IL-10+CTLA-4+ Th2 Inhibitory Cells Form in a Foxp3-Independent, IL-2−Dependent Manner from Th2 Effectors during Chronic Inflammation This information is current as of September 26, 2021. John A. Altin, Chris C. Goodnow and Matthew C. Cook J Immunol published online 30 April 2012 http://www.jimmunol.org/content/early/2012/04/30/jimmun ol.1102994 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published April 30, 2012, doi:10.4049/jimmunol.1102994 The Journal of Immunology

IL-10+CTLA-4+ Th2 Inhibitory Cells Form in a Foxp3- Independent, IL-2–Dependent Manner from Th2 Effectors during Chronic Inflammation

John A. Altin,* Chris C. Goodnow,*,1 and Matthew C. Cook†,1

Activated Th cells influence other T cells via positive feedback circuits that expand and polarize particular types of response, but little is known about how they may also initiate negative feedback against immunopathological reactions. In this study, we dem- onstrate the emergence, during chronic inflammation, of GATA-3+ Th2 inhibitory (Th2i) cells that express high levels of inhibitory proteins including IL-10, CTLA-4, and granzyme B, but do so independently of Foxp3. Whereas other Th2 effectors promote proliferation and IL-4 production by naive T cells, Th2i cells suppress proliferation and IL-4 production. We show that Th2i cells develop directly from Th2 effectors, in a manner that can be promoted by effector cytokines including IL-2, IL-10, and IL-21 ex Downloaded from vivo and that requires activation through CD28, Card11, and IL-2 in vivo. Formation of Th2i cells may act as an inbuilt activation-induced feedback inhibition mechanism against excessive or chronic Th2 responses. The Journal of Immunology, 2012, 188: 000–000.

ctivated Th cells orchestrate lymphocyte responses by lated T cells in the thymus (natural Tregs) (13, 14) or periphery influencing both B and T cells: they drive B cells to (induced Tregs) (15), and their deficiency results in widespread A produce Abs and create positive feedback activation inflammation, including unrestrained Th2 responses (16, 17). http://www.jimmunol.org/ loops that reinforce particular types of T cell response (1). In the Tregs regulate Th cell responses in various ways using a suite of case of the Th2 response, IL-4–producing T cells promote their immunosuppressive proteins, including the CD86/CD80 counter- own production by means of a molecular circuit mediated by the receptor CTLA-4 (18, 19) and the cytokine IL-10 (20–22). These transcription factors STAT-6 and GATA-3 and the cytokine IL-4 molecules reduce the immunogenicity of dendritic cells and mac- (2–5). IL-4 also acts to augment immunogenic Ag presentation to rophages for CD4 T cells by downregulating MHC class II and other T cells by potently inducing MHC class II and CD86 ex- costimulatory molecules and the production of proinflammatory pression on APCs (6–8). These polarizing and amplifying effects cytokines such as IL-12 (8, 23). +

facilitate rapid and coherent T cell mobilization against a patho- Although IL-10 production by Foxp3 natural Tregs is part of by guest on September 26, 2021 gen insult; however, they also carry the risk of immunopathol- a dedicated intrathymic differentiation program that creates “an- ogy, especially in the case of a persistent Ag such as a parasite ticipatory” regulatory cells (20–22), the cytokine is also expressed or allergen. Hence, an important unanswered question is whether by activated effector T cells (24–28). There is compelling evi- Th2 cells can also initiate a counterbalancing negative feedback dence that effector T cell-derived IL-10 limits inflammation in mechanism to inhibit further recruitment of helper cells into the the setting of chronic Th1-inducing infections (29–33). However, response. whereas IL-10 was first described as a product of Th2 clones (34), A large body of work demonstrates the critical function of it remains unknown how the balance of immunostimulatory and Foxp3+ regulatory T cells (Tregs) that form before the response suppressive actions of IL-10 and IL-4 are coordinated during Th2 initiates (9–12). Foxp3+ Tregs develop