Limits...
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.

Show MeSH

Related in: MedlinePlus

Memory T cells exhibit greater effector function than naïve T cells. (a) Fluorescence-activated cell sorting analysis of CD4+ subpopulations. Resting CD4+ lymphocytes in normal peripheral blood contains CD45RA+CCR7+ (naïve) lymphocytes (left panel) together with CD45RO+CCR7+ central memory (TCM; right panel) and CD45RO+CCR7- effector memory populations (TEM; right panel). (b) Effector function resides mainly within the CD4+CD45RO+ population. Lymphocytes were sorted into naïve (CD4+CD45RA+) and memory (CD4+CD45RO+) populations at time zero, before stimulation for 8 days with IL-2, IL-6 and tumour necrosis factor (TNF)-α. Lymphocytes were then co-cultured with monocytes at a ratio of 0.5:1 and 1:1 for 18 hours and the supernatants assayed in triplicate for TNF-α production by ELISA. ***P < 0.001 comparing CD4+CD45RA+ versus CD4+CD45RO+ populations using one-way analysis of variance (ANOVA) with Bonferroni's multiple comparison. (c) Effector function resides predominates within CD4+ effector memory population. CD45RO+ cells were further sorted by flow cytometry into CCR7+ and CCR7- populations at day 0 (and stimulated for 8 days with cytokines) or at day 8 (after cytokine expansion) and then co-cultured with monocytes at a ratio of 1:1 for 18 hours, and the supernatants assayed in triplicate for TNF-α production by ELISA. ***P < 0.001 comparing CD4+CD45RO+CCR7- versus CD4+CD45RO+CCR7+ after sorting at day 0 sort or at day 8 using one-way ANOVA with Bonferroni's multiple comparison. In panels b and c monocytes and T cells cultured alone as negative controls produced under 20 pg/ml TNF-α. Results are shown from one donor representative of five different experiments (panel b) and three different experiments (panel c). CCR, CC chemokine receptor.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2452984&req=5

Figure 3: Memory T cells exhibit greater effector function than naïve T cells. (a) Fluorescence-activated cell sorting analysis of CD4+ subpopulations. Resting CD4+ lymphocytes in normal peripheral blood contains CD45RA+CCR7+ (naïve) lymphocytes (left panel) together with CD45RO+CCR7+ central memory (TCM; right panel) and CD45RO+CCR7- effector memory populations (TEM; right panel). (b) Effector function resides mainly within the CD4+CD45RO+ population. Lymphocytes were sorted into naïve (CD4+CD45RA+) and memory (CD4+CD45RO+) populations at time zero, before stimulation for 8 days with IL-2, IL-6 and tumour necrosis factor (TNF)-α. Lymphocytes were then co-cultured with monocytes at a ratio of 0.5:1 and 1:1 for 18 hours and the supernatants assayed in triplicate for TNF-α production by ELISA. ***P < 0.001 comparing CD4+CD45RA+ versus CD4+CD45RO+ populations using one-way analysis of variance (ANOVA) with Bonferroni's multiple comparison. (c) Effector function resides predominates within CD4+ effector memory population. CD45RO+ cells were further sorted by flow cytometry into CCR7+ and CCR7- populations at day 0 (and stimulated for 8 days with cytokines) or at day 8 (after cytokine expansion) and then co-cultured with monocytes at a ratio of 1:1 for 18 hours, and the supernatants assayed in triplicate for TNF-α production by ELISA. ***P < 0.001 comparing CD4+CD45RO+CCR7- versus CD4+CD45RO+CCR7+ after sorting at day 0 sort or at day 8 using one-way ANOVA with Bonferroni's multiple comparison. In panels b and c monocytes and T cells cultured alone as negative controls produced under 20 pg/ml TNF-α. Results are shown from one donor representative of five different experiments (panel b) and three different experiments (panel c). CCR, CC chemokine receptor.

Mentions: CD4+CD45RO+ (memory) T cells and CD4+CD45RA+ (naïve) T cells [28], enriched at day 0 (Figure 3a), were sorted by flow cytometry from the same donor based on CD45RA expression and expanded for 8 days. Figure 3b demonstrates that the memory T cells when cultured at a ratio of 1:1 with monocytes induced significantly higher levels (P < 0.001) of TNF-α (6.7-fold more) than did the naïve T cells at the comparable ratio. This result was confirmed in five independent experiments. The most potent inducers of monocyte TNF-α production within the CD4+ memory population was found to reside within the effector memory population (CD4+CD45RO+CCR7-), irrespective of whether the cells were sorted before ex vivo cytokine expansion or afterward (Figure 3c). Sixfold more TNF-α was produced by the effector memory cells (CCR7-) separated at day 0 (P < 0.001) compared with central memory cells (CCR7+) and about 13-fold more TNF-α produced by effector memory cells separated at day 8 (P < 0.001) compared with central memory cells. This result was confirmed in three independent experiments.


