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Killing of myeloid APCs via HLA class I, CD2 and CD226 defines a novel mechanism of suppression by human Tr1 cells.

Magnani CF, Alberigo G, Bacchetta R, Serafini G, Andreani M, Roncarolo MG, Gregori S - Eur. J. Immunol. (2011)

Bottom Line: Notably, interaction between CD226 on Tr1 cells and their ligands on myeloid cells, leading to Tr1-cell activation, is necessary for defining Tr1-cell target specificity.We also showed that high frequency of GZB-expressing CD4(+) T cells is detected in tolerant patients and correlates with elevated occurrence of IL-10-producing CD4(+) T cells.In conclusion, the modulatory activities of Tr1 cells are not only due to suppressive cytokines but also to specific cell-to-cell interactions that lead to selective killing of myeloid cells and possibly bystander suppression.

View Article: PubMed Central - PubMed

Affiliation: San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Division of Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Scientific Institute, Milan, Italy.

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Tr1 cells express and release GZB. (A) GZB expression was determined in Th0- and Tr1-cell lines unstimulated (unstim) or stimulated with PMA (10 ng/mL; Sigma) plus IONO (ionomycin) (150 ng/mL; Sigma) for 6 h. One donor representative of 11 unstimulated donors and of five stimulated donors tested is shown. Numbers represent percentage of positive cells and MFI in bracket. (B) Alternatively, IL-10, IL-4, and GZB expression was determined upon stimulation with Leukocyte Activation Cocktail (BD Pharmingen) for 5 h. One donor out of six donors tested is shown. Numbers represent percentage of positive cells. (C) GZB expression was determined in four unstimulated Tr1- and Th0-cell clones. Numbers represent percentage of positive cells and MFI in bracket. (D) Plot represents IL-10 production expressed as pg/mL versus MFI of GZB expression in each of ten Tr1-cell clones and of nine Th0-cell clones tested. The line represents the linear regression. The p value of the correlation and the coefficient of determination (r2) are reported (two-tailed test). (E) GZB release by unstimulated (unstim) and stimulated with PMA/IONO Tr1- and Th0-cell lines was measured by ELISPOT. The Y-axis represents the number of SFU/106 cells. (F) GZB release by original Tr1-cell lines, and purified IL-10-producing cells was measured by ELISPOT. Mean±SE of GZB spots normalized to 106 cells of five (unstim), three (PMA/IONO), and three (F) independent experiments performed in duplicate is shown. SFU, spot forming units *p≤0.05, and **p≤0.005 (one-tailed test).
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fig01: Tr1 cells express and release GZB. (A) GZB expression was determined in Th0- and Tr1-cell lines unstimulated (unstim) or stimulated with PMA (10 ng/mL; Sigma) plus IONO (ionomycin) (150 ng/mL; Sigma) for 6 h. One donor representative of 11 unstimulated donors and of five stimulated donors tested is shown. Numbers represent percentage of positive cells and MFI in bracket. (B) Alternatively, IL-10, IL-4, and GZB expression was determined upon stimulation with Leukocyte Activation Cocktail (BD Pharmingen) for 5 h. One donor out of six donors tested is shown. Numbers represent percentage of positive cells. (C) GZB expression was determined in four unstimulated Tr1- and Th0-cell clones. Numbers represent percentage of positive cells and MFI in bracket. (D) Plot represents IL-10 production expressed as pg/mL versus MFI of GZB expression in each of ten Tr1-cell clones and of nine Th0-cell clones tested. The line represents the linear regression. The p value of the correlation and the coefficient of determination (r2) are reported (two-tailed test). (E) GZB release by unstimulated (unstim) and stimulated with PMA/IONO Tr1- and Th0-cell lines was measured by ELISPOT. The Y-axis represents the number of SFU/106 cells. (F) GZB release by original Tr1-cell lines, and purified IL-10-producing cells was measured by ELISPOT. Mean±SE of GZB spots normalized to 106 cells of five (unstim), three (PMA/IONO), and three (F) independent experiments performed in duplicate is shown. SFU, spot forming units *p≤0.05, and **p≤0.005 (one-tailed test).

Mentions: Tr1 polarized cell lines expressed significantly higher levels of GZB compared to Th0-cell lines (97.3 versus 12.9%, n=11, p<0.0001, Fig. 1A). Notably, IL-10-producing Tr1 cells represent 10–15% of the polarized population, thus GZB expression is not restricted to this population of cells (Fig. 1B). Tr1-cell lines express also significantly higher levels of GZA compared to Th0-cell lines (58.7% versus 9%, n=8, p<0.0001, not shown), nevertheless its expression was consistently lower than that of GZB. Tr1-cell lines contained a significantly higher percentage of PRF+ cells compared to Th0-cell lines before (8.8 versus 1.8%, n=7, p=0.015) and after stimulation (13.3 versus 5.1%, n=7, p=0.007, not shown).


