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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 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).
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Fig4: 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).

Mentions: Figure 4A-B shows representative perforin/GrzB flow cytometry dotplots (Figure 4A) and the mean distribution of intracellular perforin/GrzB subpopulations (Figure 4B) for memory T cells and NK cells (n = 4). For memory CD4 T cells, only perforin-/GrzB+ cells were mainly observed in all conditions (~4-6%, with and without brefeldin), and a small percentage of perforin+/GrzB+ cells were observed by IL2 stimulation (1.3 ± 0.6%, without brefeldin). However, memory CD8 T cells expressed more intracellular perforin and GrzB. In the absence of brefeldin, UT memory CD8 T cells were 1.1 ± 0.9% perforin+/GrzB-, 25.5 ± 1.5% perforin-/GrzB+, and 2.6 ± 1.3% perforin+/GrzB+. IL2 stimulation changed these perforin/GrzB distributions by increasing perforin expression (10.0 ± 6.5% perforin+/GrzB-, and 15.9 ± 7.3% perforin+/GrzB+), whereas CD3 stimulation did not affect the perforin/GrzB expression patterns. In the presence of brefeldin, perforin expression was mitigated and memory CD8 T cells were mostly perforin-/GrzB+ (~28% for all conditions). As expected, intracellular perforin and GrzB expression was substantially higher for NK cells compared to memory T cells. In the absence of brefeldin, NK cells were mostly perforin+/GrzB+ (UT NK cells were 14.1 ± 4.0% perforin-/GrzB+ and 66.1 ± 7.3% perforin+/GrzB+, and IL2-stimulated cells were 11.9 ± 3.2% perforin-/GrzB+ and 80.7 ± 6.1% perforin+/GrzB+), by contrast to memory T cells which were mostly perforin-/GrzB+. But in the presence of brefeldin, this distribution changed to mostly perforin-/GrzB+ (59.3 ± 12.1% perforin-/GrzB+ and 18.9 ± 10.5% perforin+/GrzB+ by UT cells, and 51.6 ± 17.5% perforin-/GrzB+ and 32.5 ± 18.4% perforin+/GrzB+ by IL2-stimulated cells), which was similar to the perforin mitigation by brefeldin of memory CD8 T cells. Thus, intracellular perforin is marginally expressed with GrzB by stimulated memory CD4 T cells, whereas perforin is more associated with GrzB in memory CD8 T cells and NK cells.Figure 4


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 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).
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Fig4: 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).
Mentions: Figure 4A-B shows representative perforin/GrzB flow cytometry dotplots (Figure 4A) and the mean distribution of intracellular perforin/GrzB subpopulations (Figure 4B) for memory T cells and NK cells (n = 4). For memory CD4 T cells, only perforin-/GrzB+ cells were mainly observed in all conditions (~4-6%, with and without brefeldin), and a small percentage of perforin+/GrzB+ cells were observed by IL2 stimulation (1.3 ± 0.6%, without brefeldin). However, memory CD8 T cells expressed more intracellular perforin and GrzB. In the absence of brefeldin, UT memory CD8 T cells were 1.1 ± 0.9% perforin+/GrzB-, 25.5 ± 1.5% perforin-/GrzB+, and 2.6 ± 1.3% perforin+/GrzB+. IL2 stimulation changed these perforin/GrzB distributions by increasing perforin expression (10.0 ± 6.5% perforin+/GrzB-, and 15.9 ± 7.3% perforin+/GrzB+), whereas CD3 stimulation did not affect the perforin/GrzB expression patterns. In the presence of brefeldin, perforin expression was mitigated and memory CD8 T cells were mostly perforin-/GrzB+ (~28% for all conditions). As expected, intracellular perforin and GrzB expression was substantially higher for NK cells compared to memory T cells. In the absence of brefeldin, NK cells were mostly perforin+/GrzB+ (UT NK cells were 14.1 ± 4.0% perforin-/GrzB+ and 66.1 ± 7.3% perforin+/GrzB+, and IL2-stimulated cells were 11.9 ± 3.2% perforin-/GrzB+ and 80.7 ± 6.1% perforin+/GrzB+), by contrast to memory T cells which were mostly perforin-/GrzB+. But in the presence of brefeldin, this distribution changed to mostly perforin-/GrzB+ (59.3 ± 12.1% perforin-/GrzB+ and 18.9 ± 10.5% perforin+/GrzB+ by UT cells, and 51.6 ± 17.5% perforin-/GrzB+ and 32.5 ± 18.4% perforin+/GrzB+ by IL2-stimulated cells), which was similar to the perforin mitigation by brefeldin of memory CD8 T cells. Thus, intracellular perforin is marginally expressed with GrzB by stimulated memory CD4 T cells, whereas perforin is more associated with GrzB in memory CD8 T cells and NK cells.Figure 4

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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