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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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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.
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Fig5: 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.

Mentions: 24 hrs costimulation or PMA/IO treatment of memory CD4 T cells resulted in GrzB expression by phenotypes consistent with Treg and Th1 cells (Figure 5A-C). Tregs were examined by measuring intracellular GrzB expression by CD25+/Foxp3+ costimulated memory CD4 T cells. Activated memory CD4 T cells expressed ~18% CD25 (13% CD25-Dim and 4.6% CD25-Bright, Figure 5A). GrzB was coexpressed with Foxp3 by CD25-Dim cells (24.2 ± 3.6% Foxp3+/GrzB-, 4.6 ± 1.0% Foxp3-/GrzB+, and 1.4 ± 0.4% Foxp3+/GrzB+), and more so by CD25-Bright cells (52.5 ± 4.0% Foxp3+/GrzB-, 4.8 ± 0.8% Foxp3-/GrzB+, and 4.3 ± 0.6% Foxp3+/GrzB+, Figure 5B). This is consistent with reports that demonstrate Treg expression of GrzB for suppressive functions [35-37]. IFNγ was modestly induced and coexpressed with GrzB by costimulated memory CD4 T cells (0.3 ± 0.1% IFNγ+/GrzB- and 0.5 ± 0.3% IFNγ+/GrzB+), but more robustly by PMA/IO treatment (29.6 ± 3.4% IFNγ+/GrzB- and 2.4 ± 0.8% IFNγ+/GrzB+, Figure 5C), indicating that memory CD4 Th1 cells express GrzB, consistent with other reports [38-41]. By contrast, IL4 was not induced by costimulated or PMA/IO-treated memory CD4 T cells (0.1% IL4+/GrzB+), suggesting that GrzB is not expressed by memory CD4 Th2 cells. IL17A was induced by costimulation and PMA/IO, but not coexpresssed with GrzB (0.1% IL17A+/GrzB+), suggesting that in these short-term conditions memory CD4 Th17 cells may not express GrzB. Th17 cells are reported to express GrzB in vivo [42,43], which likely involves more complex stimuli, timing or differentiation, and more specific phenotypic characterization (ie. CCR6, CD161, and IL-23R). Thus, Tregs and Th1 cells are subsets of peripheral blood memory CD4 T cells that produce GrzB in the present study, although other GrzB-producing subsets, particularly CD4 CTL’s, are likely involved as well [8-10,14].Figure 5


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)

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.
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Fig5: 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.
Mentions: 24 hrs costimulation or PMA/IO treatment of memory CD4 T cells resulted in GrzB expression by phenotypes consistent with Treg and Th1 cells (Figure 5A-C). Tregs were examined by measuring intracellular GrzB expression by CD25+/Foxp3+ costimulated memory CD4 T cells. Activated memory CD4 T cells expressed ~18% CD25 (13% CD25-Dim and 4.6% CD25-Bright, Figure 5A). GrzB was coexpressed with Foxp3 by CD25-Dim cells (24.2 ± 3.6% Foxp3+/GrzB-, 4.6 ± 1.0% Foxp3-/GrzB+, and 1.4 ± 0.4% Foxp3+/GrzB+), and more so by CD25-Bright cells (52.5 ± 4.0% Foxp3+/GrzB-, 4.8 ± 0.8% Foxp3-/GrzB+, and 4.3 ± 0.6% Foxp3+/GrzB+, Figure 5B). This is consistent with reports that demonstrate Treg expression of GrzB for suppressive functions [35-37]. IFNγ was modestly induced and coexpressed with GrzB by costimulated memory CD4 T cells (0.3 ± 0.1% IFNγ+/GrzB- and 0.5 ± 0.3% IFNγ+/GrzB+), but more robustly by PMA/IO treatment (29.6 ± 3.4% IFNγ+/GrzB- and 2.4 ± 0.8% IFNγ+/GrzB+, Figure 5C), indicating that memory CD4 Th1 cells express GrzB, consistent with other reports [38-41]. By contrast, IL4 was not induced by costimulated or PMA/IO-treated memory CD4 T cells (0.1% IL4+/GrzB+), suggesting that GrzB is not expressed by memory CD4 Th2 cells. IL17A was induced by costimulation and PMA/IO, but not coexpresssed with GrzB (0.1% IL17A+/GrzB+), suggesting that in these short-term conditions memory CD4 Th17 cells may not express GrzB. Th17 cells are reported to express GrzB in vivo [42,43], which likely involves more complex stimuli, timing or differentiation, and more specific phenotypic characterization (ie. CCR6, CD161, and IL-23R). Thus, Tregs and Th1 cells are subsets of peripheral blood memory CD4 T cells that produce GrzB in the present study, although other GrzB-producing subsets, particularly CD4 CTL’s, are likely involved as well [8-10,14].Figure 5

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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