Interleukin-6 Induces the Generation of IL-10-Producing Tr1 Cells and Suppresses Autoimmune Tissue Inflammation

Abstract

Compared with its pro-inflammatory function, the mechanisms underlying the anti-inflammatory effect of IL-6 are poorly understood. IL-6 can cooperate with TGF-β to induce IL-10 production in Th17 cells in vitro. However, the effect of IL-6 on generation of Tr1 cells and the in vivo importance of this effect are mostly uncharacterized. In this study, we showed that in vitro, IL-6 can induce the generation of IL-10-producing Tr1 cells from naïve CD4 T cells, independently of IL-27 and TGF-β. IL-6 induces IL-21 production in CD4 T cells and IL-10-inducing effect of IL-6 requires both IL-21 and IL-2. Although IL-6 cannot induce IL-10 production in CD8 T cells in a cell-autonomous manner, it can do so indirectly through promoting CD4 T cell IL-21 production. The IL-10-producing T cells induced by IL-6 have phenotypic, genetic and functional traits of Tr1 cells and can suppress LPS-induced in vivo inflammatory response in an IL-10-dependent fashion. Blockade of IL-6 in two autoimmune inflammation models, induced respectively by anti-CD3 antibody or Treg-depletion, led to reduction in IL-10-producing T cells and exacerbated inflammation of lung and intestine. Thus, we delineated critical pathways involved in IL-6-induced generation of Tr1 cells and demonstrated the importance of this event in restraining autoimmune tissue inflammation.

1. Introduction

Interleukin-10 (IL-10) is a key anti-inflammatory cytokine which down-modulates both innate and adaptive immune response elicited by pathogens or self-antigens and suppresses tissue inflammation and damage [1, 2]. IL-10 potently suppresses the maturation of antigen presenting cells and also inhibits the production of pro-inflammatory cytokines by these cells [1–3]. In addition, IL-10 also can directly suppress T cell differentiation into IL-17 and IFN-γ-producing effector subsets [4, 5]. The essential role of IL-10 in suppressing inflammatory immune response and autoimmune response has been clearly demonstrated in IL-10-deficient mice, which spontaneously develop autoimmune inflammatory bowel disease largely due to heightened T helper 1 (Th1) and T helper 17 (Th17) activities and defective regulatory T cell function. These mice also have increased susceptibility to immune-related tissue inflammation and pathology in response to various microbial infections and chemical irritations [3, 6, 7].

Although IL-10 can be produced by diverse lineages of immune cells [1, 2, 8], T cell-derived IL-10 is essential for suppressing T cell hyperactivity, intestinal inflammation [7] and influenza virus-elicited lung inflammation and mortality [9]. Almost all the major T cell subsets can produce IL-10 when instructed by appropriate signals [1, 2, 8]. Regulatory type 1 helper T (Tr1) cells, an inducible subset of regulatory T cells which lack Foxp3, produce high levels of IL-10 and depend on IL-10 for their differentiation and function [1, 10]. The Foxp3⁺ regulatory T cells (Tregs) also can produce IL-10 [2, 8]. Both types of regulatory T cells employ IL-10 as a critical mechanism to suppress intestinal inflammation. Furthermore, effector Th1, Th2, Th17 and CD8 T cells also produce various amount of IL-10, which helps to limit the magnitude of respective immune response against pathogens and minimize the consequent host tissue damage [2, 8, 11].

A number of signals and transcription factors that are important for induction of IL-10 gene expression in T cells have been identified. IL-27, an IL-12 family cytokine mainly derived from dendritic cells, plays a dominant role in IL-10 production in Tr1 cells by promoting the expression of transcription factors c-Maf and AhR, which cooperate to activate IL-10 promoter [10, 12, 13]. IL-27-induced IL-10 production is critically dependent on IL-27-induced IL-21 [14, 15]. In addition, IL-2 was reported to play a crucial role in promoting IL-10 production in concert with IL-27 or complement regulator CD46 [11, 16, 17]. However, the interplay of IL-2 with other cytokines or signals in the induction of T cell IL-10 production has not been defined.

IL-6 is a pleotropic cytokine that has both pro- and anti-inflammtory function [18]. It is one of the earliest inflammatory cytokines upregulated in many infectious and inflammatory conditions. By promoting the differentiation or function of Th17 cells, T follicular helper cells and B cells, IL-6 facilitates the pathogenesis of a number of autoimmune and inflammatory diseases including experimental autoimmune encephalomyelitis, rheumatoid arthritis and poriasis [19–21]. However, in quite a few inflammatory disease conditions, such as inflammatory bone destruction, type-1 diabetes, DSS-induced colitis and streptococal septic shock syndrome, IL-6 can exert protective effects [22–25]. Hence, IL-6 can either enhance or suppress tissue inflammation and damage depending the disease, tissues, environmental signals and cytokine milieu. Compared with its pro-inflammatory effect, the anti-inflammatory function and immune-suppressive effect of IL-6 and the underlying mechanisms are much less understood. Recent studies showed that IL-6 can cooperate with TGF-β to induce IL-10 production in Th17 cells but has only moderate effect by itself [10, 26]. However, the precise action of IL-6, especially that independently of TGF-β, on T cell IL-10 production and the characteristics of the IL-10-producing T cells induced by IL-6, is not delineated. More importantly, the in vivo role of IL-6 in T cell IL-10 production is not established. A number of reports have shown that IL-6 induces IL-21 production in CD4 T cells [27–29], which suggests that IL-6 may have the ability to induce Tr1 cells through promoting IL-21 production.

In the current work, we aimed to understand the precise function and in vivo importance of IL-6 in the induction of IL-10 production in both CD4 and CD8 T cells. We showed that IL-6 promotes the differentiation of IL-10-producing Tr1-like cells from naïve CD4 T cells in the absence of both IL-27 and TGF-β. IL-6 induces IL-21 production from CD4 T cells, which in turn cooperates with IL-2 to induce IL-10-production in both CD4 and CD8 T cells. IL-6-induced IL-10-producing T cells possess functional traits of Tr1 cells in that they can suppress LPS-induced innate immune cell-mediated inflammatory response in vivo. Moreover, in two in vivo models of autoimmune inflammation, we demonstrated that IL-6 is required for IL-10 production by T cells and for suppressing tissue inflammation. We thereby delineate a new immune-regulatory mechanism underlying the anti-inflammatory effect of IL-6 that has been reported in a number of autoimmune or inflammatory diseases.

2. Materials and Methods

2.1 Mice

C57BL/6 and RAG1^−/− mice were purchased from the Jackson Laboratory and kept under pathogen-free conditions. At the time of experiments mice were 6 to 10 weeks of age. All experiments were carried out under the guidelines of the Institutional Animal Care and Use Committee at the Forsyth Institute.

2.2 Antibodies and cytokines

Cells were stained and analyzed on a FACSAria III cell sorter (Becton Dickinson). Dead cells were excluded by forward light scatter. Fluorescence-conjugated Abs with the following specificities were used for staining: CD4 (GK1.5), CD8α (536-7), CD25 (PC61), CD122 (5H4), IL-10 (JES5-16E3) and IL-17 (TC11-18H10.1) were from BioLegend; IL-2 (JES6-5H4), IFN-γ (XMG1.2), Foxp3 (FJK-16s) and purified anti-CD3ε (145-2C11) were from eBioscience; and AlexaFluor-488-pSTAT5 was from BD Pharmingen. The following monoclonal neutralizing antibodies were used for blocking cytokine activity: rat-anti-mouse IL-2 (S4B6) and its isotype control rat IgG2a (2A3), anti-mouse IL-6 (MP5-20F3) and its isotype control rat IgG1(HRPN) were from BioXcell; mouse-anti-mouse TGF-β (TW7-20B9), rat-anti-mouse IFN-γ (XMG1.2), rat-anti-mouse IL-10 (JES5-16E3) and its isotype control rat IgG2b (RTK4530) were from BioLegend. Mouse IL-21R-human IgG1 Fc fusion protein and control human IgG1 Fc, and polyclonal goat anti-mouse IL-27 were from R&D Systems. The following recombinant cytokines were used: mouse IL-6, IL-21 and human TGF-β were from PeproTech; mouse IL-2 and IL-27 were from R&D Systems.