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)

Memory T cells exhibit greater effector function than naïve T cells. (a) Fluorescence-activated cell sorting analysis of CD4+ subpopulations. Resting CD4+ lymphocytes in normal peripheral blood contains CD45RA+CCR7+ (naïve) lymphocytes (left panel) together with CD45RO+CCR7+ central memory (TCM; right panel) and CD45RO+CCR7- effector memory populations (TEM; right panel). (b) Effector function resides mainly within the CD4+CD45RO+ population. Lymphocytes were sorted into naïve (CD4+CD45RA+) and memory (CD4+CD45RO+) populations at time zero, before stimulation for 8 days with IL-2, IL-6 and tumour necrosis factor (TNF)-α. Lymphocytes were then co-cultured with monocytes at a ratio of 0.5:1 and 1:1 for 18 hours and the supernatants assayed in triplicate for TNF-α production by ELISA. ***P < 0.001 comparing CD4+CD45RA+ versus CD4+CD45RO+ populations using one-way analysis of variance (ANOVA) with Bonferroni's multiple comparison. (c) Effector function resides predominates within CD4+ effector memory population. CD45RO+ cells were further sorted by flow cytometry into CCR7+ and CCR7- populations at day 0 (and stimulated for 8 days with cytokines) or at day 8 (after cytokine expansion) and then co-cultured with monocytes at a ratio of 1:1 for 18 hours, and the supernatants assayed in triplicate for TNF-α production by ELISA. ***P < 0.001 comparing CD4+CD45RO+CCR7- versus CD4+CD45RO+CCR7+ after sorting at day 0 sort or at day 8 using one-way ANOVA with Bonferroni's multiple comparison. In panels b and c monocytes and T cells cultured alone as negative controls produced under 20 pg/ml TNF-α. Results are shown from one donor representative of five different experiments (panel b) and three different experiments (panel c). CCR, CC chemokine receptor.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2452984&req=5

Figure 3: Memory T cells exhibit greater effector function than naïve T cells. (a) Fluorescence-activated cell sorting analysis of CD4+ subpopulations. Resting CD4+ lymphocytes in normal peripheral blood contains CD45RA+CCR7+ (naïve) lymphocytes (left panel) together with CD45RO+CCR7+ central memory (TCM; right panel) and CD45RO+CCR7- effector memory populations (TEM; right panel). (b) Effector function resides mainly within the CD4+CD45RO+ population. Lymphocytes were sorted into naïve (CD4+CD45RA+) and memory (CD4+CD45RO+) populations at time zero, before stimulation for 8 days with IL-2, IL-6 and tumour necrosis factor (TNF)-α. Lymphocytes were then co-cultured with monocytes at a ratio of 0.5:1 and 1:1 for 18 hours and the supernatants assayed in triplicate for TNF-α production by ELISA. ***P < 0.001 comparing CD4+CD45RA+ versus CD4+CD45RO+ populations using one-way analysis of variance (ANOVA) with Bonferroni's multiple comparison. (c) Effector function resides predominates within CD4+ effector memory population. CD45RO+ cells were further sorted by flow cytometry into CCR7+ and CCR7- populations at day 0 (and stimulated for 8 days with cytokines) or at day 8 (after cytokine expansion) and then co-cultured with monocytes at a ratio of 1:1 for 18 hours, and the supernatants assayed in triplicate for TNF-α production by ELISA. ***P < 0.001 comparing CD4+CD45RO+CCR7- versus CD4+CD45RO+CCR7+ after sorting at day 0 sort or at day 8 using one-way ANOVA with Bonferroni's multiple comparison. In panels b and c monocytes and T cells cultured alone as negative controls produced under 20 pg/ml TNF-α. Results are shown from one donor representative of five different experiments (panel b) and three different experiments (panel c). CCR, CC chemokine receptor.
Mentions: CD4+CD45RO+ (memory) T cells and CD4+CD45RA+ (naïve) T cells [28], enriched at day 0 (Figure 3a), were sorted by flow cytometry from the same donor based on CD45RA expression and expanded for 8 days. Figure 3b demonstrates that the memory T cells when cultured at a ratio of 1:1 with monocytes induced significantly higher levels (P < 0.001) of TNF-α (6.7-fold more) than did the naïve T cells at the comparable ratio. This result was confirmed in five independent experiments. The most potent inducers of monocyte TNF-α production within the CD4+ memory population was found to reside within the effector memory population (CD4+CD45RO+CCR7-), irrespective of whether the cells were sorted before ex vivo cytokine expansion or afterward (Figure 3c). Sixfold more TNF-α was produced by the effector memory cells (CCR7-) separated at day 0 (P < 0.001) compared with central memory cells (CCR7+) and about 13-fold more TNF-α produced by effector memory cells separated at day 8 (P < 0.001) compared with central memory cells. This result was confirmed in three independent experiments.

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