from appropriately stimu- differentiation. In this study, we describe how IL-10 is synthesized by a discrete *Department of Immunology, John Curtin School of Medical Research, Australian Th2 subset that forms from Th2 effector cells during chronic in- + National University, Canberra, Australian Capital Territory 0200, Australia; and flammation in vivo. Remarkably, these IL-10 Th2 cells are also † Department of Immunology, Canberra Hospital, Canberra, Australian Capital Ter- distinguished by their upregulation of a broader program of trans- ritory 2606, Australia 1 acting T cell inhibitory molecules including CTLA-4 and gran- C.C.G. and M.C.C. contributed equally to this work. zyme B and by their ability to inhibit naive CD4+ T cell prolif- Received for publication October 10, 2011. Accepted for publication April 3, 2012. eration and IL-4 production in vitro. We show that these IL-10+ This work was supported by National Health and Medical Research Council Program Th2 inhibitory (Th2i) cells can arise directly from nonsuppressive Grant 427620 (to C.C.G. and M.C.C.). Th2 effector precursors under the influence of effector cytokines The sequences presented in this article have been submitted to ArrayExpress (http:// ebi.ac.uk/arrayexpress/) under accession number E-MTAB-948. including IL-2 and IL-21. These results clarify IL-10 production by Th2 cells in vivo and illuminate a cellular mechanism by which Address correspondence and reprint requests to Dr. Matthew C. Cook or Dr. Chris C. Goodnow, Department of Immunology, Canberra Hospital, Canberra, Australian Th2 responses may exert feedback to prevent further recruitment Capital Territory 2606, Australia (M.C.C.) or Department of Immunology, John of helper cells and immunopathological responses. Curtin School of Medical Research, Australian National University, Canberra, Aus- tralian Capital Territory 0200, Australia (C.C.G.). E-mail addresses: Matthew. [email protected] (M.C.C.) and [email protected] (C.C.G.) Materials and Methods The online version of this article contains supplemental material. Mice Abbreviations used in this article: FC, mean fold change; HEL, hen egg lysozyme; null Tg, transgenic; Th2i, Th2 inhibitory; Treg, . All mice were on the C57BL/6 background. Foxp3 males were analyzed at 20–30 d of age, and Foxp3+/null carrier females were used for breeding Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 (35). Card11unm mice were generated by ENU mutagenesis on the C57BL/

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102994 2 Th2 EFFECTORS FORM Th2 INHIBITORY CELLS DURING INFLAMMATION

6 background and have been described (36, 37). Ndfip10/0 mice (on the Suppression assays C57BL/6 background) carry an ENU-induced point mutation in a splice CFSE-labeled, sorted CD4+CD44loCD62LhiCD25– lymphocytes from donor site of the Ndfip1 gene that leads to a severely truncated Ndfip1 a/a transcript and the development of disease resembling that seen in Ndfip1null Ly5 mice (responders) were cocultured in 96-well plates with or without sorted CD3–CD4–CD8– splenocytes from Ly5a/b mice (APCs) and secreted mice (38). All animal procedures were approved by the Australian Na- + – hi + + + tional University Animal Ethics and Experimentation Committee. cytokine surface capture-sorted IL-4 IL-10 CD44 CD4 or IL-4 IL-10 CD44hiCD4+ lymphocytes from Ly5b/b Foxp3null mice (regulators) in the Ex vivo stimulation and intracellular staining presence of soluble anti-mouse CD3. Cells were plated at a ratio of APCs/ responders/regulators of 5:2:1 (normal) or 10:2:1 (stronger Ag presenta- Lymphocytes from pooled lymph nodes (cervical, inguinal, axillary, bra- tion). Total numbers of APCs per well was within the range of 1.5 3 105 to chial) were suspended in RPMI 1640 media containing 10% FCS (Life 3.75 3 105, being a constant value within a given experiment. In the IL-10 3 5 Technologies). Cells were plated at 1 10 per well in 96-well tissue neutralization experiment, IL-10 neutralizing Ab (clone JES5-2A5; BD culture plates in 200 ml of media containing PMA (50 ng/ml; Sigma), Biosciences) or isotype control (clone