2.3 T cell purification and in vitro stimulation

Splenocytes (2 × 10⁶/ml) from C57BL/6 mice were stimulated with soluble anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) for 3 days. Where indicated, naïve-enriched CD4 and CD8 T cells were purified from spleen and lymph nodes by negative selection using mouse CD4⁺ or CD8⁺ T cell isolation kits (Miltenyi Biotec) and placed in 24-well plate that were coated with anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) at 1 × 10⁶ per well. Cytokines or cytokine-blockade agents were added in culture as indicated. IL-6, IL-21 and IL-2 were used at 20 ng/ml, 50 ng/ml and 10 ng/ml, respectively, and blocking antibodies were used at 10 μg/ml. Anti-IL-2, anti-IL-6, IL-21R-Fc and anti-IL-27 as well as their respective control IgGs were all used at 10 μg/ml. Where indicated, a cell-permeable STAT3 inhibitor peptide or STAT5 inhibitor compound (both from Calbiochem) were added in culture at 50 μM, 15 min before the addition of IL-6. Cells were cultured for 1 or 3 days and harvested for analysis.

2.4 Intracellular staining for cytokines, Foxp3 and pSTAT5

For intracellular cytokine staining, cells were stained for surface molecules first, then fixed and permeabilized with Cytofix/Cytoperm buffer (eBioscience) and subsequently incubated with indicated anti-cytokine antibodies in Perm/Wash buffer (eBioscience) for 30 min. Intracellular Foxp3 staining was performed by using a Foxp3 Fixation/Permeabilization kit following the manufacturer’s instruction. For intracellular phosphorylated STAT5a (pSTAT5) staining, cells were fixed and permeabilized with 4% paraformaldehyde followed by methanol/acetone mixture (1:1, v/v) before incubated with anti-pSTAT5 antibody for 1 hour at room temperature. Control staining with isotype control IgGs was performed in all the experiments.

2.5 In vivo immune suppression assay

Splenocytes were stimulated and cultured with IL-6 or IL-27 plus TGF-β in vitro for 3 days. At the end of the culture, live cells, which were almost entirely T cells, were sorted with the FACSaria III. Control naïve T cells were purified from C57BL/6 mice by negative selection using MACS system (Miltenyi Biotech). 2.5 × 10⁶ Cells were transferred intravenously (i.v.) into each RAG1^−/− mice, which also received intraperitoneal (i.p.) injection of 100 μg monoclonal rat-anti-mouse IFN-γ concomitantly. Where indicated, 100 μg neutralizing monoclonal rat-anti-mouse IL-10 or its isotype control rat IgG2b were also injected i.p. to RAG1^−/− mice. 24 Hours later, LPS (200 ng, sigma) was i.p. administered into these mice and mice were sacrificed 3 hours later to collect blood and spleen.

2.6 In vivo T cell stimulation by anti-CD3

C57BL/6 mice were injected i.p. with 100 μg of a neutralizing monoclonal rat-anti-mouse IL-6 or its isotype control rat IgG1. Six hours later, all mice were injected i.p. with 20 μg monoclonal rat-anti-mouse CD3 (145-2C11). 40 hours later, the mice were injected again with same amount of anti-CD3 plus respective anti-cytokine antibodies. 4 hours after the second injection, mice were sacrificed and splenocytes and mesenteric lymph node cells were isolated and restimulated in vitro for 4 hours with phorbol 12-myristate 13-acetate (50 ng/ml) and ionomycin (1 μM; both from Calbiochem), with the addition of monensin (eBioscience) during the final 2 hours. Cells were then stained for intracellular cytokines as well as surface markers with the intracellular cytokine staining kit (eBioscience and BioLegend).

2.7 Induction of multi-organ inflammation in RAG1-deficient mice

The mixtures of splenocytes and lymph node cells from donor C57B/6 mice were incubated with biotinylated anti-CD25 (PC61.5) and anti-CD122 (TM-β1), followed by anti-biotin-conjugated magnetic beads. CD25⁻CD122⁻cells were collected by negative selection using MACS system (Miltenyi Biotec). 6 × 10⁶ CD25⁻CD122⁻cells were transferred into 6– 8 week old RAG1^−/− mice by i.v. injection. After 2 weeks, poly I:C (20 ug) was administered i.p. to mice together with 50 ug monoclonal rat-anti-IL-6 Ab or its isotype control rat IgG1, 3 times per week for 2 additional weeks. Mice were then sacrificed and organs harvested for analysis.

2.8 Preparation of tissue cell suspension

Lung, colon, liver or extra-orbital lacrimal gland were cut into small fragments and placed in a grinder and processed in a tissue homogenizer. Tissue homogenates were filtered through a 100 μm nylon mesh, washed twice in PBS and finally resuspended in culture medium.

2.9 Histology and immunofluorescence staining

Tissue samples were fixed in 4 % paraformaldehyde, embedded in paraffin and sectioned to 5 μm thickness from different areas across the tissue. Sections were then stained with hematoxylin and eosin (H&E) and examined for the presence of leukocytes under a microscope. Some paraffin sections were subjected to immunofluorescence staining after deparaffinization, re-hydration and antigen retrieval. The sections were incubated with primary biotin conjugated anti-CD3 at 4°C overnight, followed by FITC-conjugated streptavidin. The stained samples were examined with a Leica laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany). Images were average projections of three optical sections and processed with the Leica confocal software (Leica Microsystems, Wetzlar, Germany).

2.10 In situ apoptosis detection

Paraffin embedded tissue sections were de-paraffinized, hydrated and then subjected to in situ apoptosis assay using Trevigen TACS.XL In Situ Apoptosis Detection kit (purchased from R&D Systems) according to the manufacturer’s instruction. Briefly, re-hydrated tissue sections were partially digested with proteinase-K for 20 min and then endogenous peroxidases were inactivated by incubation in 3% H₂O₂. After that, DNA fragmentation was detected following the manufacturer’s protocol.

2.11 Real-time PCR

Total RNA was reverse-transcribed into cDNA using Oligo(dT) and Superscript III (Invitrogen) or M-MLV reverse transcriptase (Promega). The cDNA was subjected to real-time PCR amplification (Qiagen) for 40 cycles with annealing and extension temperature at 60°C, on a LightCycler 480 Reat-Time PCR System (Roche). Primer sequences are: c-Maf forward (F), 5′-AGCAGTTGGTGACCATGTCG-3′ ; reverse (R), 5′-TGGAGATCTCCTGCTTGAGG-3′. AhR F, 5′-CAACATCACCTATGCCAGCC-3′ ; R, 5′-TCTGTGTTCAGCCGGTCTCT-3′. IL-10 F, 5′-GCTCTTACTGACTGGCATAG-3′ ; R, 5′-CGCAGCTCTAGGAGCATGTG-3′. Prdm1 F, 5′-CCT GCT TTT CAA GTA TGC TGC -3′; R, 5′-TCA CCG ATG AGG GGT CCA AA -3′. IL-21 F, 5′-CGC CTC CTG ATT AGA CTT CG -3′; R, 5′-AAG CTG CAT GCT CAC AGT GC -3′. IRF-4 F, 5′-GGG CAA GCA GGA CTA CAA TC-3′; R, 5′-AGG ATC TGG CTT GTC GAT CC-3′. TNF-α F, 5′-CCT TTC ACT CAC TGG CCC AA -3′; R, 5′-AGT GCC TCT TCT GCC AGT TC -3′. IL-12p40 F, 5′-CAC ATC TGC TGC TCC ACA AG -3′; R, 5′-CCG TCC GGA GTA ATT TGG TG -3′. IL-23p19 F, 5′-CTC TCG GAA TCT CTG CAT GC-3′; R, 5′-ACC ATC TTC ACA CTG GAT ACG -3′.

