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Resting CD4+ effector memory T cells are precursors of bystander-activated effectors: a surrogate model of rheumatoid arthritis synovial T-cell function.

Brennan FM, Smith NM, Owen S, Li C, Amjadi P, Green P, Andersson A, Palfreeman AC, Hillyer P, Foey A, Beech JT, Feldmann M - Arthritis Res. Ther. (2008)

Bottom Line: Here, using magnetic beads and fluorescence-activated cell sorting, we extensively phenotype the Tck effector cells and conclude that effector function resides within the CD4+CD45RO+, CCR7-, CD49dhigh population, and that these cells are derived from the effector memory CD4+ T cells in resting blood.Moreover, blockade of these ligands also resulted in inhibition of spontaneous tumour necrosis factor-alpha production in RA synovial mononuclear cell cultures.Taken together, these data strengthen our understanding of T-cell effector function, highlight the multiple involvement of different cell surface ligands in cell-cell contact and, provide novel insights into the pathogenesis of inflammatory RA disease.

View Article: PubMed Central - HTML - PubMed

Affiliation: Kennedy Institute of Rheumatology Division, Imperial College Faculty of Medicine, Aspenlea Road, London, W6 8LH, UK. f.brennan@imperial.ac.uk

ABSTRACT

Background: Previously we described a system whereby human peripheral blood T cells stimulated for 8 days in a cytokine cocktail acquired effector function for contact-dependent induction of proinflammatory cytokines from monocytes. We termed these cells cytokine-activated (Tck) cells and found that the signalling pathways elicited in the responding monocytes were identical whether they were placed in contact with Tck cells or with T cells isolated from rheumatoid arthritis (RA) synovial tissue.

Methods: Here, using magnetic beads and fluorescence-activated cell sorting, we extensively phenotype the Tck effector cells and conclude that effector function resides within the CD4+CD45RO+, CCR7-, CD49dhigh population, and that these cells are derived from the effector memory CD4+ T cells in resting blood.

Results: After stimulation in culture, these cells produce a wide range of T-cell cytokines, undergo proliferation and differentiate to acquire an extensively activated phenotype resembling RA synovial T cells. Blocking antibodies against CD69, CD18, or CD49d resulted in a reduction of tumour necrosis factor-alpha production from monocytes stimulated with CD4+CD45RO+ Tck cells in the co-culture assay. Moreover, blockade of these ligands also resulted in inhibition of spontaneous tumour necrosis factor-alpha production in RA synovial mononuclear cell cultures.

Conclusion: Taken together, these data strengthen our understanding of T-cell effector function, highlight the multiple involvement of different cell surface ligands in cell-cell contact and, provide novel insights into the pathogenesis of inflammatory RA disease.

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The effector function of cytokine-stimulated lymphocytes resides within the CD4+ population. (a) Effector function of the CD4+ cytokine-activated T (Tck) cell population compared with that of the CD4- Tck population. Lymphocytes were positively separated using CD4+ magnetic beads (Dynal) before stimulation for 8 days with IL-2, IL-6 and tumour necrosis factor (TNF)-α. On day 8 lymphocytes were co-cultured with autologous monocytes (at the indicated ratios) for 18 hours before supernatants were removed and assayed for TNF-α by ELISA. Supernatants from cultures of monocytes and T cells alone as negative controls contained under 50 pg/ml. This experiment is representative of seven different donors. ***P < 0.001 comparing CD4+ versus CD4- at a ratio of 3:1, or comparing CD4+ versus CD4- at a ratio of 5:1 using one-way analysis of variance (ANOVA) with Bonferroni's multiple comparison. Because of limited CD4- cell numbers, a comparison at a ratio of 7:1 of could not be performed in this experiment. (b) Effector function of CD8+ Tck population compared with that of CD8- Tck population. Lymphocytes were positively separated using CD8+ magnetic beads (Dynal) before stimulation for 8 days with IL-2, IL-6 and TNF-α. On day 8 lymphocytes were co-cultured with autologous monocytes (at the indicated ratios) for 18 hours before the supernatants were removed and assayed for TNF-α by ELISA. Supernatants from cultures of monocytes and T cells alone as negative controls contained under 50 pg/ml. This experiment is representative of seven different donors. ***P < 0.001 comparing CD8+ versus CD8- at a ratio of 5:1, or comparing CD8+ at versus CD8- at a ratio of 7:1 using one-way ANOVA with Bonferroni's multiple comparison.
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Figure 2: The effector function of cytokine-stimulated lymphocytes resides within the CD4+ population. (a) Effector function of the CD4+ cytokine-activated T (Tck) cell population compared with that of the CD4- Tck population. Lymphocytes were positively separated using CD4+ magnetic beads (Dynal) before stimulation for 8 days with IL-2, IL-6 and tumour necrosis factor (TNF)-α. On day 8 lymphocytes were co-cultured with autologous monocytes (at the indicated ratios) for 18 hours before supernatants were removed and assayed for TNF-α by ELISA. Supernatants from cultures of monocytes and T cells alone as negative controls contained under 50 pg/ml. This experiment is representative of seven different donors. ***P < 0.001 comparing CD4+ versus CD4- at a ratio of 3:1, or comparing CD4+ versus CD4- at a ratio of 5:1 using one-way analysis of variance (ANOVA) with Bonferroni's multiple comparison. Because of limited CD4- cell numbers, a comparison at a ratio of 7:1 of could not be performed in this experiment. (b) Effector function of CD8+ Tck population compared with that of CD8- Tck population. Lymphocytes were positively separated using CD8+ magnetic beads (Dynal) before stimulation for 8 days with IL-2, IL-6 and TNF-α. On day 8 lymphocytes were co-cultured with autologous monocytes (at the indicated ratios) for 18 hours before the supernatants were removed and assayed for TNF-α by ELISA. Supernatants from cultures of monocytes and T cells alone as negative controls contained under 50 pg/ml. This experiment is representative of seven different donors. ***P < 0.001 comparing CD8+ versus CD8- at a ratio of 5:1, or comparing CD8+ at versus CD8- at a ratio of 7:1 using one-way ANOVA with Bonferroni's multiple comparison.

