Isolation and characterization of human interleukin-10-secreting T cells from peripheral blood

Abstract

Recent studies have expanded our understanding of the role of the anti-inflammatory cytokine interleukin (IL)-10, produced by multiple lineages of both human and murine T cells, in regulating the immune response. Here, we demonstrate that the small percentage of circulating CD4(+) T cells that secrete IL-10 can be isolated from human peripheral blood and, importantly, we have optimized a protocol to expand these cells in both antigen-specific and polyclonal manners. Expanded CD4(+)IL-10(+) T cells abrogate proliferation and T helper (Th) 1-like cytokine production in an antigen-specific manner, and to a lesser extent exhibit bystander suppressive capacity. CD4(+)IL-10(+) T cells are suppressive in a cell contact-dependent way, though they do not require secretion of IL-10 for their suppressive role in vitro. CD4(+)IL-10(+) T cells have an activated phenotype, with high expression of CD25, CD69, and cytotoxic T-lymphocyte antigen-4, and are largely FoxP3 negative. This novel method for the isolation and expansion of suppressive IL-10-secreting T cells has important implications both for further research and clinical therapeutic development.

Fig. 1

Column selection of IL-10–secreting T cells leads to accumulation of dead cells. CytoStim-treated PBMCs were harvested after 16 hours and subjected to a Dead Cell Removal Kit. IL-10–positive T cells (and IL-10–negative fraction) were separated by MS columns after an IL-10 Secretion Assay. Cells were sampled at each stage for immunophenotyping. (A) Representative FACS plots show FSC/SSC of human PBMCs after CytoStim treatment (top), in the IL-10–negative fraction (middle), and in the IL-10–positive fraction (bottom). R1-gated cell staining for surface CD4FITC/IL-10PE is also depicted. Plots are representative of one of six experiments. Circled gates are R1; arrow in bottom FSC/SSC plot denotes low FSC/SSC, “dead” cell population. (B) 7-AAD staining was performed on each cell fraction to assess viability. The FACS histogram depicts lymphocyte-gated 7-AAD staining for whole naive PMBCs (thin black line), CytoStim-treated PBMCs (thin black dotted line), IL-10–negative fraction (thick dark gray dashed line) and IL-10⁺ cells (filled light gray histogram). The data are representative of one of five experiments. (C) The FACS histogram depicts lymphocyte-gated CD4 PE staining after negative selection for CD4⁺ cells. The dot plots show side scatter (SSC) versus IL-10–PE and 7-AAD versus AnnexinV FITC post-IL-10 flow cytometry sorting. The data are representative of one of five experiments.

Fig. 2

PPD-specific IL-10⁺ T cells are anergic and suppressive in a dose-dependent fashion. IL-10⁺ T cells were selected from human peripheral blood and expanded with PPD, IL-2, and IL-15 for 10 days. Autologous naive PBMCs (1 × 10⁶ cells/condition) were activated in 1-mL cultures in 48-well plates in the presence (+) or absence (–) of (A) 50 μg/mL PPD, (B) anti-CD3/CD28 beads (1.25 μl/10⁶ cells), or (C) 50 μg/mL MBP (after one additional round of restimulation with PPD). 1 × 10⁶ IL-10⁺ T cells were titrated into wells with naive PBMCs in a dilution series from 5 × 10⁵ cells/well to 6.25 × 10⁴ cells/well in 2-fold dilutions (three PBMCs: one IL-10⁺ T-cell ratio [1 × 10⁶:3 × 10⁵] used for MBP treatment). IL-10⁺ T cells (1 × 10⁶ cells/well) were also stimulated with irradiated APCs (3 × 10⁶ cells/well), with or without PPD, or alone with anti-CD3/CD28 beads. At the peak response days (day 7 for PPD; day 4 for anti-CD3/CD28 beads; and day 9 for MBP), 100-μl duplicates were harvested into 96-well plates and pulsed with 0.5 μCi ³[H]-thymidine. Results are expressed as corrected counts per minute (ccpm); error bars represent standard error (SE). TCL, T-cell line. *p < 0.01; NS, not significant. Data for PPD and anti-CD3/CD28 stimulation representative of three experiments; MBP data are from one experiment (one distinct individual donor per experiment).