2.12 ELISA

TNF-α and IL-12/23p40 concentration in mouse serum and TGF-β concentration in supernatants from in vitro cell cultures were determined with ELISA Max^™ kits (BioLegend) according to the manufacturer’s protocols.

2.13 Statistical analysis

All statistical significance was determined by Student’s t-test (two-tailed, two-sample equal variance). P values smaller than 0.05 were considered as statistically significant.

3. Results

3.1 IL-6 promotes generation of IL-10-producing CD4 and CD8 T cells with Tr1-like characteristics

IL-21 has been shown to promote differentiation of IL-10 production in both CD4 and CD8 T cells. In comparison, the effect of IL-6 in such events is not well characterized. We therefore assessed the effects of IL-6 on IL-10 production in both CD4 and CD8 T cells by using an in vitro T cell stimulation system that enabled us to examine cytokine production by both CD4 and CD8 T cells. We stimulated total splenocytes from C57BL/6 mice with soluble anti-CD3 plus anti-CD28 antibodies, in the absence or presence of exogenous IL-6. To compare the effect of IL-6 with IL-21, we also treated the cells with IL-21. After 3 days of culture, we noticed that IL-6 and IL-21 both preferentially promoted the expansion of CD4 T cells, as indicated by an increase in the proportion of these cells compared to cells cultured in medium alone (Fig. 1a, upper panels). Intracellular cytokine staining showed that IL-6 and IL-21 both led to a marked increase in the percentage of CD4 T cells and CD8 T cells that produced IL-10 (Fig. 1a and 1b). The absolute number of IL-10⁺ CD4 and CD8 T cells in culture was also considerably increased by both IL-6 and IL-21 (Fig. 1b, lower panels). However, unlike IL-21, IL-6 reduced the percentage of CD4 and CD8 T cells that produced IFN-γ, whereas IL-21 did not significantly affect IFN-γ production (Fig. 1b, upper panels). As a result, a markedly higher proportion of IL-10-producing CD4 or CD8 T cells induced by IL-6 are IL-10⁺IFN-γ⁻ compared with those induced by IL-21. In both culture conditions, few IL-17⁺ T cells were generated (data not shown), indicating that the endogenous TGF-β in the culture is either absent or at very low level that is not sufficient to induce IL-17 production in conjunction with IL-6. Indeed, ELISA assay showed that endogenous TGF-β levels in the culture supernatants were below 10 pg/ml (Supplemental Fig. 1). The great majority of the IL-10⁺IFN-γ⁻ cells in IL-6-treated cultures were IL-2⁻IL-4⁻IL-17⁻Foxp3⁻, thus possessing characteristics of Tr1 cells (data not shown). At molecular level, IL-6 increased IL-10 mRNA level to similar extent as IL-21 without affecting Foxp3 expression (Fig. 1C). Moreover, similarly as IL-21, IL-6 increased the expression of c-Maf and AhR, two critical transcription factors for IL-10 production and Tr1 differentiation (Fig. 1C). Hence, similarly as IL-21, IL-6 promotes the generation of IL-10-producing CD4 and CD8 T cells with phenotypic and genetic traits of Tr1 cells and it does so independently of TGF-β.

Figure 1. IL-6 and IL-21 promote IL-10 production in both CD4 and CD8 T cells.

C57BL/6 splenocytes were stimulated with anti-CD3 plus anti-CD28 for 72 hours in the presence or absence of IL-6 or IL-21. (A) Flow cytometry of surface CD4, CD8 and intracellular IL-10 and IFN-γ expression in gated CD4 T cells and CD8 T cells. Numbers indicate percentage of IL-10⁺ or IFN-γ⁺ cells within CD4 and CD8 T cells. (B) Mean percentage (upper panels) and absolute numbers (lower panels) of IL-10⁺ or IFN-γ⁺ cells within CD4 and CD8 T cells at the end of the culture. Data are the average of analyses of 12 independent samples (4 samples per experiment, total 3 independent experiments). (C) Real-time PCR analysis of gene expression, presented relative to that of β-actin, in splenocytes stimulated for 24 hours with anti-CD3 plus anti-CD28 with or without exogenous IL-6 or IL-21. Data are the average of 6 independent samples (2 samples per experiment, total 3 independent experiments).

3.2 Induction of IL-10 by IL-6 requires both IL-21 and IL-2 but not IL-27

We next wanted to determine whether IL-6 effect is dependent on IL-27, a dominant inducer of T cell IL-10 production in various immune responses. The results showed that the promoting effect of IL-6 on IL-10 was not affected by the IL-27 blockade (Fig. 2). The efficiency of anti-IL-27 antibody was confirmed by the effective inhibition of IL-27-induced IL-10 production (Supplemental Figure 2). As expected, IL-10-production induced by IL-6 was blocked by the addition of a neutralizing anti-IL-6 antibody (Supplemental Figure 2). Since a few recent reports showed that IL-2, in concert with IL-27, promotes IL-10 production in T cells, we next assessed the requirement for IL-2 in IL-6-induced IL-10 production. Interestingly, IL-10-production induced by IL-6 was completely abrogated by a neutralizing anti-IL-2 antibody (Fig. 2A). Moreover, the effect of IL-6 on IL-10 induction was almost completely abolished by IL-21R-Fc, a fusion protein that inhibits endogenous IL-21R signaling (Fig. 2A). The inhibitor function of IL-21R-Fc was confirmed by the blockade of IL-21-induced IL-10 production (Supplemental Fig. 2). Thus, IL-6 promotes the generation of IL-10-producing T cells in an IL-21- and IL-2-dependent fashion. In contrast, the inhibitory effect of IL-6 on IFN-γ production was not affected by the blockade of either IL-2 or IL-21 (Fig. 2A and 2B), and thus independent of both of these cytokines.

Figure 2. IL-6 or IL-21 induction of IL-10-producing T cells was dependent on IL-2 but not IL-27.

(A) Splenocytes were stimulated as described in Figure 1 with IL-6 in the presence of indicated blocking agents. Intracellular IL-10 and IFN-γ expression in gated CD4 T cells and CD8 T cells were analyzed on a flow cytometer. Mean percentage of IL-10⁺ or IFN-γ⁺ cells within CD4 and CD8 T cells is shown. (B) Splenocytes were stimulated and cultured with IL-21 in the presence of indicated blocking antibodies. Intracellular IL-10 and IFN-γ expression in gated CD4 T cells and CD8 T cells were analyzed on a flow cytometer. Mean percentage of IL-10⁺ or IFN-γ⁺ cells within CD4 and CD8 T cells is shown. (C) Splenocytes were stimulated and cultured with exogenous IL-2 together with indicated blocking agents. Flow cytometry of IL-10 and IFN-γ expression in gated CD4 T cells and CD8 T cells is shown. Data in A-C are representative of or the average of 6 independent samples for each group (2 samples per experiment, total 3 independent experiments). (D) Flow cytomery of surface IL-2Rα and IL-2Rβ and intracellular pSTAT5 in gated CD4 and CD8 T cells from splenocytes stimulated for 24 hours under indicated conditions. Data are representative of analyses of 4 independent samples for each group (2 samples per experiment, total 2 independent experiments).