MG1-45; BioLegend) was added at ionomycin (500 ng/ml; Sigma), and GolgiStop (1/1000; BD) and left to 10 mg/ml. On day 3, cells were restimulated with PMA and ionomycin and accumulate cytokines for 3–4 h at 37˚C. After stimulation, cells were stained with Abs to detect Ly5a, Ly5b, IL-4, IL-10, and CTLA-4 using the harvested and surface stained for 20 min at 4˚C with allophycocyanin– method described earlier. Cy7–conjugated anti-mouse CD4 (clone GK1.5; BD), Pacific blue-con- jugated anti-mouse CD44 (clone IM7; BioLegend), Alexa Fluor 700- Cell culture conjugated anti-mouse CD45.1 (clone A20; BioLegend), PerCP–Cy5.5– Sorted IL-4+IL-10–CD44hiCD4+ cells (1 3 104) from Foxp3null mice were conjugated anti-mouse CD45.2 (clone 104; BD), FITC-conjugated anti- cultured in 96-well plates in 200 ml RPMI 1640 media containing 10% mouse ST2 (clone D.8; MD Bioproducts), and/or allophycocyanin- FCS (Life Technologies) with anti-mouse IL-2 (10 mg/ml) or the following conjugated anti-mouse CTLA-4 (clone UC10-4B9; eBioscience). Cells recombinant cytokines: IL-2 (5 ng/ml), IL-4 (10 ng/ml), IL-5 (20 ng/ml), were fixed and permeabilized using the eBioscience intracellular staining IL-6 (20 ng/ml), IL-7 (10 ng/ml), IL-10 (10 ng/ml), IL-12 (10 ng/ml), IL- Downloaded from kit and then stained for 20 min at 4˚C with PE–Cy7–conjugated anti-mouse 15 (10 ng/ml), IL-21 (10 ng/ml), or TGF-b (2 ng/ml). After 0, 20, or 44 h IL-4 (clone BVD6-24G2; eBioscience), PE- or allophycocyanin-conju- culture, cells were restimulated with PMA and ionomycin and stained with gated anti-mouse IL-10 (clone JES5-16E3; BD), allophycocyanin- Abs to detect IL-4, IL-10, and CTLA-4 using the method described earlier. conjugated anti-mouse IFN-g (clone XMG1.2; eBioscience), FITC- conjugated anti-mouse IL-17A (clone TC11-18H10.1; BioLegend), Statistical analysis FITC-conjugated anti-mouse Foxp3 (clone FJK-16a; eBioscience), Alexa Fluor 488-conjugated anti-mouse GATA3 (clone L50-823; BD), Unpaired or paired t tests were used to compare two groups of data. For allophycocyanin-conjugated anti-mouse CTLA-4 (clone UC10-4B9; situations where more than two groups were compared, ANOVA followed http://www.jimmunol.org/ eBioscience), allophycocyanin- or PE-conjugated anti-mouse IL-5 (clone by pairwise posttests were used. The a value applied for significance was TRFK5; BD), Dylight 488-conjugated anti-mouse Blimp-1 (clone 3H2-E8; 0.05. GraphPad Prism (GraphPad Software) was used for statistical analysis. Novus Biologicals), and/or allophycocyanin- or PE-conjugated anti-human granzyme B (clone GB12; Invitrogen). After staining, cells were washed Results and then read on an LSR II flow cytometer (BD). FlowJo software (Tree Star) was used for analysis. Identification of an IL-10–producing Th2 cell subset during chronic inflammation in vivo Secreted cytokine surface capture In Foxp3-deficient mice, IL-4–producing CD4 T (Th2) cells form Lymphocytes from pooled lymph nodes were suspended at 4 3 107/ml in spontaneously, typically composing .20% of the CD44hiCD4+ PBS (2 mM EDTA and 0.5% FCS) containing 20% of a premixed 1:1 activated effector/memory T cell pool present in lymph nodes (Fig. by guest on September 26, 2021 solution of the mouse IL-4 and IL-10 Catch Reagents (Miltenyi Biotec). After a 10-min incubation on ice, cells were diluted 1:200 into prewarmed 1A, 1B). We took advantage of this robustly detectable Th2 pop- RPMI 1640 media containing 10% FCS (Life Technologies), PMA (50 ng/ ulation to test whether discrete subsets of Th2 cells exist in the ml; Sigma), and ionomycin (500 ng/ml; Sigma) and then incubated for 2 h context of chronic inflammation in vivo. Single-cell analysis re- at 37˚C with constant slow rotation. Cells were then pelleted and resus- vealed two prominent Th2 subsets: one producing IL-4 without pended in PBS (2 mM EDTA and 0.5% FCS) containing the mouse IL-10 IL-10, and the other producing both IL-4 and IL-10 (Fig. 1A). Both allophycocyanin detection reagent (Miltenyi Biotec), Alexa 488-conju- – + + gated anti-mouse IL-4 (clone 11B11; BD Biosciences), allophycocyanin– IL-10 and IL-10 populations of IL-4 cells expressed