Mentions: Figure 2a demonstrates that effector function (induction of monocyte TNF-α in a dose-dependent manner from 500 to 800 pg/ml by day 8 Tcks) resided in cells derived at day 0 from the CD4+ (P < 0.001) but not the CD4- population (which included NK cells, CD8+ cells and NK T cells). Less TNF-α (<100 pg/ml) was induced with day 8 Tck cells derived from a starting population of CD4- cells (CD8+, NK T and NK cells). To exclude the possibility that ligation of CD4 by the anti-CD4 magnetic beads affected the result, Figure 2b shows that the CD8+ cells enriched at day 0 (which included CD8+ T cells and NK T cells) did not induce an effector response at day 8 (<50 pg/ml). In contrast, the CD8- enriched cells (which were predominantly CD4+ along with CD56+ NK cells) induced a potent Tck effector response (P < 0.001) in a dose-dependent manner at day 8 (75 to 875 pg/ml).


Resting CD4+ effector memory T cells are precursors of bystander-activated effectors: a surrogate model of rheumatoid arthritis synovial T-cell function.

Brennan FM, Smith NM, Owen S, Li C, Amjadi P, Green P, Andersson A, Palfreeman AC, Hillyer P, Foey A, Beech JT, Feldmann M - Arthritis Res. Ther. (2008)

The effector function of cytokine-stimulated lymphocytes resides within the CD4+ population. (a) Effector function of the CD4+ cytokine-activated T (Tck) cell population compared with that of the CD4- Tck population. Lymphocytes were positively separated using CD4+ magnetic beads (Dynal) before stimulation for 8 days with IL-2, IL-6 and tumour necrosis factor (TNF)-α. On day 8 lymphocytes were co-cultured with autologous monocytes (at the indicated ratios) for 18 hours before supernatants were removed and assayed for TNF-α by ELISA. Supernatants from cultures of monocytes and T cells alone as negative controls contained under 50 pg/ml. This experiment is representative of seven different donors. ***P < 0.001 comparing CD4+ versus CD4- at a ratio of 3:1, or comparing CD4+ versus CD4- at a ratio of 5:1 using one-way analysis of variance (ANOVA) with Bonferroni's multiple comparison. Because of limited CD4- cell numbers, a comparison at a ratio of 7:1 of could not be performed in this experiment. (b) Effector function of CD8+ Tck population compared with that of CD8- Tck population. Lymphocytes were positively separated using CD8+ magnetic beads (Dynal) before stimulation for 8 days with IL-2, IL-6 and TNF-α. On day 8 lymphocytes were co-cultured with autologous monocytes (at the indicated ratios) for 18 hours before the supernatants were removed and assayed for TNF-α by ELISA. Supernatants from cultures of monocytes and T cells alone as negative controls contained under 50 pg/ml. This experiment is representative of seven different donors. ***P < 0.001 comparing CD8+ versus CD8- at a ratio of 5:1, or comparing CD8+ at versus CD8- at a ratio of 7:1 using one-way ANOVA with Bonferroni's multiple comparison.
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Related In: Results  -  Collection

