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

Lin L, Couturier J, Yu X, Medina MA, Kozinetz CA, Lewis DE - BMC Immunol. (2014)

Bottom Line: 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.Secretion of GrzB by activated CD8 T cells may be more tightly controlled compared to CD4 T cells.

View Article: PubMed Central - PubMed

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.

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.

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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).
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Fig1: 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).

Mentions: Measurement of intracellular GrzB by flow cytometry showed that resting and activated memory CD8 T cells stored significantly more GrzB compared to memory CD4 T cells (Figure 1A). Resting memory CD8 T cells expressed 25.2 ± 4.7% intracellular GrzB protein, which did not significantly increase after 24 hrs costimulation to 29.0 ± 3.3% (p = 0.38, n = 10). Resting memory CD4 T cells expressed little to no intracellular GrzB (2.8 ± 0.5%), but GrzB expression was significantly increased by costimulation to 8.9 ± 1.9% (p = 0.0002, n = 13). Additionally, resting and activated memory CD8 T cells expressed significantly more intracellular GrzB compared to resting and activated memory CD4 T cells (p = 0.002). These data corroborate previous reports of intracellular GrzB protein measurements by flow cytometry or western blot showing higher expression by CD8 compared to CD4 T cells [11-15]. However, unlike memory CD8 T cells, GrzB synthesis is actually upregulated by memory CD4 T cells during T cell activation. These data suggest that activated memory CD8 T cells secrete pre-synthesized GrzB (stored in lysosomes) directly from granules, whereas activated memory CD4 T cells secrete pre-stored and newly synthesized GrzB. This GrzB synthesis step by memory CD4 T cells may also be consistent with a previously reported observation of a less rapid, but ultimately comparable, CTL activity (target cell lysis in a 6 hrs assay) by antigen-specific CD4 T cells compared to CD8 T cells in a murine model of LCMV infection [14].Figure 1


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

Lin L, Couturier J, Yu X, Medina MA, Kozinetz CA, Lewis DE - BMC Immunol. (2014)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4195902&req=5

Fig1: 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).
Mentions: Measurement of intracellular GrzB by flow cytometry showed that resting and activated memory CD8 T cells stored significantly more GrzB compared to memory CD4 T cells (Figure 1A). Resting memory CD8 T cells expressed 25.2 ± 4.7% intracellular GrzB protein, which did not significantly increase after 24 hrs costimulation to 29.0 ± 3.3% (p = 0.38, n = 10). Resting memory CD4 T cells expressed little to no intracellular GrzB (2.8 ± 0.5%), but GrzB expression was significantly increased by costimulation to 8.9 ± 1.9% (p = 0.0002, n = 13). Additionally, resting and activated memory CD8 T cells expressed significantly more intracellular GrzB compared to resting and activated memory CD4 T cells (p = 0.002). These data corroborate previous reports of intracellular GrzB protein measurements by flow cytometry or western blot showing higher expression by CD8 compared to CD4 T cells [11-15]. However, unlike memory CD8 T cells, GrzB synthesis is actually upregulated by memory CD4 T cells during T cell activation. These data suggest that activated memory CD8 T cells secrete pre-synthesized GrzB (stored in lysosomes) directly from granules, whereas activated memory CD4 T cells secrete pre-stored and newly synthesized GrzB. This GrzB synthesis step by memory CD4 T cells may also be consistent with a previously reported observation of a less rapid, but ultimately comparable, CTL activity (target cell lysis in a 6 hrs assay) by antigen-specific CD4 T cells compared to CD8 T cells in a murine model of LCMV infection [14].Figure 1

Bottom Line: 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.Secretion of GrzB by activated CD8 T cells may be more tightly controlled compared to CD4 T cells.

View Article: PubMed Central - PubMed

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.

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.

Show MeSH
Related in: MedlinePlus