 ng/ml) or anti-CD3 only mabs (1 μg/ml) for 24 hrs +/- brefeldin. (B) Mean (n = 4) distribution of perforin/GrzB subsets of memory CD4, memory CD8, and NK cells. (C-D) Extracellular GrzB and perforin production by memory CD4, memory CD8, and NK cells after 24 hrs culture +/- brefeldin (mean ± sem, n = 3-4, ^ap < 0.05 comparing UT NK cells to UT memory CD4 and memory CD8 T cells, ^bp < 0.05 comparing IL2-treated NK cells to IL2-treated memory CD4 and memory CD8 T cells, ^cp < 0.05 comparing UT or IL2-treated NK cells to UT or IL2-treated memory CD4 T cells).

Figure 5

GrzB production by memory CD4 T cell subsets and coexpression with cytokines. (A-B) GrzB expression by Tregs. Memory CD4 T cells were purified from peripheral blood, and 5×10⁵ cells (in 1ml medium) were untreated (UT) or activated (CD3/CD28 costimulation) for 24 hrs. Cells were then stained for CD25, Foxp3, and GrzB. (A) CD25 expression levels (Negative, Dim, or Bright) by memory CD4 T cells after 24 hrs culture (mean ± sem, n = 3). (B) Foxp3/GrzB expression by CD25-Negative, CD25-Dim, or CD25-Bright memory CD4 T cells after 24 hrs culture. Graph shows the mean (n = 3) distribution of Foxp3/GrzB subsets gated on CD25-Negative, -Dim, or -Bright cells. (C) GrzB coexpression with IFNγ, IL4, or IL17A by memory CD4 T cells. Memory CD4 T cells (5×10⁵ cells in 1ml medium) were untreated for activated (CD3/CD28 costimulation or PMA/IO in the presence of brefeldin) for 24 hrs, then stained for GrzB and either IFNγ, IL4, or IL17A. Graphs show the mean (n = 3) distribution of cytokine/GrzB subsets. (D) GrzB/IFNγ coexpression by memory CD8 T cells. Purified memory CD8 T cells (5×10⁵ cells in 1 ml medium) were untreated or activated (CD3/CD28 costimulation or PMA/IO in the presence of brefeldin) for 24 hrs. Graph shows the mean (n = 3) distribution of IFNγ/GrzB subsets.