The plasticity of regulatory T cell function

Abstract

Regulatory T cells (T(regs)) can suppress a wide variety of cell types, in diverse organ sites and inflammatory conditions. Whereas T(regs) possess multiple suppressive mechanisms, the number required for maximal function is unclear. Furthermore, whether any interrelationship or cross-regulatory mechanisms exist to orchestrate and control their utilization is unknown. In this study, we assessed the functional capacity of T(regs) lacking the ability to secrete both IL-10 and IL-35, which individually are required for maximal T(reg) activity. Surprisingly, IL-10/IL-35 double-deficient T(regs) were fully functional in vitro and in vivo. Loss of IL-10 and IL-35 was compensated for by a concurrent increase in cathepsin E (Ctse) expression, enhanced TRAIL (Tnfsf10) expression, and soluble TRAIL release, rendering IL-10/IL-35 double-deficient T(regs) functionally dependent on TRAIL in vitro and in vivo. Lastly, whereas C57BL/6 T(regs) are normally IL-10/IL-35 dependent, BALB/c T(regs), which express high levels of cathepsin E and enhanced TRAIL expression, are partially TRAIL dependent by default. These data reveal that cross-regulatory pathways exist that control the utilization of suppressive mechanisms, thereby providing T(reg) functional plasticity.

Figure 1. Ebi3^−/−Il10^−/− T_regs are suppressive in vitro and in vivo

(A) Wild type or knock out T_reg purified by FACS were titrated in a standard T_reg assay with T_conv cells and anti-CD3 and anti-CD28 coated latex beads. Proliferation of T_conv responder cells was determined by [³H]-thymidine incorporation (p-value: wild type T_regs compared to Ebi3^−/−Il10^−/−, and Il12a^−/−Il10^−/− T_regs, Not significant (NS)). (B) Wild type or knock out T_reg were cultured with anti-CD3 and anti-CD28 coated latex beads and T_conv cells in the inserts of a Transwell™ culture plate. Third party, wild type responder T_conv was activated in the bottom chamber of the plate. Proliferation of responder cells was determined by [³H]-thymidine incorporation. Proliferation ranged from 30,000–60,000 cpm. p-value: *: <0.05, NS: Not significant. (C) Congenically marked wild type T_conv cells, wild type or knock out T_regs purified by FACS were injected at 4:1 ratio into Rag1^−/− mice. CD4⁺ cell numbers in the spleen were analyzed after 7 days by flow cytometry. p-value: * <0.05. (D) Wild type T_conv cells (0.5×10⁶) were injected into Rag1^−/− mice. The weight of the mice was monitored weekly for weight loss. Once the mice had lost 5% of its body weight wild type or knock out T_regs were injected. Mice were monitored for percent weight change calculated based on the weight at the time of T_reg injection. p-value: * <0.05 and NS: Not significant. (E) Colonic tissue sections stained with H & E stain were scored in a blinded manner. Representative images of sections from 3 independent experiments are shown with histological score in parentheses. Data represent the mean ± SEM of (A) 3, (B) 3–5, (C) 4–9 mice per group, (D&E) 3 independent experiments.

Figure 2. Ebi3^−/−Il10^−/− T_regs that developed in a mixed bone marrow chimera can function in vitro and in vivo

Congenically labeled wild type bone marrow and knock out bone marrow was mixed at a 1:1 ratio and injected into sub lethally irradiated Rag1^−/− mice. (A) After 8 weeks Thy.1.2⁺ wild type T_reg cells or knock out T_reg cells were purified by FACS from the bone marrow chimeric mice and cultured in the inserts of a Transwell™ plate in the presence of wild type T_conv cells and anti-CD3 and anti-CD28 coated latex beads. Wild type naive T_conv cells were activated in the presence of anti-CD3 and anti-CD28 coated beads in the bottom chamber of a Transwell™ for 72 h. Proliferation was determined by [³H]-thymidine incorporation. Data represents the mean± SEM of two independent experiments. (p-value; NS: Not significant). (B) Purified wild type or Ebi3^−/−Il10^−/− bone marrow chimera-derived T_regs were injected into Rag1^−/− mice in the presence of congenically marked naïve T_conv cells. The expansion of naïve Thy1.1 CD4⁺ T cells were assessed by flow cytometry. Data represents the mean ± SEM of two independent experiments with 3–4 mice per group (p-value *=0.06).

Figure 3. Up-regulation of CTSE by Ebi3^−/−Il10^−/− T_regs

(A) mRNA was isolated from wild type or knock out T_reg purified by FACS and used for Affymetrix analysis. Modulated genes in knock out T_reg compared to wild type T_regs is depicted in a heat map. (B) Volcano plot comparing wild type and Ebi3^−/− Il10^−/−, T_regs. Highest modulated genes are marked. (C) mRNA was isolated from wild type or knock out T_reg purified by FACS, cDNA synthesized and Ctse expression assessed by qPCR. Data are the mean of 2 independent experiments. (D) Wild type or knock out T_reg were stained for intracellular CTSE [grey - 2^nd antibody control, open histograms; in green - wild type T_regs and in blue; Ebi3^−/− Il10^−/− T_regs]. (E) Equal numbers of FACS purified wild type or knock out T_reg were lysed, CTSE immunoprecipitated and analysed by SDS-PAGE/western blot. Data are representative (A, B, D and E) of three independent experiments.

