IL-33 Receptor-Expressing Regulatory T Cells Are Highly Activated, Th2 Biased and Suppress CD4 T Cell Proliferation through IL-10 and TGFβ Release

Immunomodulatory Foxp3+ regulatory T cells (Tregs) form a heterogeneous population consisting of subsets with different activation states, migratory properties and suppressive functions. Recently, expression of the IL-33 receptor ST2 was shown on Tregs in inflammatory settings. Here we report that ST2 expression identifies highly activated Tregs in mice even under homeostatic conditions. ST2+ Tregs preferentially accumulate at non-lymphoid sites, likely mediated by their high expression of several chemokine receptors facilitating tissue homing. ST2+ Tregs exhibit a Th2-biased character, expressing GATA-3 and producing the Th2 cytokines IL-5 and IL-13 –especially in response to IL-33. Yet, IL-33 is dispensable for the generation and maintenance of these cells in vivo. Furthermore, ST2+ Tregs are superior to ST2− Tregs in suppressing CD4+ T cell proliferation in vitro independent of IL-33. This higher suppressive capacity is partially mediated by enhanced production and activation of the anti-inflammatory cytokines IL-10 and TGFβ. Thus, ST2 expression identifies a highly activated, strongly suppressive Treg subset preferentially located in non-lymphoid tissues. Here ST2+ Tregs may be well positioned to immediately react to IL-33 alarm signals. Their specific properties may render ST2+ Tregs useful targets for immunomodulatory therapies

ST2⁺ Tregs preferentially home outside of secondary lymphoid organs and exhibit a highly activated phenotype.

<p>Flow cytometric analysis of the phenotype and frequency of WT ST2⁺ and ST2⁻ Foxp3⁺ Tregs in spleen, pLN, blood, lung, lamina propria of the small intestine (siLP) and colon (coLP): <i>(A)</i> Frequency of ST2⁺ Tregs <i>(left)</i> and MFI of the ST2 staining on the ST2⁺ Treg fraction <i>(right)</i>. <i>(B)</i> MFI of chemokine receptor and α4β7 staining on ST2⁺ and ST2⁻ Tregs. <i>(C)</i> KLRG1 and CD103 expression in ST2⁺ <i>(top)</i> and ST2⁻ <i>(bottom)</i> Tregs from spleen; quantified frequencies from indicated organs <i>(right)</i>. <i>(D)</i> Frequency of CD44^hi, CD62L^lo and CTLA-4⁺ T cells within ST2⁺ and ST2⁻ Treg populations. <i>(E)</i> MFI of the Foxp3 staining <i>(left)</i> and geometric mean index of GATA-3 <i>(right)</i> in ST2⁺ and ST2⁻ Tregs. <i>(F)</i> Quantification of mRNA expression of the indicated genes from FACS-sorted ST2⁺ and ST2⁻ CD25⁺ Tregs from spleen and pLN <i>ex vivo</i>. mRNA expression normalized to <i>Hprt</i> endogenous control. <i>(G)</i> Frequency of ST2⁺ and ST2⁻ Tregs with IL-10 production capability as detected by GFP expression from <i>B6</i>.<i>Foxp3</i>^<i>hCD2</i> <i>xIl10</i>^<i>gfp</i> reporter mice. Fig <i>2A</i>: Data are representative of at least 2 independent experiments. Bar graphs show the mean ± SD of at least 5 biological replicates. Fig <i>2B</i>: pooled data from 2 independent experiments with 3–5 biological replicates each. Bar graphs show the mean ± SD. Fig <i>2C–2E</i> and <i>2G</i>: Data are representative of at least 2 independent experiments. Scatter plots depict one mouse as individual dot with mean ± SD. Fig <i>2F</i>: pooled data from 2 independent experiments. Significance was tested using unpaired Student’s t test. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; non-significant (ns) p > 0.05.</p

ST2⁺ Tregs express Th2 cytokines and suppress CD4⁺ T cell proliferation via IL-10 and TGFβ.