high levels Cy7–conjugated anti-mouse CD4 (clone GK1.5; BD), and Pacific blue- of the master Th2 transcription factor GATA-3. Double-producing conjugated anti-mouse CD44 (clone IM7; BioLegend). IL-4+IL-10– IL-4+IL-10+ cells composed an average of ∼6% of the total CD44hi hi + + + hi + CD44 CD4 and/or IL-4 IL-10 CD44 CD4 cells were then sorted using CD4+ population and ∼15% of the total IL-4+ CD44hiCD4+ pop- a FACSAria II (BD). ulation (Fig. 1B). Foxp3null mice also contained expanded subsets Microarray analysis of CD44hiCD4+ T cells producing IFN-g (Th1) or IL-17 (Th17), + – hi + + + but these subsets contained few IL-10-producers (Fig. 1C). For the discovery experiment, IL-4 IL-10 CD44 CD4 and IL-4 IL-10 + CD44hiCD4+ cells were sorted from Foxp3null mice using secreted cytokine To determine whether formation of the identified IL-10 Th2 surface capture. Sorted cell pellets were snap-frozen and shipped on dry ice subset depends on Foxp3+ Treg deficiency or is a more general to the Miltenyi Biotec Genomic Service Department for Agilent Whole feature of chronic Th2 inflammation in vivo, we examined a sec- Genome Microarray Service. For the verification experiment, samples from ond mouse model of chronic Th2 inflammation. Ndfip1 encodes different mice were sorted in an independent experiment and the RNA extracted using the TRIzol reagent (Invitrogen). RNA quality was verified a protein that binds to the E3 ubiquitin ligase Itch, and deficiency using a Bioanalyzer (Agilent) and pellets then submitted to the Biomo- of Ndfip1 or Itch causes chronic inflammatory disease with ac- lecular Resource Facility at the John Curtin School of Medical Research cumulation of the Th2 transcription factor JunB in T cells (38–40). for reverse transcription, amplification, and hybridization to Affymetrix Analysis of CD44hiCD4+ T cells from Ndfip1-deficient mice GeneChip Mouse Gene 1.0 ST Arrays. The complete microarray data has revealed an expanded IL-4–producing population in vivo despite been deposited in ArrayExpress (http://ebi.ac.uk/arrayexpress/) under ac- + cession number E-MTAB-948. normal frequencies of Foxp3 Tregs (Fig. 1D, 1E). Moreover, the Th2 population arising in Ndfip1-deficient mice was similar to that Stimulation with HEL protein in vivo observed in Foxp3null mice as it included distinct populations of + – + + hi + Suspensions containing an equal mixture of 106 3A9-expressing CD4+ IL-4 IL-10 and IL-4 IL-10 CD44 CD4 T cells, both express- T cells each from the spleens of CD451/2 wild-type 3A9 TCR transgenic ing high levels of GATA-3, but not Foxp3 (Fig. 1E). Thus, a dis- 2/2 0/0 (Tg) and CD45 Ndfip1 3A9 TCR Tg mice were transferred intrave- tinct IL-10+ Th2 population arises during chronic Th2 nously into CD451/1 wild-type recipients. Recipients subsequently received inflammation in vivo, and this occurs within Foxp3–IL-4+ cells in i.p. injection of 100 mg hen egg lysozyme (HEL) protein in PBS or were + left uninjected, and their spleens were collected on day 6 for flow cyto- the presence or absence of the Foxp3 gene and co-residing Foxp3 metric analysis. Tregs. The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/

FIGURE 1. Identification of IL-10– and IL-10+ Th2 cell subsets in mice with chronic Th2 inflammation. (A) Production of IL-4 and IL-10 and expression of GATA-3 by gated CD44hiCD4+ lymph node T cells from wild-type and Foxp3null mice, measured by 3-h ex vivo restimulation of lymph node cells with PMA and ionomycin followed by intracellular staining. (B) Frequency of IL-4+IL-10– and IL-4+IL-10+ cells among gated CD44hiCD4+ lymph node T cells from wild-type by guest on September 26, 2021 and Foxp3null mice. (C) Production of IFN-g, IL-17, and IL-10 by gated CD44hiCD4+ lymph node T cells from wild-type and Foxp3 null mice, measured as in (A). (D) Frequency of Foxp3+ cells among gated CD4+ lymph node T cells from wild-type and Ndfip10/0 mice measured by intracellular staining. (E) Production of IL-4 and IL-10 and expression of GATA-3 and Foxp3 by gated CD44hiCD4+ lymph node T cells from Ndfip10/0 mice. Data are from at least three independent experiments.