Figure 4. Cathepsin E enhances the suppression of T_conv cells by TRAIL

293T cells were transfected either with Ctse and Tnfsf10 alone or together. The cells were irradiated with 3000 rads 48h post transfection and seeded at a density of 7000 cells per well in a 96 well flat bottom plate. Freshly isolated C57BL/6 T_conv cells were added to the seeded plate at 8×10⁴ per well and stimulated with anti-CD3 and anti-CD28 coated beads for 72 hours. Proliferation of responder cells was determined by [³H]-thymidine incorporation. T_conv cell proliferation was calculated by subtracting the basal [3H]-thymidine incorporation of irradiated 293T plus T cells without anti-CD3 and anti-CD28 stimulation. Data represent the average of three independent experiments. p-value, * : <0.05.

Figure 5. TRAIL dependence and modulation in Ebi3^−/− Il10^−/−T_regs

Wild type or knock out T_reg purified by FACS were activated in presence of anti-CD3 and anti-CD28 coated latex beads with IL2 for 16 and 24h. (A) Cells were collected and surface TRAIL expression detected by flow cytometry using an anti-mouse TRAIL antibody. Data are representative of 3 independent experiments. (B) Mean fluorescence intensity (MFI) of surface TRAIL expression following activation from 3–4 independent experiments were plotted. Students t Test; p-value ** = <0.05. (C) Wild type or knock out T_reg were cultured in the insert of a Transwell™ culture plate in the presence of wild type T_conv cells. zVAD or DMSO control was added to the Transwell™ assay. Freshly purified wild type responder T_conv cells were activated in the bottom chamber of the plate. Proliferation of responder cells was determined by [³H]-thymidine incorporation. Data represent the mean ± SEM of 2 independent experiments. p-value * = 0.07.

Figure 6. Ebi3^−/−Il10^−/− T_reg mediated suppression is TRAIL-dependent

Wild type or knock out T_regs purified by FACS (A) were titrated in a T_reg assay with wild type or DR5^−/− T_conv cells and stimulated with anti-CD3 and anti-CD28 coated latex beads or (B) were cultured with wild type T_conv cells in the insert of a Transwell™ culture plate. Wild type or DR5^−/− T_conv cells were activated in the bottom chamber of the plate with anti-CD3 and anti-CD28 coated latex beads. Proliferation of responder wild type or DR5^−/− T_conv cells was determined by [³H]-thymidine incorporation. CPM ranged between 30,000–65,000. Results shown here are average of 4–5 independent experiments. (C) Wild type and Ebi3^−/−Il10^−/− T_regs were stimulated with anti-CD3 and anti-CD28 coated latex beads in the presence of T_conv cells in the insert of a Transwell™ culture plate. Freshly purified wild type responder T_conv cells were activated in the bottom wells in the presence of a titrated amount of DR5-Fc. Data is average of 2–3 independent experiments, One way ANCOVA p-value *= 0.01 (D) Congenically marked wild type T_conv cells and wild type or knock out T_regs were injected at 4:1 ratio in to Rag^−/− mice. On days 1 and 3 TRAIL neutralizing mAb or isotype control were injected by i.p. CD4, Thy1.1 and Thy 1.2 T cell numbers in the spleen were analyzed after 7 days by flow cytometry. Data includes 3–6 mice per group from 3 independent experiments. (E) Wild type littermate control T_conv cells or (F) DR5^−/− T_conv cells (0.5 × 10⁶ cells) were injected into Rag1^−/− mice. The weight of the mice was monitored weekly for weight loss. Percent weight change was calculated based on the weight at the time of T_reg injection. (G) Wild type or knock out T_regs purified by FACS were cultured with wild type T_conv cells in the insert of a Transwell™ culture plate. Wild type T_conv cells were activated in the bottom chamber of the plate with anti-CD3 and anti-CD28-coated latex beads. Proliferation of responder T_conv cells was determined by [³H]-thymidine incorporation. CPM ranged between 30,000–70,000. Results shown here are mean ± SEM of 3 independent experiments. p-value - A–G; *: < 0.05, ** < 0.005, NS: Not significant.

Figure 7. BALB/c T_reg preferentially use TRAIL-mediated pathways compared to C57BL/6 T_regs

(A) mRNA was isolated from freshly purified C57BL/6 or BALB/c T_conv cells and T_regs, cDNA synthesized and qPCR performed to assess Ctse expression. (B) Intracellular staining for CTSE was performed with purified C57BL/6 or BALB/c T_reg [Grey filled - secondary antibody only control; open histogram - C57BL/6 T_regs and closed histogram BALB/c T_regs]. (C) TRAIL staining was performed with Tnfsf10^−/−, wild type C57BL/6 or BALB/c T_regs, activated in presence of anti-CD3 and anti-CD28 coated latex beads with IL2 for 16h and surface TRAIL expression was detected by flow cytometry using a anti-mouse TRAIL antibody (MFI from three independent experiments, p-value:0.07). (D) Wild type C57BL/6 or BALB/c T_regs were mixed at 1:2 ratio with naïve wild type T_conv cells in the presence of anti-CD3 and anti-CD28 coated beads in the insert of a Transwell™ culture plate for 72h. Neutralizing antibodies against IL-10, IL-35 or a DR5-Fc protein were added to the Transwell™ assay at pre-determined concentrations as described in the methods. Freshly purified wild type responder T_conv were activated in the bottom chamber of a Transwell™ culture plate. Proliferation of the responder cells was determined by [³H]-thymidine incorporation. p-value *: < 0.05. Data represent 3–4 independent experiments.