Similarly as IL-6, the promoting effect of IL-21 on IL-10-producing T cells was not dependent on IL-27 but was dependent on IL-2 (Fig. 2B). Moreover, the effect of IL-21 did not require IL-6 (Fig. 2B), supporting the conclusion that IL-21 acts downstream of IL-6 to promote generation of IL-10-producing T cells. To assess the effect of IL-2, we added exogenous IL-2 (10 ng/ml) to the T cell stimulation culture, which led to a considerable increase in IL-10 production by both CD4 and CD8 T cells (Fig. 2C). The effect of exogenous IL-2 on IL-10 production was not affected by addition of a neutralizing anti-IL-6 or IL-21R-Fc (Fig. 2C). The neutralizing activity of anti-IL-6 was confirmed by the inhibition of IL-6-mediated IL-10 production (Supplemental Fig 2). Therefore, high level of IL-2 can induce both IL-10 and IFN-γ production independently of either IL-6 or IL-21. We next tested whether IL-6 and IL-21 induce IL-10 by enhancing IL-2 receptor expression or signaling in T cells. We stimulated splenocytes in vitro with anti-CD3 plus anti-CD28 in the absence or presence of IL-6 or IL-21 for 1 day, and assessed the surface levels of IL-2Rα or IL-2Rβ chain. Neither IL-6 nor IL-21 affected the level of these IL-2R subunits. Analysis of phosphorylated STAT5 (pSTAT5) level showed that neither IL-6 nor IL-21 significantly altered pSTAT5 level in CD4 or CD8 T cells (Fig. 2D). Hence, IL-6 and IL-21 do not affect expression or signaling of IL-2R. Thus, the effect of IL-6 and IL-21 to promote IL-10 is not achieved by enhancing IL-2 signaling, but instead by cooperating with IL-2 in a synergistic mode.

3.3 IL-6 directly affects CD4 T cells but indirectly affects CD8 T cells to promote IL-10 production

To directly assess the effect of IL-6 on CD4 T cells, we stimulated purified naïve-enriched CD4 T cells with plate bound anti-CD3 plus anti-CD28 in medium alone or in the presence of IL-6 for 3 days. We confirmed that the starting CD4 T cells were of high purity (> 99%) and more than 97% of them were CD25⁻CD44⁻CD62L^hi naïve T cells (Supplemental Fig. 3A). The naïve property of these CD4 T cells was also confirmed by the absence of IFN-γ-, IL-4- or IL-17-producing effector cells (Supplemental Fig. 3A). IL-6 considerably increased the IL-10-producing cells in purified CD4 T cells, and this effect was independent of endogenous TGF-β because blocking TGF-β by a neutralizing antibody did not affect induction of IL-10 by IL-6 (Fig. 3A). Consistent with this, the endogenous TGF-β levels in CD4 T cell culture supernatants were extremely low and well below 15 pg/ml (Supplemental Fig. 3B). The great majority of the IL-10⁺ cells induced by IL-6 were IFN-γ ⁻IL-4⁻IL-17⁻Foxp3⁻ (Supple Fig. 3C, and data not shown), and thus possessed phenotypic characteristics of Tr1 cells. Moreover, very small proportion of cells were IL-17⁺ or Foxp3⁺ in cultures in medium alone, and addition of IL-6 did not affect these percentages (Supplemental Fig. 3C). Hence, IL-6 can promote differentiation of Tr1 cells without affecting Th17 or Tregs. We also analyzed the IL-10-producing Tr1-like cells for the expression of a panel of chemokine receptors, which are differentially expressed on various effector T cell subsets. Only a small fraction (2 – 10%) of IL-10⁺ CD4 T cells expressed CXCR3, CXCR5, CCR4, CCR5 and CCR6; a significant fraction (> 10%) of these cells expressed CCR9 and few cells (< 2%) expressed CCR7 (Supplemental Fig. 3D). These characteristics were very similar to the IL-10⁺ Tr1 cells generated in the presence of IL-27 plus TGF-β conducted in the same experiments (data not shown). Taken together, IL-6-induced IL-10-producing CD4 T cells resemble Tr1 cells, based on both their cytokine profiles and chemokine receptor expression patterns.

Figure 3. IL-6 promotes differentiation of IL-10-producing T cells from purified CD4 T cells but not CD8 T cells.

(A) Purified naïve-enriched CD4 T cells or (B) CD8 T cells were stimulated with plate-bound anti-CD3 plus anti-CD28 with indicated cytokines and blocking agents for 72 hours. Flow cytometry of IL-10 and IFN-γ expression in gated CD4 T cells and CD8 T cells is shown in upper panels and mean percentage of IL-10⁺ or IFN-γ⁺ cells within CD4 and CD8 T cells is shown in lower panels. (C) Purified CD4 or CD8 T cells were stimulated and cultured with IL-6 or IL-21 in the presence of anti-IL-2 or IL-21R-Fc. IL-10 and IFN-γ expression in gated CD4 T cells and CD8 T cells is shown. (D) Mean percentage of IL-10⁺ or IFN-γ⁺ cells within CD4 (left panels) and CD8 (right panels) T cells. All the data are representative of or the average of analyses of 6 independent samples for each group (2 samples per experiment, total 3 independent experiments).

In contrast to its effect on purified CD4 T cells, IL-6 did not induce IL-10 production by purified CD8 T cells (Fig. 3B). Thus IL-6 directly promotes the generation of IL-10-producing Tr1-like cells from naïve CD4 T cells but could not directly induce the event in CD8 T cells.

We next examined whether IL-6-induced IL-10 production in purified CD4 T cells requires IL-21 and IL-2 as we have shown in the stimulation culture of splenocytes. Indeed, the induction effect of IL-6 on IL-10 production was completely abrogated by the blockade of either IL-21 or IL-2 (Fig. 3C and 3D). Moreover, IL-21 induced IL-10 production in purified CD4 T cells and this effect was also dependent on IL-2 (Fig. 3C and 3D). IL-6 and IL-21 did not exhibit synergistic effect on promoting IL-10 production when added together in culture (Supplemental Fig. 3E), further reinforcing the conclusion that IL-21 is a downstream mediator of the IL-10-inducing effect of IL-6, instead of a cooperator.

We noted that in stimulated splenocytes which contained both CD4 and CD8 T cells, IL-6 could induce IL-10 production by CD8 T cells, but in purified CD8 T cells, IL-6 could not induce this event. We reasoned that IL-10-inducing effect of IL-6 on CD8 T cells require the presence of CD4 T cells, particularly CD4 T cell-derived IL-21. Indeed, IL-21 was able to potently induce IL-10 production by purified CD8 T cells (Fig. 3C and 3D). Similarly as in CD4 T cells, the effect of IL-21 to induce IL-10 production in CD8 T cells required IL-2 (Fig. 3C and 3D).

We also investigated the effect of IL-6 and IL-21 on IFN-γ production. In purified CD4 T cells, IL-6 did not affect IFN-γ production, whereas IL-21enhanced this event (Fig. 3A and 3D). In purified CD8 T cells, both IL-6 and IL-21 exerted inhibitory effect on IFN-γ production and they did so independently of IL-2 (Fig. 3B and 3D). Thus IL-6 and IL-21 differentially affect IFN-γ production in CD4 and CD8 T cells.