Distinct gene expression profile of IL-10+ Th2 cells contribute to their suppressive capacity. In order of decreasing FC We next investigated whether the in vivo-formed Th2 subsets were (indicated in parentheses), these are Il10 (Ref. 22) (88), Ctla4 distinguished other than by their IL-10–producing capacity. Using (Ref. 18) (9.2), Pparg (Ref. 42) (8.7), Fgl2 (Ref. 43) (7.3), Prdm1 Gzmb Ikzf2 Ahr secreted cytokine surface capture technology (41), we isolated (Ref. 44) (3.6), (Ref. 45) (3.1), (Ref. 46) (2.9), (Ref. 47) (2.6), Ccr8 (Ref. 48) (2.8). in vivo-formed IL-4+IL-10– and IL-4+IL-10+ cells from three Using intracellular staining followed by flow cytometry, we individual Foxp3 null mice without the need for fixation or per- confirmed changes in gene expression at the protein level for meabilization (Fig. 2A), extracted RNA, and compared global CTLA-4 (Ctla4), granzyme B (Gzmb), IL-5 (Il5), ST2 (Il1rl1), and gene expression on microarrays. One hundred thirty-six genes Blimp-1 (Prdm1) (Fig. 3B). CTLA-4 was found to be the most had probes whose intensity was above the array background sensitive and specific marker of in vivo IL-10+ Th2 cells after IL- (median of the normalized intensities .3 for the six samples) and + – + + + 10 itself: total CTLA-4 was detected at low levels in IL-4 IL-10 showed a mean fold change (FC) .2.5 (IL-4 IL-10 versus IL-4 + + – cells and at a high level in most of the IL-4 IL-10 cells. Surface IL-10 ), with p value ,0.1 (comparing biological replicates) – CTLA-4 was detected on ∼10% of IL-10 cells but on a majority (Fig. 2B). of the IL-10+ cells, and its expression was nearly exclusive to Th2 This list of candidate genes that distinguish IL-10+ from IL-10– cells: IL-4+ cells accounted for ∼80% of all cells expressing Th2 cells was evaluated by repeating the same comparison of null + + + – surface CTLA-4 in Foxp3 lymph nodes (Supplemental Fig. 1). sorted IL-4 IL-10 versus IL-4 IL-10 cells in an independent IL-10+ and IL-10– Th2 subsets from Ndfip10/0 mice were also experiment using a different microarray platform. Of the 136 tested by flow cytometry for expression of Ags encoded by can- genes short-listed in the first experiment, 28 showed an FC of didate genes identified in the microarray experiments. Consistent .2.5 in the second experiment (comparing two samples of each with results from the Foxp3null mice, IL-4+IL-10+ T cells in cell type) and so are considered with high confidence to be genes Ndfip10/0 mice showed a markedly elevated expression of CTLA-4 + + upregulated in the IL-4 IL-10 subset (Fig. 3A). Of these, the and granzyme B expression relative to their IL-4+IL-10– coun- most highly upregulated was Il10 itself (FC = 88 on array ex- terparts (Supplemental Fig. 2). Notably, however, ST2, Blimp-1 periment 1, FC = 84 on array experiment 2). Strikingly, 9 of the 28 and IL-5 were not highly upregulated in IL-10+ Th2 cells from the are genes known to be upregulated in Foxp3+ Tregs and/or to Ndfip10/0 mice. 4 Th2 EFFECTORS FORM Th2 INHIBITORY CELLS DURING INFLAMMATION Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 2. Expression profile of IL-10– versus IL-10+ Th2 cells: discovery. (A) Sorting gates for isolating IL-4– and IL-10–secreting subsets of CD44hi CD4+ lymph node T cells from Foxp3null mice detected by 2-h ex vivo stimulation with PMA and ionomycin in conjunction with surface cytokine capture staining. (B) Microarray analysis of IL-4+IL-10– versus IL-4+IL-10+ cells sorted according to the gates shown in (A). The heat map shows a color-scale representation of the expression values relative to the mean (blue, below mean; red, above mean) for the 136 genes that had probes whose intensity was above the array background (median of the normalized intensities .3 for the six samples) and showed an FC .2.5 (IL-4+IL-10+ versus IL-4+IL-10–), with p value ,0.1 (comparing biological replicates). Genes are ranked by decreasing FC.