<p><i>(A-D)</i> ST2⁺ and ST2⁻ Tregs from spleen and lymph nodes of WT mice activated <i>in vitro</i> by plate-bound anti-CD3/anti-CD28 antibodies in the presence of IL-2 with or without recombinant IL-33 for 60–70 hours: <i>(A)</i> Fold change in the number of viable Tregs upon IL-33 treatment. <i>(B) Tgfb1</i> mRNA expression normalized to <i>Hprt</i> endogenous control. <i>(C)</i> Cytokine concentration in the supernatants as determined by cytometric bead array. <i>(D)</i> Geometric mean index of GATA-3 in stable ST2⁺ and ST2⁻ Tregs at the end of culture. <i>(E) In vitro</i> suppression assay with ST2⁺ and ST2⁻ Tregs as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161507#pone.0161507.g003" target="_blank">Fig 3</a> (Treg:Tresp ratio 1:5) with addition of blocking anti-IL-10R antibody or TGFβRI inhibitor. The relative division index indicates the fold increase in division of Tresp upon treatment. Division index of untreated Tresp was set to 1 in each group. <i>(F)</i> Quantification of mRNA expression of the indicated genes from sorted ST2⁺ and ST2⁻ CD25⁺ Tregs <i>ex vivo</i>. mRNA expression normalized to <i>Hprt</i> endogenous control. Fig <i>4A</i>–<i>4C</i>, <i>4E</i> and <i>4F</i>, data pooled from 2–3 independent experiments each performed with 2 replicates per condition. Fig <i>4D</i> is representative of 2 independent experiments with at least 2 replicates per condition each. Bar graphs show the mean ± SD. Significance was tested using unpaired Student’s t test. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; non-significant (ns) p > 0.05.</p

ST2⁺ Tregs suppress CD4⁺ T cell proliferation more effectively than ST2⁻ Tregs <i>in vitro</i>.

<p><i>(A)</i> Proliferation profiles of CellTrace-labelled WT CD25^- CD62L^hi CD4⁺ responder T cells (Tresp) co-cultured with WT ST2⁺ (black) and ST2⁻ (grey) CD25⁺ Tregs during an <i>in vitro</i> suppression assay at day 4 of culture. T cells were stimulated by APCs and anti-CD3 antibody with <i>(right column)</i> or without <i>(left column)</i> the addition of recombinant IL-33. Treg:Tresp ratios are indicated <i>(left)</i>. Percentage of divided cells and the division index (number in brackets) are shown in each histogram in the respective color. <i>(B)</i> Proliferation profile of Tresp cultured under the same conditions as in 3A but without Tregs, either with (grey) or without (black) the addition of anti-CD3 antibody. <i>(C)</i> MFI of the ST2 staining on all Tregs recovered from the cultures described in 3A. Data are representative of 2–3 independent experiments.</p

ST2⁺ Tregs arise independently of IL-33 signals.

<p>Phenotype of Tregs in naive WT, <i>Il1rl1</i>^<i>-/-</i> and <i>Il33</i>^<i>-/-</i> mice: <i>(A)</i> ST2 and Foxp3 expression by splenic CD4⁺ T cells of one representative naive WT mouse; quadrant numbers indicate the average frequency ± SD in 4 mice. <i>(B)</i> Frequencies and total numbers of FoxP3⁺ Tregs in spleen (Spl), peripheral lymph nodes (pLN) and lung. <i>(C)</i> Frequency of ST2 expression in Tregs of spleen and lung. <i>(D)</i> Total number of ST2⁺ Tregs in spleen, pLN and lung. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161507#pone.0161507.g002" target="_blank">Fig <i>2A</i>, <i>2C</i> and <i>2D</i></a>: Data are representative of at least 2 independent experiments. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161507#pone.0161507.g002" target="_blank">Fig <i>2B</i></a>: Pooled data from 2 independent experiments, each with 4 mice per genotype. Bar graphs show the mean ± SD of at least 4 individual mice. Significance was tested using unpaired Student’s t test. Asterisks indicate significance; all others non-significant. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.</p

Sialylated Autoantigen-Reactive IgG Antibodies Attenuate Disease Development in Autoimmune Mouse Models of Lupus Nephritis and Rheumatoid Arthritis

