Expression and function of TNF and IL-1 receptors on human regulatory T cells

Abstract

Regulatory T cells (Tregs) suppress immune activation and are critical in preventing autoimmune diseases. While the ability of Tregs to inhibit proliferation of other T cells is well established, it is not yet clear whether Tregs also modulate inflammatory cytokines during an immune response. Here, we show that the expression of inflammatory cytokine receptors IL-1R1 and TNFR2 were higher on resting mature Tregs compared to naïve or memory T cells. While upon activation through the T cell receptor (TCR), expression of IL-1R1 and TNFR2 were upregulated on all T cell subsets, IL-1R1 maintained significantly higher expression on activated Tregs as compared to other T cell subsets. The decoy receptor for IL-1 (IL-1R2) was not expressed by any of the resting T cells but was rapidly upregulated and preferentially expressed upon TCR-stimulation on Tregs. In addition, we found that Tregs also expressed high levels of mRNA for IL-1 antagonist, IL-1RA. TCR-stimulation of naïve T cells in the presence of TGFbeta, which induces FOXP3 expression, however did not result in upregulation of IL-1R1 or IL-1R2. In addition, ectopic expression of FOXP3 in non-Tregs, while causing significant upregulation of IL-1R1 and IL-1R2, did not achieve the levels seen in bona fide Tregs. We also determined that resting human Tregs expressing IL-1R1 did not have higher suppressive capacity compared to IL-1R1- Tregs, suggesting that IL-1R1 does not discriminate suppressive resting Tregs in healthy individuals. Functionally, activated human Tregs displayed a capacity to neutralize IL-1beta, which suggests a physiological significance for the expression of IL-1 decoy receptor on Tregs. In conclusion, our findings that human Tregs preferentially express receptors for TNF and IL-1 suggest a potential function in sensing and dampening local inflammation.

Figure 1. Expression of IL-1R1 and TNFR2 on human T cell subsets.

(A) Total PBMC from healthy donors were stained with for CD3, CD4, CD25, CD45RO, IL-1R1 and TNFR2 after resting overnight in culture with or without IL-2. Lymphocytes were gated on CD3+CD4+, which were further gated into naïve (T_N CD45RO−CD25−), memory (T_M, CD45RO+CD25−), naïve Treg (TNreg, CD45RO− CD25+), and Treg (CD45RO+CD25+). (B) Expression of IL-1R1 and TNFR2 is shown for each subset. Data is representative of 6 different donors. (C) Results calculated as in (B) are shown for 6 donors with statistical analysis.

Figure 2. Expression of IL-1R1, TNFR2 and IL-1R2 on activated CD4+ T cell subsets.

(A) Total CD4+ cells were activated with anti-CD3/CD28 coated beads for two days and stained for GARP, CD45RO, IL-1R1, IL-1R2, and TNFR2. Live cells were gated into T_N (CD45RO−GARP−), T_M (CD45RO+GARP−), TNreg (CD45RO−GARP+), and Treg (CD45RO+GARP+). (B) Expression of IL-1R2, IL-1R1 and TNFR2 are shown for each population. A representative donor is shown. (C) Statistical analysis of results shown in (B) from multiple donor blood.

Figure 3. Time-course of IL-1R1, IL-1R2 and TNFR2 expression on activated CD4+ T cell subsets.

(A) Total resting CD4+ cells were sorted based on CD25 and CD45RO expression into 4 subsets as described above. The subsets were then stimulated through the TCR and stained for IL-1R1, TNFR2 and IL-1R2 daily. Histogram overlays of expression on resting (red), day 1 (blue) and day 2 (green) post activation are shown. (B–D) Percentages of IL-1R1, IL-1R2 and TNFR2-expressing T cells over several days post- TCR-activation. (E) The amount of soluble IL-1R2 detected in the supernatant from T cells washed and re-plated at 0.5×10⁶/ml daily, as determined by CBA.

Figure 4. Time-course of IL-1R1, IL-1R2 and TNFR2 expression on in vitro expanded and reactivated T cell subsets.

Sorted TNregs and T_N cells were reactivated at least 14 days after initial stimulation to produce expanded Treg and Teff cells, respectively. (A–C) Cells were stained at day 14 post first activation and then daily after re-stimulation through TCR (day +1, etc). (D) Supernatants were harvested from the cells restimulated washed and plated at 0.5×10⁶/ml daily and the level of soluble IL-1R2 was analyzed.

Figure 5. Regulation of IL-1R1 and IL-1R2 expression by FOXP3.

(A) Cells ectopically overexpressing FOXP3 were generated using lentiviral transduction as described in the methods. Cells were restimulated, and stained two days later for GARP and IL-1R1 expression. (B) Expression of IL-R1, IL-1R2 and secretion of soluble IL-1R2 in empty vector transduced, FOXP3 transduced, TGFβ treated, and expanded Tregs. TGFβ treated cells were reactivated in the presence of TGFβ1 (20 ng/ml) one week after initial activation. Expanded Tregs were gated on GARP+ cells to exclude non-Tregs in the same cultures from the analysis. Data are average of three donors. (C) Comparison of IL-1R1 and soluble IL-1R2 expression on FOXP3 shRNA lentivirus treated cells. After initial infection, expansion, and sorting, cells were reactivated and stained one day later. Supernatants were analyzed for sIL-1R2 from cells plated at 0.5×10⁶/ml daily. Data are average of three donors.

Figure 6. Suppressive capacity of IL-1R1 expressing Tregs.

(A) Total resting CD4 cells were stained for CD25, CD45RO, and IL-1R1 expression. Cells were gated on Treg and T_M as described in figure 1 and further sorted based on expression of IL-1R1. Since T_N cells lack IL-1R1 expression, these cells were used to set the sort gate. (B) Suppression of T_N target cells by Tregs or Teff cells was measured for various concentrations of OKT3 and various suppressor: target ratios using the Treg suppression assay described in the methods. One representative OKT3 concentration is shown.

Figure 7. Can Tregs neutralize IL-1β activity?

(A) Schematic of neutralization assay, as described in the methods. (B) Jurkat cells (10,000 cells) were cultured with recombinant IL-1β (10 pg/ml), incubated for 4 hours, and then transferred to pre-plated fibroblasts (1000 cells). Twice the amount of recombinant rhIL-1R2 (9 ng/ml) secreted by Jurkat cells overexpressing IL-1R2 was similarly preincubated with IL-1уÂand added to fibroblasts. After overnight incubation, supernatants from the fibroblasts were collected and tested for IL-6 and IL-8 using CBA. Data are average of duplicate wells and are representative of three experiments. (C) Using the same set up as described in (A), expanded Treg or Teff cells were tested for neutralization. Recombinant rhIL-1R2 (5 ug/ml) was used as a positive control for neutralization of all IL-1β. Data are representative of 3 donors. (D) Percent neutralization, calculated as described in methods, was averaged for 3 donors.