3.4 Molecular mechanisms underlying the effect of IL-6

To understand the molecular mechanisms underlying the action of IL-6, we stimulated purified naïve-enriched CD4 or CD8 T cells in different conditions for 1 day and analyzed gene expression of critical transcriptional regulators of IL-10 production and Tr1 cell differentiation by real-time RT-PCR. In CD4 T cells, IL-6 significantly upregulated mRNA levels of IL-10 in an IL-2- and IL-21-dependent manner. Consistent with previous reports, IL-6 greatly upregulated expression of IL-21 and this event was partially dependent on IL-2 and independent of IL-21 (Fig. 4A). At molecular level, IL-6 dramatically upregulated expression of c-Maf, Ahr and IRF4, three crucial transcriptional activators of IL-10 gene (Fig. 4A). Moreover, the induction of these genes was completely abrogated by blockade of either IL-2 or IL-21. In conclusion, IL-6 induces expression and production of IL-21 by CD4 T cells and IL-21 in turn cooperates with IL-2 to induce expression of c-Maf, AhR and IRF4, thereby inducing IL-10 production in CD4 T cells. The induction of IL-21 is probably the critical primary effect of IL-6 that results in IL-10 induction.

Figure 4. Molecular mechanisms underlying the effect of IL-6 on IL-10-production.

Real-time PCR analysis of gene expression in purified CD4 T cells (A) or purified CD8 T cells (B) that were stimulated and cultured with IL-6 and indicated blocking agents for 24 hours, presented relative to that of β-actin. (C) Real-time PCR analysis of IL-21mRNA levels, presented relative to that of β-actin, in splenocytes that were stimulated as described in Figure 1 with indicated cytokines for 24 hours. (D) Real-time PCR analysis of gene expression in splenocytes that were stimulated with IL-6, in the absence or presence of STAT3 and STAT5 inhibitors, for 24 hours. All the data are the average of analyses of 4 independent samples for each group (2 samples per experiment, total 2 independent experiments).

In purified CD8 T cells, due to the lack of CD4 T cells, IL-6 could not directly induce IL-10 gene expression, whereas IL-21 could greatly induce IL-10 production in cooperation with IL-2 (Fig. 4B, top panel). Interestingly, IL-6 could induce IL-21 expression in CD8 T cells; however, the relative level of IL-21 induced by IL-6 was at least 100 times lower in CD8 T cells than that in CD4 T cells (Compare Fig. 4B and 4A 2^nd panels). Thus, the failure of IL-6 to directly induce IL-10 production by CD8 T cells, in the absence of CD4 T cells, is likely due to inefficient IL-21 induction in CD8 T cells. At molecular level, IL-6 could upregulate c-Maf and AhR expression to a comparable or greater degree as IL-21, whereas neither IL-6 nor IL-21 affected IRF4 expression. Thus, none of these three transcription factors seemed to be the determining factor for the induction of IL-10 by IL-21 but not IL-6. It was shown that Blimp-1 is necessary for IL-10 production by CD8 T cells induced by cooperation between IL-27 and IL-2 [11], we thus examined the expression of prdm1, the gene encoding Blimp-1. Consistent with the pattern of IL-10 expression, prdm1 expression in CD8 T cells was only slightly upregulated by IL-6 but markedly upregulated by IL-21, in an IL-2-dependent fashion (Fig. 4C). Thus, IL-21 and IL-2 cooperate to upregulate prdm1, thereby inducing IL-10 production in CD8 T cells. In contrast, IL-6 has limited ability to induce prdm1 expression in CD8 T cells and thus cannot promote IL-10 production in CD8 T cells in a cell-autonomous manner.

The major signaling components activated by IL-6 and IL-2 are STAT3 and STAT5, respectively. To assess whether the effects of IL-6 and IL-2 require these STAT molecules, we stimulated naïve CD4 T cells with IL-6 in the absence or presence of STAT3 or STAT5 inhibitors. Induction of IL-10, c-Maf and AhR expression by IL-6 and the endogenous IL-2 was greatly reduced by the addition of either inhibitor (Fig. 4D), indicating that both IL-6-activated STAT3 and IL-2-activated STAT5 are required for these events.

3.5 IL-10-producing cells induced by IL-6 were functional Tr1-like cells that suppressed in vivo LPS-induced inflammatory response

The IL-10-producing T cells generated with IL-6 exhibited Tr1-like phenotypes. To further determine whether they had Tr1-like function, we assayed for their suppressive function in vivo. We stimulated C57BL/6 splenocytes without or with IL-6 for 3 days as described earlier. We also stimulated these cells in the presence of IL-27 plus TGF-β to generate authentic Tr1 cells, which served as positive control. On day 3 of culture, more than 95% of the cells were T cells in all culture conditions. Both IL-6 and the combination of IL-27 plus TGF-β led to the generation of IL-10⁺IFN-γ⁻ Tr1-like cells as well as IL-10⁺IFN-γ⁺ cells (Fig. 5A). We sorted live cells from these cultures based on their forward- and side-scatters (Supplemental Fig. 4A) and transferred these cells into RAG1-deficient mice, which also received i.p. injection of a neutralizing anti-IFN-γ to eliminate potential effect of IFN-γ produced by some of these cells (Supplemental Fig. 4B). In addition, we transferred naïve-enriched resting T cells into RAG1-deficient mice as negative control. One day after the transfer, the RAG1-deficient mice received i.p. injection of LPS to activate the innate immune cells, particularly antigen presenting cells. Serum and spleens were collected 3 hours after LPS administration and analyzed for pro-inflammatory cytokines (Supplemental Fig. 4B). LPS treatment led to substantial increase in mRNA levels of TNF-α, IL-12p40 and IL-23p19 in splenocytes from RAG1-deficient mice that had received either naïve-enriched resting T cells or in vitro activated T cells generated in the absence of additional cytokines (Fig. 5B). By contrast, LPS-induced increase in these pro-inflammatory cytokines was markedly reduced in RAG1-deficient mice that had received T cells generated in either IL-6 or the combination of IL-27 plus TGF-β (Fig. 5B). Furthermore, serum levels of both TNF-α and IL-12p40 were markedly lower in mice transferred with activated T cells cultured with IL-6 or IL-27 plus TGF-β compared to mice transferred with naïve-enriched resting T cells or activated T cells generated in the absence of additional cytokines (Fig. 5C). To determine whether the suppressive effect of these cells is dependent on IL-10, we transferred T cells generated with IL-6 into RAG1-KO mice that received injection of a neutralizing anti-IL-10 antibody or its control IgG. Blockade of IL-10 greatly diminished the suppression of pro-inflammatory cytokine production by transferred T cells (Fig. 5D and 5E). Hence, T cells generated with IL-6 suppress LPS-induced pro-inflammatory cytokine production in an IL-10-dependent fashion, similarly to the Tr1 cells generated with IL-27 plus TGF-β.

Figure 5. IL-6-induced IL-10-producing T cells suppress in vivo LPS-induced inflammatory response.

Splenocytes from C57B/6 mice were stimulated and cultured with IL-6 or IL-27 plus TGF-β for 3 days. (A) Flow cytometry of CD4 and CD8 expression in total live cells (based on FSC and SSC gating) and IL-10 and IFN-γ expression in gated CD4 T cells and CD8 T cells at the end of the culture. (B) Electronically sorted live cells (based on FSC and SSC gating) from the above cultures or freshly isolated naïve-enriched T cells were transferred to RAG1−/− mice that have also received injection of anti-IFN-γ. After 24 hours, mice received injection of LPS and were sacrificed 3 hours later. Real-time PCR analysis of pro-inflammatory cytokine gene expression in splenocytes is presented relative to that of β-actin. (C) Serum level of TNF-α and IL-12p40 as measured by ELISA. Data in A-C are representative of or the average of analyses of 5 mice for each group (1–2 mice per experiment, total 3 independent experiments). (D) C57B/6 splenocyts were stimulated and cultured with IL-6 for 3 days and transferred into RAG1−/− mice that have also received anti-IFN-γ together with either anti-IL-10 or its isotype control IgG. Gene expression of pro- inflammatory cytokine in splenocytes was measured. (E) Serum cytokine concentrations from same mice in D. Data in C and D are the average of analyses of 4 mice for each group (2 mice per experiment, total 2 independent experiments).