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Figure 2: The effector function of cytokine-stimulated lymphocytes resides within the CD4+ population. (a) Effector function of the CD4+ cytokine-activated T (Tck) cell population compared with that of the CD4- Tck population. Lymphocytes were positively separated using CD4+ magnetic beads (Dynal) before stimulation for 8 days with IL-2, IL-6 and tumour necrosis factor (TNF)-α. On day 8 lymphocytes were co-cultured with autologous monocytes (at the indicated ratios) for 18 hours before supernatants were removed and assayed for TNF-α by ELISA. Supernatants from cultures of monocytes and T cells alone as negative controls contained under 50 pg/ml. This experiment is representative of seven different donors. ***P < 0.001 comparing CD4+ versus CD4- at a ratio of 3:1, or comparing CD4+ versus CD4- at a ratio of 5:1 using one-way analysis of variance (ANOVA) with Bonferroni's multiple comparison. Because of limited CD4- cell numbers, a comparison at a ratio of 7:1 of could not be performed in this experiment. (b) Effector function of CD8+ Tck population compared with that of CD8- Tck population. Lymphocytes were positively separated using CD8+ magnetic beads (Dynal) before stimulation for 8 days with IL-2, IL-6 and TNF-α. On day 8 lymphocytes were co-cultured with autologous monocytes (at the indicated ratios) for 18 hours before the supernatants were removed and assayed for TNF-α by ELISA. Supernatants from cultures of monocytes and T cells alone as negative controls contained under 50 pg/ml. This experiment is representative of seven different donors. ***P < 0.001 comparing CD8+ versus CD8- at a ratio of 5:1, or comparing CD8+ at versus CD8- at a ratio of 7:1 using one-way ANOVA with Bonferroni's multiple comparison.
Mentions: Figure 2a demonstrates that effector function (induction of monocyte TNF-α in a dose-dependent manner from 500 to 800 pg/ml by day 8 Tcks) resided in cells derived at day 0 from the CD4+ (P < 0.001) but not the CD4- population (which included NK cells, CD8+ cells and NK T cells). Less TNF-α (<100 pg/ml) was induced with day 8 Tck cells derived from a starting population of CD4- cells (CD8+, NK T and NK cells). To exclude the possibility that ligation of CD4 by the anti-CD4 magnetic beads affected the result, Figure 2b shows that the CD8+ cells enriched at day 0 (which included CD8+ T cells and NK T cells) did not induce an effector response at day 8 (<50 pg/ml). In contrast, the CD8- enriched cells (which were predominantly CD4+ along with CD56+ NK cells) induced a potent Tck effector response (P < 0.001) in a dose-dependent manner at day 8 (75 to 875 pg/ml).

Bottom Line: Here, using magnetic beads and fluorescence-activated cell sorting, we extensively phenotype the Tck effector cells and conclude that effector function resides within the CD4+CD45RO+, CCR7-, CD49dhigh population, and that these cells are derived from the effector memory CD4+ T cells in resting blood.Moreover, blockade of these ligands also resulted in inhibition of spontaneous tumour necrosis factor-alpha production in RA synovial mononuclear cell cultures.Taken together, these data strengthen our understanding of T-cell effector function, highlight the multiple involvement of different cell surface ligands in cell-cell contact and, provide novel insights into the pathogenesis of inflammatory RA disease.

View Article: PubMed Central - HTML - PubMed

Affiliation: Kennedy Institute of Rheumatology Division, Imperial College Faculty of Medicine, Aspenlea Road, London, W6 8LH, UK. f.brennan@imperial.ac.uk

ABSTRACT

Background: Previously we described a system whereby human peripheral blood T cells stimulated for 8 days in a cytokine cocktail acquired effector function for contact-dependent induction of proinflammatory cytokines from monocytes. We termed these cells cytokine-activated (Tck) cells and found that the signalling pathways elicited in the responding monocytes were identical whether they were placed in contact with Tck cells or with T cells isolated from rheumatoid arthritis (RA) synovial tissue.

Methods: Here, using magnetic beads and fluorescence-activated cell sorting, we extensively phenotype the Tck effector cells and conclude that effector function resides within the CD4+CD45RO+, CCR7-, CD49dhigh population, and that these cells are derived from the effector memory CD4+ T cells in resting blood.

Results: After stimulation in culture, these cells produce a wide range of T-cell cytokines, undergo proliferation and differentiate to acquire an extensively activated phenotype resembling RA synovial T cells. Blocking antibodies against CD69, CD18, or CD49d resulted in a reduction of tumour necrosis factor-alpha production from monocytes stimulated with CD4+CD45RO+ Tck cells in the co-culture assay. Moreover, blockade of these ligands also resulted in inhibition of spontaneous tumour necrosis factor-alpha production in RA synovial mononuclear cell cultures.

Conclusion: Taken together, these data strengthen our understanding of T-cell effector function, highlight the multiple involvement of different cell surface ligands in cell-cell contact and, provide novel insights into the pathogenesis of inflammatory RA disease.

Show MeSH
Related in: MedlinePlus