To test whether cells of the IL-10+ Th2 phenotype arise during recognizes HEL peptide in the context of H2-IAk. Equal numbers a synchronized immune response to a specific Ag, we bred of naive HEL-specific CD4+ T cells from wild-type 3A9 Tg and Ndfip10/0 mice with mice bearing the 3A9 TCR transgene that Ndfip10/0 3A9 Tg donors were then transferred into congenic The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 3. Expression profile of IL-10– versus IL-10+ Th2 cells: validation. (A) Short list of genes upregulated with high confidence in IL-10+ versus IL-10– Th2 cells, determined by two independent microarray experiments. The heat map shows a color-scale representation of the expression values relative to the mean for the 28 genes that had FC .2.5 (IL-4+IL-10+/IL-4+IL-10–) in both experiments, ranked by fold-increase. (B) Expression of total CTLA-4 (icCTLA-4), surface CTLA-4 (sCTLA-4), ST2, IL-5, Blimp-1, and granzyme B on IL-4+IL-10– (red histogram) and IL-4+IL-10+ (blue his- togram) cells from Foxp3null mice detected by ex vivo stimulation and intracellular or cell surface staining. Plots show the corresponding mean fluo- rescence intensities (MFIs) from three individual Foxp3 null mice, expressed as a percentage of the MFI of IL-4–CD44hiCD4+ cells (non-Th2 effector cells).

CD451/1 hosts, some of which were injected i.p. with 100 mg and Ndfip1-deficient mice, the foreign Ag-driven IL-4+IL-10+ HEL protein (Supplemental Fig. 3). Under these immunization cells expressed high levels of CTLA-4 and granzyme B. + + conditions, no IL-4 IL-10 cells were detected among the wild- + type donor cells, whether they were naive (in uninjected hosts) or IL-10 Th2 cells suppress proliferation Ag-activated (in HEL-injected hosts). The naive Ndfip1-deficient The analyses described earlier revealed that IL-10+ Th2 cells T cells were also uniformly IL-4–IL-10–, however a prominent upregulate a range of additional molecules that are known to in- IL-4+IL-10+ population emerged among the Ag-activated Ndfip1- hibit the activation of other T cells, including CTLA-4 (49, 50), deficient T cells. Further phenotypic analysis revealed that like Gzmb (45), and Fgl2 (43, 51). To test whether IL-4+IL-10+ cells the IL-4+IL-10+ cells that arise spontaneously in Foxp3-deficient can modify the proliferation and differentiation of naive T cells, 6 Th2 EFFECTORS FORM Th2 INHIBITORY CELLS DURING INFLAMMATION

IL-4+IL-10+ or IL-4+IL-10– cells were isolated from Foxp3 null liferation and IL-4 production were still markedly reduced when mice and added as a third population to cultures of Ly5a/a IL-4+IL-10+ cells were present (Supplemental Fig. 4). We con- (CD45.1) congenically marked and CFSE-labeled naive CD4+ clude that IL-4+IL-10+ cells are functionally distinct from IL-4+ responder cells and Ly5a/b marked APCs, together with anti-CD3 IL-10– cells because they suppress the proliferation and differ- Ab (Fig. 4A, 4B). Whereas none of the naive CD4 responder cells entiation of naive T cells. This suppression is partially dependent cultured in the absence of APCs had divided after 3 d, 50% had on IL-10 because neutralization of this cytokine led to a signifi- divided $2 times in cultures where APCs were present, although cant increase in the frequency of responders that divided in the few produced IL-4. When sorted IL-4+IL-10– cells were added presence of IL-4+IL-10+ cells (Fig. 4C). as the third population, proliferation of the responder cells was + – modestly increased, and they now exhibited a division-linked IL-10 Th2 cells form from IL-10 Th2 cells differentiation to IL-4 secretion, so that ∼90% of the naive re- In the suppression cocultures described earlier, analysis of the sponder cells were IL-4+. regulator populations at the endpoint of the assay revealed that By