3.6 IL-6 is essential for in vivo IL-10 production by both CD4 and CD8 T cells in response to anti-CD3 stimulation

Repeated injections of anti-CD3 antibody in mice can induce IL-10 production from T cells [12, 30], in addition to cytokine storm and organ inflammation. To assess the role of IL-6 in IL-10 production in vivo, we injected anti-CD3 into C57BL/6 mice that have received prior injection of control IgG or anti-IL-6. After 36 hours, mice received another dose of anti-CD3 in conjunction with IgG or anti-IL-6.

Four hours after the second injection, we measured IL-10 production in T cells by intracellular staining. In both spleen and mesenteric lymph nodes, the percentages of IL-10⁺ cells within CD4 T cells were significantly reduced (P=0.01 and 0.001, respectively) by anti-IL-6 treatment (Fig. 6A and 6B). The proportion of IL-10⁺ cells within CD8 T cells in mesenteric lymph nodes was also significantly decreased (P=0.01) by anti-IL-6, but that in splenocytes was not statistically affected (Fig. 6). In contrast to IL-10, the percentage of CD4 T cells that expressed Foxp3 was not reduced by anti-IL-6, while CD8 T cells were almost devoid of Foxp3⁺ in all treatment conditions (Fig. 6A and 6B). Interestingly, anti-IL-6 mainly reduced IL-10 production in Foxp3⁻ CD4 T cells with only slight effect on Foxp3⁺ population (Fig. 6A). Finally, IFN-γ- and IL-17- producing CD4 and CD8 T cells did not show statistically significant changes upon treatment with anti-IL-6 (Fig. 6A and 6B). Consistent with the reduction in IL-10-producing T cells, the mesenteric lymph nodes and spleens from mice treated with anti-IL-6 showed enlargement both in size and weight compared with those treated with control IgG (Supplemental Fig. 5). In summary, IL-6 is required for the optimal generation of IL-10-producing CD4 T cells and CD8 T cells in vivo induced by anti-CD3.

Figure 6. IL-6 is essential for optimal IL-10 production by T cells in vivo.

C57BL/6 mice were injected with anti-CD3 plus control IgG or anti-IL-6 twice, 40 hours apart, and analyzed 4 hours after the second injection. (A) Flow cytometry of IL-10, IFN-γ and Foxp3 expression in gated CD4 T cells and CD8 T cells from mesenteric lymph nodes (right panels) or spleen (left panels). Numbers indicate percentage of cytokine⁺ or Foxp3⁺ cells within CD4 and CD8 T cells. (B) Mean percentage of cytokine⁺ or Foxp3⁺ cells within CD4 and CD8 T cells. All the data are representative of or the average of analyses of 10 mice for each group (2 mice per experiment, total 5 independent experiments).

3.7 IL-6 suppresses anti-CD3-induced lung inflammation

To examine the biological consequences of reduced IL-10-production in T cells, we examined the presence of immune cells in several organs by H&E staining of tissue sections in the same mice described above. In mice treated with anti-CD3 plus control IgG, a low degree of inflammation was observed in lung, but not in colon, liver and lacrimal glands. Blockade of IL-6 resulted in an increase in inflammation of lung, whereas no inflammation was observed in the other organs analyzed (Fig. 7A). Flow cytometric analysis of single cell suspension from lung showed a dramatic increase in the percentage of total mononuclear cells (based on forward scatter and side scatter characteristics) (Fig. 7B). Further analysis of these cells revealed that the percentages of TCRβ⁻ cells, TCRβ⁺ CD4⁻ CD8⁻ cells, TCRβ⁺CD4 and TCRβ⁺CD8 T cells in the lung were significantly higher (P=0.001, 0.01, 0.03 and 0.01, respectively) with anti-IL-6 treatment (Fig. 7B). Immunofluorescence staining also demonstrated a considerably higher number of lung infiltrating T cells, characterized by surface CD3, in mice treated with anti-IL-6 (Fig. 7C). Finally, analysis of cytokine production by lung infiltrating T cells demonstrated that blockade of IL-6 led to a marked decrease in IL-10-producing CD4 and CD8 T cells that infiltrated lung (Fig. 7D), similarly as that observed in spleen and mesenteric lymph nodes. Thus, the reduction in IL-10 upon blockade of IL-6 is associated with enhanced numbers of T cells and other mononuclear cells in lung. We conclude that IL-6 induces IL-10 production in T cells in response to in vivo anti-CD3 stimulation and restrains lung inflammation.

Figure 7. IL-6 is essential for suppressing lung inflammation in vivo.

C57BL/6 mice were treated as described in Figure 6 with additional anti-CD3 plus IgG or anti-IL-6. (A) Hematoxylin and eosin staining of lung, colon, liver and lacrimal gland sections. (B) Upper panels, forward-scatter and side-scatter profile of cells from lung tissue. Gated cells are mononuclear cells that could contain lymphocytes, monocytes, dendritic cells and NK cells. Lower panels, percentage of TCR-β ⁻ cells, TCR⁺ CD4⁻CD8⁻ cells or TCR⁺ CD4⁺ and CD8⁺ T cells are shown in the lung. (C) Immunofluorescence staining of lung sections for CD3⁺ cells (T cells). (D) Right panels, flow cytometry of surface CD4, CD8 and intracellular IL-10 and IFN-γ expression in gated CD4 T cells and CD8 T cells in lung tissue. Left panels, mean percentage of IL-10⁺, IFN-γ⁺ or IL-17⁺ cells within CD4 and CD8 T cells. All the data are representative of or the average of analyses of 6 mice for each group (2 mice per experiment, total 3 independent experiments).

3.8 IL-6 promotes T cell IL-10 production and limits inflammation of lung and colon in a transfer-induced multi-organ inflammation model

We next evaluated the importance of IL-6 in in vivo generation of IL-10-producing T cells in a transfer-induced multi-organ inflammation model. We transferred splenocytes and lymph node cell mixture that have been depleted of both CD25⁺ and CD122⁺ regulatory T cells into RAG1-deficient mice (Supplemental Figure 6). Two weeks after the transfer, we administered low dose poly I:C to mice to activate innate immune cells (Supplemental Figure 6). The activation of both T cells and innate immune cells by regulatory T cell depletion and poly I:C injection resulted in considerable inflammation of lung and colon and a lower degree of inflammation in liver and lacrimal gland 4 weeks after the adoptive transfer, shown by H&E staining of tissue sections (Fig. 8A, upper panels). Blockade of IL-6 in the last 2 weeks, simultaneously with poly I:C injection, significantly exacerbated inflammation of all the organs examined (Fig. 8A, lower panels). Flow cytometric analysis of cytokine production by T cells in spleen and mesenteric lymph nodes showed that blockade of IL-6 resulted in a considerable decrease in IL-10-producing CD4 and CD8 T cells, without affecting percentages of IFN-γ- and IL-17-producing T cells (Supplemental Figure 7). IL-6 blockaded also led to a dramatic reduction in IL-10 production by CD4 and CD8 T cells that infiltrated lung and colon (Fig. 8B and 8C). Additionally, IL-6 blockade resulted in a decrease in IFN-γ and IL-17 production by colon-infiltrating T cells, but not lung-infiltrating T cells (Fig. 8B and 8C). To determine whether blockade of IL-6 leads to enhanced tissue damage, we analyzed apoptosis in tissue sections by in situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. There was no apoptotic cells detected in the lung in either treatment group (Supplemental Fig. 8). However, blockade of IL-6 led to a moderate increase in apoptosis in the colon and a marked increase in apoptosis in the lacrimal glands (Supplemental Fig. 8). Therefore, IL-6 promotes optimal generation of IL-10-producing T cells and restrains inflammation of multiple organs in a transfer-induced multi-organ inflammation model.