Pro- and anti-inflammatory effector functions of IgG antibodies (Abs) depend on their subclass and Fc glycosylation pattern. Accumulation of non-galactosylated (agalactosylated; G0) IgG Abs in the serum of rheumatoid arthritis and systemic lupus erythematosus (SLE) patients reflects severity of the diseases. In contrast, sialylated IgG Abs are responsible for anti-inflammatory effects of the intravenous immunoglobulin (pooled human serum IgG from healthy donors), administered in high doses (2 g/kg) to treat autoimmune patients. However, whether low amounts of sialylated autoantigen-reactive IgG Abs can also inhibit autoimmune diseases is hardly investigated. Here, we explore whether sialylated autoantigen-reactive IgG Abs can inhibit autoimmune pathology in different mouse models. We found that sialylated IgG auto-Abs fail to induce inflammation and lupus nephritis in a B cell receptor (BCR) transgenic lupus model, but instead are associated with lower frequencies of pathogenic Th1, Th17 and B cell responses. In accordance, the transfer of small amounts of immune complexes containing sialylated IgG Abs was sufficient to attenuate the development of nephritis. We further showed that administration of sialylated collagen type II (Col II)-specific IgG Abs attenuated the disease symptoms in a model of Col II-induced arthritis and reduced pathogenic Th17 cell and autoantigen-specific IgG Ab responses. We conclude that sialylated autoantigen-specific IgG Abs may represent a promising tool for treating pathogenic T and B cell immune responses in autoimmune diseases

Tolerance induction with T cell-dependent protein antigens induces regulatory sialylated IgGs

Background: Under inflammatory conditions, T cell-dependent (TD) protein antigens induce proinflammatory T-and B-cell responses. In contrast, tolerance induction by TD antigens without costimulation triggers the development of regulatory T cells. Under both conditions, IgG antibodies are generated, but whether they have different immunoregulatory functions remains elusive. Objective: It was shown recently that proinflammatory or anti-inflammatory effector functions of IgG molecules are determined by different Fc N-linked glycosylation patterns. We sought to examine the Fc glycosylation and anti-inflammatory quality of IgG molecules formed on TD tolerance induction. Methods: We administered chicken ovalbumin (OVA) with or without costimulus to mice and analyzed OVA-reactive IgG Fc glycosylation. The anti-inflammatory function of differentially glycosylated anti-OVA IgGs was further investigated in studies with dendritic cell cultures and in an in vivo model of allergic airway disease. Additionally, we analyzed the Fc glycosylation pattern of birch pollen-reactive serum IgGs after successful allergen-specific immunotherapy in patients. Results: Stimulation with TD antigens under inflammatory conditions induces plasma cells expressing low levels of alpha 2,6-sialyltransferase and producing desialylated IgGs. In contrast, plasma cells induced on tolerance induction did not downregulate alpha 2,6-sialyltransferase expression and secreted immunosuppressive sialylated IgGs that were sufficient to block antigen-specific T- and B-cell responses, dendritic cell maturation, and allergic airway inflammation. Importantly, successful specific immunotherapy in allergic patients also induced sialylated allergen-specific IgGs. Conclusions: Our data show a novel antigen-specific immunoregulatory mechanism mediated by anti-inflammatory sialylated IgGs that are formed on TD tolerance induction. These findings might help to develop novel antigen-specific therapies for the treatment of allergy and autoimmunity. (J Allergy Clin Immunol 2012;129:1647-55.

Presentation_1_Sialylated Autoantigen-Reactive IgG Antibodies Attenuate Disease Development in Autoimmune Mouse Models of Lupus Nephritis and Rheumatoid Arthritis.PDF

<p>Pro- and anti-inflammatory effector functions of IgG antibodies (Abs) depend on their subclass and Fc glycosylation pattern. Accumulation of non-galactosylated (agalactosylated; G0) IgG Abs in the serum of rheumatoid arthritis and systemic lupus erythematosus (SLE) patients reflects severity of the diseases. In contrast, sialylated IgG Abs are responsible for anti-inflammatory effects of the intravenous immunoglobulin (pooled human serum IgG from healthy donors), administered in high doses (2 g/kg) to treat autoimmune patients. However, whether low amounts of sialylated autoantigen-reactive IgG Abs can also inhibit autoimmune diseases is hardly investigated. Here, we explore whether sialylated autoantigen-reactive IgG Abs can inhibit autoimmune pathology in different mouse models. We found that sialylated IgG auto-Abs fail to induce inflammation and lupus nephritis in a B cell receptor (BCR) transgenic lupus model, but instead are associated with lower frequencies of pathogenic Th1, Th17 and B cell responses. In accordance, the transfer of small amounts of immune complexes containing sialylated IgG Abs was sufficient to attenuate the development of nephritis. We further showed that administration of sialylated collagen type II (Col II)-specific IgG Abs attenuated the disease symptoms in a model of Col II-induced arthritis and reduced pathogenic Th17 cell and autoantigen-specific IgG Ab responses. We conclude that sialylated autoantigen-specific IgG Abs may represent a promising tool for treating pathogenic T and B cell immune responses in autoimmune diseases.</p