contrast, when IL-4+IL-10+ cells were added as the third many of the sorted IL-4+IL-10– cells had adopted an IL-4+IL-10+ population, division of the naive CD4 responder cells was mark- phenotype by the end of the culture period, but that the converse edly suppressed so that 25% had divided $2 times. IL-4 expres- was not apparent (Fig. 4A), raising the possibility of a precursor– sion by the naive responder cells was induced by the IL-4+IL-10+ progeny relation between IL-10– and IL-10+ Th2 cells. To test this cells but to a lower level than that induced by IL-4+IL-10– cells, possibility, sorted IL-4+IL-10– cells from Foxp3null donors were gauged either by overall frequency or assessed in relation to cell labeled with CFSE and cultured alone or with exogenous cyto- division number (Fig. 4A, 4B). A similar effect was observed kines before being briefly restimulated with PMA and ionomycin Downloaded from when the assay was modified by doubling the frequency of APCs: and stained intracellularly to test their cytokine-producing po- this led to more robust proliferation among responders, but pro- tential (Fig. 5A, 5B). http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 4. Effect of IL-10+ versus IL-10– Th2 cells on proliferation and differentiation of naive CD4 T cells. (A) CFSE-labeled Ly5a/a naive CD4+ T cells were analyzed as “responder” cells after 72 h of culture with anti-CD3 under four conditions: alone; together with Ly5a/b APCs (comprising T cell- depleted splenocytes); or together with APCs and Ly5b/b IL-4+IL-10–CD44hiCD4+ or IL-4+IL-10+CD44hiCD4+ cells sorted from Foxp3null mice (“regu- lators”). Left panels show Ly5 gating for responders and regulators, middle panels are gated on Ly5a/a responder cells, and right panels are gated on the Ly5b/b regulators after stimulation with PMA and ionomycin for the final 3 h. (B) Quantification of the percentage of naive CD4 responder cells that had undergone $2 cell divisions and that were IL-4+ in independent replicate cultures with the conditions in (A). (C) Quantification of the percentage of naive CD4 responder cells that had undergone $2 cell divisions in an independent suppression assay conducted in the presence of IL-10 neutralizing Ab or an isotype control. *p , 0.05. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 5. Acquisition of IL-10–producing ability, CTLA-4 expression, and suppressive function by IL-10– Th2 cells. (A) Viable IL-4+IL-10–CD44hi CD4+ cells were sorted from Foxp3null mice using secreted cytokine surface capture technology, labeled with CFSE to measure cell division, and analyzed by intracellular staining for IL-4, IL-10, and CTLA-4 after 0, 20, or 44 h of culture in the indicated conditions followed by 3-h stimulation with PMA and ionomycin. In each set of plots from 20- or 44-h cultures, the first and third columns show all cells, and the second column shows gated IL-4+ cells. The dashed vertical line indicates the median CFSE fluorescence intensity of undivided cells. (B) Quantitation of the frequency of IL-10+ cells among IL-4+ by guest on September 26, 2021 cells in independent 20-h cultures of sorted IL-4+IL-10–CD44hiCD4+ cells from three individual Foxp3null mice in the presence of the indicated cytokines and analyzed as in (A). Columns indicate the arithmetic means, and dashed lines provide a reference for the mean frequencies for control cells at 0 or 20 h in the absence of exogenous cytokines (*p , 0.05). (C) Percentage of gated naive CD4 “responder” T cells that had undergone $2 cell divisions in a sup- pression assay performed as in Fig. 4. Regulator cells added were either IL-4+IL-10+CD44hiCD4+ cells sorted from Foxp3null mice or equal numbers of sorted IL-4+IL-10–CD44hiCD4+ cells that had first been cultured with IL-2 for 0, 6, 24, or 48 h.