Killing of myeloid APCs via HLA class I, CD2 and CD226 defines a novel mechanism of suppression by human Tr1 cells.

Magnani CF, Alberigo G, Bacchetta R, Serafini G, Andreani M, Roncarolo MG, Gregori S - Eur. J. Immunol. (2011)

Tr1 cells express and release GZB. (A) GZB expression was determined in Th0- and Tr1-cell lines unstimulated (unstim) or stimulated with PMA (10 ng/mL; Sigma) plus IONO (ionomycin) (150 ng/mL; Sigma) for 6 h. One donor representative of 11 unstimulated donors and of five stimulated donors tested is shown. Numbers represent percentage of positive cells and MFI in bracket. (B) Alternatively, IL-10, IL-4, and GZB expression was determined upon stimulation with Leukocyte Activation Cocktail (BD Pharmingen) for 5 h. One donor out of six donors tested is shown. Numbers represent percentage of positive cells. (C) GZB expression was determined in four unstimulated Tr1- and Th0-cell clones. Numbers represent percentage of positive cells and MFI in bracket. (D) Plot represents IL-10 production expressed as pg/mL versus MFI of GZB expression in each of ten Tr1-cell clones and of nine Th0-cell clones tested. The line represents the linear regression. The p value of the correlation and the coefficient of determination (r2) are reported (two-tailed test). (E) GZB release by unstimulated (unstim) and stimulated with PMA/IONO Tr1- and Th0-cell lines was measured by ELISPOT. The Y-axis represents the number of SFU/106 cells. (F) GZB release by original Tr1-cell lines, and purified IL-10-producing cells was measured by ELISPOT. Mean±SE of GZB spots normalized to 106 cells of five (unstim), three (PMA/IONO), and three (F) independent experiments performed in duplicate is shown. SFU, spot forming units *p≤0.05, and **p≤0.005 (one-tailed test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig01: Tr1 cells express and release GZB. (A) GZB expression was determined in Th0- and Tr1-cell lines unstimulated (unstim) or stimulated with PMA (10 ng/mL; Sigma) plus IONO (ionomycin) (150 ng/mL; Sigma) for 6 h. One donor representative of 11 unstimulated donors and of five stimulated donors tested is shown. Numbers represent percentage of positive cells and MFI in bracket. (B) Alternatively, IL-10, IL-4, and GZB expression was determined upon stimulation with Leukocyte Activation Cocktail (BD Pharmingen) for 5 h. One donor out of six donors tested is shown. Numbers represent percentage of positive cells. (C) GZB expression was determined in four unstimulated Tr1- and Th0-cell clones. Numbers represent percentage of positive cells and MFI in bracket. (D) Plot represents IL-10 production expressed as pg/mL versus MFI of GZB expression in each of ten Tr1-cell clones and of nine Th0-cell clones tested. The line represents the linear regression. The p value of the correlation and the coefficient of determination (r2) are reported (two-tailed test). (E) GZB release by unstimulated (unstim) and stimulated with PMA/IONO Tr1- and Th0-cell lines was measured by ELISPOT. The Y-axis represents the number of SFU/106 cells. (F) GZB release by original Tr1-cell lines, and purified IL-10-producing cells was measured by ELISPOT. Mean±SE of GZB spots normalized to 106 cells of five (unstim), three (PMA/IONO), and three (F) independent experiments performed in duplicate is shown. SFU, spot forming units *p≤0.05, and **p≤0.005 (one-tailed test).
Mentions: Tr1 polarized cell lines expressed significantly higher levels of GZB compared to Th0-cell lines (97.3 versus 12.9%, n=11, p<0.0001, Fig. 1A). Notably, IL-10-producing Tr1 cells represent 10–15% of the polarized population, thus GZB expression is not restricted to this population of cells (Fig. 1B). Tr1-cell lines express also significantly higher levels of GZA compared to Th0-cell lines (58.7% versus 9%, n=8, p<0.0001, not shown), nevertheless its expression was consistently lower than that of GZB. Tr1-cell lines contained a significantly higher percentage of PRF+ cells compared to Th0-cell lines before (8.8 versus 1.8%, n=7, p=0.015) and after stimulation (13.3 versus 5.1%, n=7, p=0.007, not shown).

Bottom Line: Notably, interaction between CD226 on Tr1 cells and their ligands on myeloid cells, leading to Tr1-cell activation, is necessary for defining Tr1-cell target specificity.We also showed that high frequency of GZB-expressing CD4(+) T cells is detected in tolerant patients and correlates with elevated occurrence of IL-10-producing CD4(+) T cells.In conclusion, the modulatory activities of Tr1 cells are not only due to suppressive cytokines but also to specific cell-to-cell interactions that lead to selective killing of myeloid cells and possibly bystander suppression.

View Article: PubMed Central - PubMed

Affiliation: San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Division of Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Scientific Institute, Milan, Italy.

Show MeSH
Related in: MedlinePlus