Figure 8. IL-6 restrains multi-organ inflammation in an autoimmune inflammation model.

CD25⁻CD122⁻ cells isolated from spleen and lymph nodes from C57BL/6 mice were transferred to RAG1^−/− mice. Two weeks later, the recipient mice received repeated injections of poly I:C plus IgG or anti-IL-6 for another 2 weeks. (A) Hematoxylin and eosin staining of tissue sections (scale bar = 100 μm). (B) Flow cytometry of surface CD4, CD8 and intracellular IL-10 and IFN-γ expression in gated CD4 T cells and CD8 T cells in lung tissue (right panels) or in colon tissue (left panels). (C) Mean percentage of IL-10⁺, IFN-γ⁺ or IL-17⁺ cells within CD4 and CD8 T cells. All the data are representative of or the average of analyses of 6 mice for each group (2 mice per experiment, total 3 independent experiments).

3.9 Model: IL-6 induces generation of IL-10-producing CD4 and CD8 T cells by inducing IL-21 and cooperating with IL-2

We propose a model in which IL-6 induces IL-21 production in CD4 T cells. IL-6-induced IL-21 in turn cooperates with T cell-derived IL-2 to promote IL-10-production in CD4 T cells through transcriptional factors c-Maf, AhR and IRF4. Moreover, IL-6-induced IL-21 from CD4 T cell also collaborates with IL-2 to enhance IL-10-production in CD8 T cells by upregulating Blimp1. Hence, IL-6 promotes IL-10-production in CD4 T cells in a cell intrinsic fashion, whereas it induces IL-10 in CD8 T cells in a non-cell-intrinsic fashion. In both types of cells, IL-6 effect is dependent on IL-21 and IL-2. In comparison, IL-27 employs largely similar mechanisms as IL-6 to induce IL-10 production in CD4 T cells (Fig. 9), in that they both rely on IL-21, IL-2 and the same transcriptional factors [12, 14, 16]. However, in CD8 T cells, unlike IL-6, IL-27 can cooperate with IL-2 to induce IL-10 production in an IL-21-independent manner (illustrated in Fig. 9) [11].

Figure 9. Summary of IL-6 effect on inducing IL-10-producing T cells.

IL-6 induces IL-21 production in CD4 T cells, and IL-21 in turn cooperates with T cell-produced IL-2 to promote IL-10-production in CD4 T cells by upregulating expression of c-Maf, AhR and IRF4. CD4 T cell-derived IL-21, in collaboration with IL-2, enhances IL-10-production in CD8 T cells by upregulating Blimp1. IL-27 also induces IL-21 production in CD4 T cells, through activated STAT3 and STAT1, and thus induces IL-10-production by CD4 and CD8 T cells. In addition, in CD8 T cells, IL-27 can also cooperate with IL-2 to induce Blimp1 expression and IL-10 production in an IL-21-independent manner.

4. Discussion

In this study, we demonstrated that IL-6 promotes IL-10-producing Tr1-like cells independently of both IL-27 and TGF-β. IL-6 induces IL-21 production from CD4 T cells, which in turn cooperates with IL-2 to induce IL-10-production by both CD4 and CD8 T cells through different transcriptional factors. IL-6-induced IL-10-producing T cells exhibit phenotypic, genetic and functional characteristics of authentic Tr1 cells. In two in vivo models of T cell initiated multi-organ inflammation, IL-6 is required for the optimal IL-10 production by T cells and for inhibition of tissue inflammation. Hence, IL-6 is an important promoter of T cell IL-10 production in vivo and limits tissue inflammation in certain diseases. We hereby revealed a new immune-regulatory mechanism underlying the anti-inflammatory effect of IL-6 that has been reported in a number of autoimmune or inflammatory diseases.

IL-6 was previously reported to cooperate with TGF-β to promote IL-10 production in Th17 cells [10, 31]. Here we provided first evidence that IL-6 can promote generation of functional IL-10-producing Tr1 cells and it can do so in the absence of both IL-27 and TGF-β. Compared to the authentic Tr1 cells generated in IL-27 plus TGF-β and reported in the literature, we showed that IL-6-induced IL-10-producing CD4 T cells have the same phenotypic and functional characteristics, as determined by their cytokine production, chemokine receptor expression and in vivo suppressive function. Nevertheless, it will be important to characterize other important traits of these cells, such as expression of CD25 and ICOS-1, in future studies, and compare these traits with authentic Tr1 cells. Several reports have shown that IL-6 alone appears inefficient in promoting IL-10 production in CD4 T cells [10, 15, 26]. We also found that in naïve CD4 T cells activated in vitro, IL-6 only induces about 4–5% of cells to produce IL-10. This percentage is similar to what was reported previously [10]. Comparing to the percentages of IL-10-producing cells generated with the combination of IL-6 and TGF-β, IL-6 alone appears inefficient. However, our analysis showed that although more IL-10-producing cells are generated by the combination of IL-6 and TGF-β, most of those IL-10-producing cells also simultaneously produce IL-17. In contrast, although small in amount, the IL-10-producing cells induced by IL-6 alone contain mostly cells bearing the phenotype and function of bona fide Tr1 cells, in that they do not produce any other common effector cytokines and have immune-suppressive effect in vivo.

Both IL-21 and IL-2 derived from CD4 T cells are required for IL-6-induced Tr1 generation and the induction of IL-21 in CD4 T cells is the primary effect of IL-6 in this event. In contrast, IL-6 is not sufficient to induce IL-10 production from purified CD8 T cells and it can only do so when CD4 T cells are present. The missing factor in CD8 T cells seems to be CD4 T cell-derived IL-21, because the level of IL-21 expression by purified CD8 T cells in response to IL-6 is more than 100 times lower than that produced by CD4 T cells. Moreover, provision of exogenous IL-21 is sufficient to induce IL-10 production in purified CD8 T cells. Interestingly, CD8 T cell-derived IL-2 is of sufficient amount to cooperate with IL-21 to induce IL-10 production. Taken these data together, we conclude that the primary effect of IL-6 in the generation of Tr1 cells is the induction of IL-21 production from CD4 T cells. Hence, IL-21 is a critical downstream effector for both IL-27 and IL-6 in the generation of Tr1 cells. Importantly, the effect of IL-21 requires the cooperation with IL-2 derived from activated T cells.

