ECM components guide IL-10 producing regulatory T-cell (TR1) induction from effector memory T-cell precursors

Abstract

We describe a role for ECM as a biosensor for inflammatory microenvironments that plays a critical role in peripheral immune tolerance. We show that hyaluronan (HA) promotes induction of Foxp3- IL-10-producing regulatory T cells (TR1) from conventional T-cell precursors in both murine and human systems. This is, to our knowledge, the first description of an ECM component inducing regulatory T cells. Intact HA, characteristic of healing tissues, promotes induction of TR1 capable of abrogating disease in an IL-10-dependent mouse colitis model whereas fragmentary HA, typical of inflamed tissues, does not, indicating a decisive role for tissue integrity in this system. The TR1 precursor cells in this system are CD4(+)CD62L(-)FoxP3(-), suggesting that effector memory cells assume a regulatory phenotype when they encounter their cognate antigen in the context of intact HA. Matrix integrity cues might thereby play a central role in maintaining peripheral tolerance. This TR1 induction is mediated by CD44 cross-linking and signaling through p38 and ERK1/2. This induction is suppressed, also in a CD44-dependent manner, by osteopontin, a component of chronically inflamed ECM, indicating that CD44 signaling serves as a nexus for fate decisions regarding TR1 induction. Finally, we demonstrate that TR1 induction signals can be recapitulated using synthetic matrices. These results reveal important roles for the matrix microenvironment in immune regulation and suggest unique strategies for immunomodulation.

Fig. 1.

HMW-HA costimulation induces a TR1-like phenotype in CD4⁺GFP/FoxP3⁻CD62L⁻ effector memory T cells. (A) IL-10 production upon activation with aCD3/28 alone or in conjunction with the ECM components HMW-HA, COL, and fibrinogen (FB). (B) Effects of HMW-HA or LMW-HA treatment on IL-10 production. (C) Fold change in TH1, TH2, and TH17 cytokines levels upon HMW-HA costimulation (n ≥ 5 independent experiments each for A–C). (D) IL-10 production upon activation with aCD3/28, HMW-HA alone, or the two reagents in combination. Each condition was performed in triplicate; the image is representative of three experiments. (E) Fold change in IL-10 concentration in cell culture supernatants taken from mouse CD4⁺GFP/FoxP3⁻ T cells sorted on the basis of CD62L expression and activated with or without HMW-HA (n = 6). For A, C, and E, data were normalized to the aCD3/28 condition, with this value equaling 1 for each experiment.

Fig. 2.

Generated TR1 can suppress the development of colitis. (A) Impact of putative TR1 and controls on survival in an IL-10–dependent mouse colitis model (average n = 12 mice per group). (B) Histology scores for the colitis seen in the mice in A. (C) Representative histology of colon sections taken from the mice in A demonstrates goblet cell depletion, inflammatory infiltrate, epithelial shedding, and crypt microabscesses only in mice receiving aCD3/28-treated T cells or PBS solution.

Fig. 3.

CD44 cross-linking promotes MAP kinase-dependent IL-10 production. (A) HMW-HA–induced IL-10 production using WT or CD44^−/− precursor T cells (n = 7). (B) IL-10 production upon costimulation with plate-bound or soluble aCD44 (n = 5). (C) Effects of CD44 cross-linking on IL-10 production, normalized to proliferation. Data are expressed as pg/mL of IL-10 produced on a per-cpm basis. (D) Suppression assay using TR1 cells induced with aCD44 costimulation. C and D are each representative of two experiments. Representative intracellular staining (E) and fold change in IL-10 production (F) following treatment with aCD44 and selective inhibitors of p38 (SB202190), ERK1/2 (UO126), and MEK (PD98059; n = 5). (G) Fold change in IL-10 concentration in cell culture supernatants taken from mouse CD4⁺GFP/FoxP3⁻ T cells sorted on the basis of CD62L expression and activated with or without aCD44 Ab. Data were normalized to the aCD3/28 condition of the CD62L⁻ population, with this value equaling 1 for each experiment (n = 4).

Fig. 4.

OPN abrogates HMW-HA–mediated IL-10 production. (A) Effects of OPN concentration on IL-10 production (n = 3). (B) Effect of OPN on IL-10 production by WT and CD44^−/− T cells. (C) Effect of RGD or RGE peptides on OPN-mediated suppression of IL-10 production. Data for B and C are each representative of three experiments. (D) Effects of integrin antibodies on HMW-HA–mediated IL-10 production (n = 5).

Fig. 5.

A synthetic matrix promotes TR1 induction. (A) Schematic of a hydrogel that delivers a set of stimuli capable of inducing TR1 from conventional T-cell precursors. (B) IL-10 production following plate-based or hydrogel-based activation (n = 5). (C) The impact of supplemental IL-2 on IL-10 production in the setting of plate-based or HA/COL (Extracel) or HA/COL/HS (Extracel-HP) hydrogels (n = 5). (D) Representative intracellular staining for IL-10 under these same conditions. (E) Levels of TH1, TH2, and TH17 cytokines upon hydrogel-based activation (n = 3).