Fig. 3

Anti-CD3/CD28 expanded IL-10⁺ T cells suppress naive PBMC proliferation and effector cytokine production. IL-10⁺ T cells were expanded with anti-CD3/CD28 beads, rhIL-2 and rhIL-15 for two rounds of stimulation; cells were used at day 9 of RII. Autologous naive PBMCs (1 × 10⁶ cells/condition) were activated in the presence (+) or absence (–) of anti-CD3/CD28 beads. IL-10⁺ T cells were titrated into wells with naive PBMCs in a dilution series from 5 × 10⁵ (A) or 1 × 10⁶ (B–F) cells/well to 3.125 × 10⁴ cells/well in 2-fold dilutions. IL-10⁺ T cells (1 × 10⁶ cells/well) were also stimulated with or without anti-CD3/CD28 beads alone. (A) At the peak response (day 4), 100-μl duplicates were harvested into 96-well plates and pulsed with 0.5 μCi ³[H]-thymidine. Results are expressed as ccpm; error bars represent SE. (B-F) supernatant was collected from the cultures at day 2 and used in a cytometric bead array (CBA) to measure cytokine secretion. Results for (B) IL-2, (C) IFN–γ, (D) TNF–α, (E) IL-5, and (F) IL-10 are expressed in pg/mL. Data representative of three experiments (one distinct individual donor per experiment). TCL, T-cell line. In A, ratios represented as # PBMC: # TCL. For B–F, ratios are written as # TCL/# PBMC.

Fig. 4

Cell contact, but neither IL-10 nor TGF-β, is required for IL-10⁺ T–cell–mediated suppression in vitro. (A) IL-10⁺ T cells were expanded with anti-CD3/CD28 beads, rhIL-2 and rhIL-15 for two rounds of stimulation; cells were used at day 9 of RII. Autologous naive PBMCs (1 × 10⁶ cells/condition) were activated in the presence (+) or absence (−) of anti-CD3/CD28 beads. IL-10⁺ T cells were titrated into wells with naive PBMCs at a 3:1 ratio (PBMCs: IL-10⁺ T cells). rhIL-10sRα (1 μg/mL) and/or rhTGF-βsRII (100 ng/mL) were added to cultures where indicated. At the peak response (day 3), 100-μl duplicates were harvested into 96-well plates and pulsed with 0.5 μCi ³[H]-thymidine. Results are expressed as ccpm; error bars represent SE. (B) IL-10⁺ and IL-10⁻ T cells (selected and sorted from the same donor) were stimulated with PPD, IL-2 and IL-15 for 13 days. Autologous naive PBMCs (1 × 10⁶/ chamber) were added to the top and bottom chambers of transwells (separated by a porous membrane in 24-well plates), with or without PPD (50 μg/mL) in both chambers. Resting day 13 PPD-specific IL-10⁺ or IL-10⁻ T cells were added to chambers where indicated at 3 × 10⁵ cells/chamber. On day 7, 100-μl duplicates were harvested into 96-well plates and pulsed with 0.5 μCi ³[H]-thymidine. Results are expressed as ccpm; error bars represent standard error. *p < 0.01; **p < 0.001. Data representative of three experiments (one distinct individual donor per experiment). TCL, T-cell line; Top, top well; bot, bottom well.

Fig. 5

Anti-CD3/CD28 stimulated IL-10⁺ T cells have an activated cell phenotype. IL-10⁺ T cells (selected from human PBMCs) were expanded by stimulation with anti-CD3/CD28 (in the presence of IL-2 and IL-15) for one round, and then frozen. Thawed cells were subsequently washed and reactivated with anti-CD3/CD28 for 1, 5, or 13 days; stains depicted show autologous naive PBMC controls, d1 IL-10 TCL, day 5 IL-10 TCL, and day 13 IL-10 TCL. These cells were harvested and stained at each time point. Top two row histograms represent cell-surface staining for CD4 and CD69. Dot plots show CD4-gated cell surface stains for CD25 (third row), CD127 (fourth row), and CTLA-4 (fifth row). Bottom row shows dot plots for CD4-gated, intracellularly stained FoxP3 expression. Protein expression was measured by flow cytometry (BD FACS Scan) and data were analyzed with FlowJo software. Numbers represent the percentage expression of the described protein populations on the IL-10⁺ T-cell line (TCL) at indicated time points. Data are representative of two experiments.