IL-6 is one of the first and the key inflammatory cytokines that are upregulated in various inflammatory conditions. We demonstrated that in two in vivo models of inflammatory diseases, IL-6 plays an indispensable role in optimal IL-10 production by both CD4 and CD8 T cells and consequently suppresses infiltration of tissues by T cells and other mononuclear cells. Hence, we showed a critical immune-regulatory property of IL-6 in two inflammatory disease conditions, which is achieved by promoting T cell production of IL-10. Consistent with enhanced tissue inflammation upon IL-6 blockade, increased amount of apoptotic cells was detected in colon and lacrimal glands. We did not detect cell apoptosis in the lung either with control IgG or anti-IL-6 treatment. Future studies will test whether IL-6 blockade exacerbate lung dysfunction by measuring airway hyperresponsiveness and lung protease activity. We also noted that IL-6 blockade led to various degrees of reduction in IL-17-producing T cells in either lung or colon in the two in vivo inflammation models. However IL-17-producing T cells in spleen and lymph nodes are not affected. These results suggest that IL-6 may promote the survival or expansion of IL-17-producing T cells within the target organs or facilitate the tissue migration of IL-17-producing T cells. Future studies will investigate these possibilities. However, despite a reduction in IL-17-producing T cells, IL-6 blockade leads to enhanced tissue inflammation, which suggests that the impaired IL-10-production was the dominant consequence of IL-6 blockade. It was reported that IL-6 plays a protective role in DSS-induced colitis and inhibits inflammation of colon and suppresses T cell-mediated hepatitis through negatively regulating NKT cells [24, 25]. It will be important to assess whether the enhancement of IL-10 production by IL-6 also contributes to the above protective effect of IL-6 and whether it is in fact the key mechanism. Moreover, recent studies have shown a critical role of T cell-derived IL-10 in limiting lung inflammation upon influenza virus infection [9]. IL-6 is one of the most prominent cytokines induced by TLR signals elicited by many viruses [32, 33]. Hence, it will be imperative to test whether IL-6-induced Tr1 cells can ameliorate autoimmune diseases such as colitis and viral infection-induced lung inflammation upon adoptive transfer into diseased mice. It will also be important to determine whether IL-6 induction of Tr1 cells normally plays an immune-regulatory and tissue-protective role in these diseases, by using both antibody blockade approach and IL-10- and IL-6-deficient mice. We will test these possibilities in our future studies.

Several reports have shown that IL-27 cooperates with IL-2 to promote IL-10-producing Tr1 cells and CD8 T cells [11, 16]. Moreover, IL-2 is required for anti-CD46-induced IL-10 production by human CD4 T cells [17]. Here we demonstrated that IL-6 and IL-21 also require collaboration from T cell-derived IL-2 to promote the generation of Tr1 cells. We also found blockade of IL-2 leads to similar reduction in IL-10-producing T cells and exacerbation of tissue inflammation as blockade of IL-6 in the same in vivo models (data not shown). Thus, IL-2 can employ several different mechanisms to contribute to immune suppression and tolerance, by positively regulating both Foxp3⁺ Tregs and IL-10-producing Tr1 cells.

Transcription factor c-Maf is a critical regulator of IL-10 transcription in both CD4 and CD8 T cells [12, 34]. AhR and Blimp-1 are important for IL-10 expression in CD4 and CD8 T cells, respectively [11, 12, 35]. In addition, IRF4 was reported to promote IL-10 expression in Treg and Th2 cells [36, 37]. We found that IL-6 treatment rapidly upregulates the mRNA levels of c-Maf, AhR and IRF4 in activated CD4 T cells, which correlates with the enhanced IL-10 expression. These molecular changes are dependent on both IL-21 and IL-2. Consistent with this, IL-6 was previously shown to cooperate with IL-2 to induce c-Maf and cooperates with TGF-β to induce AhR expression in CD4 T cells [38, 39]. We also demonstrate that the effects of IL-6 and IL-2 are dependent on both STAT3 and STAT5, as inhibiting the activity of either molecule severely diminishes the induction of IL-10, c-Maf and AhR. Importantly, both activated STAT3 and STAT5 can directly bind to c-Maf and IL-10 promoter, and thus cooperation between STAT5 and STAT3 may synergize to optimally activate these promoters [38, 40–42] as well as promoters of AhR and IRF4. This perhaps is the chief mechanism that underlies the synergy between STAT3- and STAT5-activating cytokines in general. However, it will be interesting to investigate whether IL-2-induced activated NFκB, in addition to STAT5, also cooperates with activated STAT3 to induce optimal IL-10 production. In CD8 T cells, IL-10 expression, which is induced by IL-21 but not by IL-6, does not correlate with the expression of the above three genes, but correlates with that of prdm1, which encodes Blimp-1. IL-21 and IL-2 cooperate to upregulate prdm1 expression and consequently IL-10 production. This is in line with previous reports that Blimp-1 is a critical promoter of IL-10 expression in CD8 T cells downstream of IL-27 [11]. Hence, CD4 and CD8 T cells employ different transcriptional machinery to control the expression of IL-10 gene, but both machineries can be activated by the cooperation of IL-21 and IL-2.

It is also worth noting that IL-6 and IL-27 utilize similar mechanisms to induce IL-10 production in CD4 T cells, in that they both rely on IL-21, IL-2 and the same transcriptional factors (illustrated in Fig. 9) [12, 14, 16]. However, unlike IL-6, which cannot induce IL-10 expression in CD8 T cells in the absence of CD4 T cell-derived IL-21, IL-27 can cooperate with IL-2 to induce IL-10 production in CD8 T cells in a CD4 T cell- and IL-21-independent manner (illustrated in Fig. 9) [11].

In this work, we also made formal demonstration that both IL-6 and IL-21 inhibit IFN-γ expression by CD8 T cells in a cell-autonomous manner. Unlike its IL-10-promoting effect, the IFN-γ-inhibiting effect of IL-6 is independently of IL-21. It is possible that since IL-6 has been shown to upregulate SOCS-1 expression in activated T cells [43], the elevated SOCS-1 level in CD8 T cells lowered the intensity or duration of IL-2 and IFN-γ signaling, thereby impairing IFN-γ expression. However, IL-6 and IL-21 exhibited different effect on IFN-γ production in purified and activated CD4 T cells, in that IL-21 promotes IFN-γ production whereas IL-6 dose not directly affect this process. The mechanisms underlying the differential effect of these two STAT3-activating cytokines require further investigation. It is possible that although both cytokines mainly act through STAT3 in the event of IL-10-induction, the difference in the intensity or duration of STAT1 activation determines the effect of these cytokines on IFN-γ gene expression. When both CD4 and CD8 T cells are present in the same culture system, IL-6 inhibits IFN-γ production in CD4 T cells in a non-cell autonomous fashion, probably by decreasing IFN-γ derived from CD8 T cells that can facilitate IFN-γ production by CD4 T cells. The negative effect of IL-6 on IFN-γ production by T cells may serve as an additional immune-regulatory function of IL-6, in addition to induction of IL-10-producing T cells.

Our findings defined a new immune-regulatory mechanism for the tissue-protective effect of IL-6, which was reported in a number of immune-mediated diseases. We showed that IL-6 is capable of inducing Tr1 cells from CD4 T cells in a cell-autonomous fashion, through induction of IL-21 and it can also promote IL-10 production by CD8 T cell indirectly through CD4 T cell-derived IL-21. Additionally, IL-6 negatively regulates IFN-γ in CD8 T cells independently of IL-21, and can inhibit IFN-γ expression by CD4 T cells indirectly through CD8 T cell-derived IFN-γ. We showed that IL-6 plays a crucial role in in vivo generation of IL-10-producing T cells and inhibition of tissue inflammation in two models of autoimmune inflammation. IL-27 and IL-2 cooperative pathway is no doubt the predominant mechanism for generation of IL-10-producing T cells in many types of infections and autoimmune responses. Our work suggests that under certain circumstances, alteration of IL-6 levels can lead to changes in T cell IL-10 production even when IL-27 and TGF-β are absent or unaffected. A deeper understanding about the anti-inflammatory potential of IL-6 could lead to development of novel treatment strategies to combat immune-mediated tissue pathologies in human.

• IL-6 can induce the generation of functional Tr1 cells from naïve CD4 T cells.

• The effect of IL-6 requires IL-21 and IL-2 but not IL-27 or TGF-β.

• IL-6-induced IL-21 cooperates with IL-2 to activate Ahr and c-Maf gene expression.

• Blockade of IL-6 impairs IL-10 production in two autoimmune inflammation models.

• Blockade of IL-6 exacerbates autoimmune inflammation in multiple organs.