Consistent with their isolation on the basis of secretion of IL-4 time point could only be accounted for by division-independent but not IL-10, cells that were restimulated immediately after sorting conversion of IL-4+IL-10–CTLAlo precursors. Nevertheless, at the (without a period in culture) included a large fraction that produced 44-h time point, the cultured cells underwent some division, by IL-4, of which less than 3% coproduced IL-10. In contrast, after which time 35, 40, and 60% of the IL-2–, IL-10–, and IL-21– 20-h culture in media alone, a clear subset of IL-4+IL-10+ cells had treated cells, respectively, had become IL-10–producing, includ- developed in the absence of any cell division. These accounted for ing many that had upregulated CTLA-4 considerably relative to 15% of IL-4+ cells and were enriched for CTLA-4hi cells. The IL-4+IL-10– cells in the same cultures. formation of this subset was inhibited by addition of neutralizing We tested whether conversion of the Th2 cells into IL-10+CTLA- Ab to IL-2 (anti–IL-2) and markedly augmented by addition of 4hi cells was accompanied by acquisition of suppressive activity exogenous IL-2, both effects being observed in the absence of any against naive T cell proliferation. Sorted IL-4+IL-10– CD4 cells cell division (Fig. 5A). from Foxp3null donors were cultured as above with IL-2 for 0, 6, We tested the ability of various other factors known to influence 24, or 48 h prior to being tested for their ability to suppress the T cell survival and/or differentiation (IL-4, IL-5, IL-6, IL-7, IL-10, proliferation of naive T cells (Fig. 5C). Over these time points, the IL-12, IL-15, IL-21, TGF-b) to drive the conversion of IL-4+IL- fraction of cultured cells that had adopted an IL-10+CTLA-4hi 10– cells into IL-4+IL-10+ cells (Fig. 5B). Supplementation with phenotype increased steadily and, depending on the time point, IL-4, IL-5, IL-6, IL-7, IL-12, or IL-15 did not lead to an altered this was either associated (48 h) or not associated (0, 6, 24 h) with proportion of IL-10+ cells relative to the “media only” condition cell proliferation (data not shown). In parallel, the cultured IL-4+ after 20 h of culture. In contrast, IL-10 and IL-21 each robustly IL-10– cells gained the capacity to suppress proliferation of naive increased the proportion of IL-10+ cells to 30–40% of IL-4+ cells, responder T cells. and this was accompanied by striking increases in CTLA-4 ex- + pression on the IL-10+ cells. Addition of TGF-b led to a small, but Formation of the IL-10 Th2 subset depends on IL-2, Card11, statistically significant, decrease in the proportion of IL-10+ cells. and CD28 in vivo Because CFSE remained undiluted in all treatments at the 20-h We next investigated whether formation of the suppressive IL-10+ time point, the emergence of IL-4+IL-10+CTLA-4hi cells at this Th2 subset requires a distinctly regulated event in vivo. A sto- 8 Th2 EFFECTORS FORM Th2 INHIBITORY CELLS DURING INFLAMMATION chastic model would predict that the IL-10+ subset forms during on formation of Th2 subsets. To this end, we introduced loss-of- any Th2 response. A regulated conversion model, by contrast, function alleles of Card11 or CD28 onto the Foxp3null back- predicts that there exist stimuli necessary for the formation of the ground. Card11 is a critical intracellular scaffolding protein that IL-10+ subset from IL-10– Th2 effectors and that removal of these couples TCR and CD28 receptor signals to activation of canonical stimuli allows inflammatory states where Th2 effectors accumulate NF-kB transcription factors required for T cell activation and IL-2 but their IL-10+ Th2 progeny are selectively reduced or absent. production (36, 52–54). Homozygosity for the hypomorphic allele In view of its ability to promote IL-4+IL-10– cell formation ex Card11unm (36, 37) abolished selectively the formation of the IL- vivo, we began by testing whether IL-2 was required for the 4+IL-10+ cells while preserving the high frequency of IL-4+IL-10– subset’s formation in vivo. We generated mice that were doubly cells and severe inflammatory disease in Foxp3-deficient mice: deficient in Foxp3 and Il2. Consistent with an important role for whereas 10–18% of IL-4+ cells coproduce IL-10 in Foxp3null IL-2 in Th2 cell differentiation generally, the frequency of IL-4+ mice, this was reduced to 1–2% of IL-4 producers in Card11unm 3 IL-10– cells was ∼10-fold lower in these mice than in Foxp3null Foxp3 null mice (Fig. 6B). Mice heterozygous for Card11unm controls (Fig. 6A). However, there was an ∼100-fold decrease in showed an intermediate decrease, with 7% of IL-4+ cells pro- the frequency of IL-4+IL-10+ cells in the IL-2–deficient Foxp3null ducing IL-10 on average. Although not as striking as Card11unm, mice, indicating that formation of this subset is even more de- which interferes with both TCR and CD28 signaling, deficiency of pendent on IL-2 than other Th2 cells. CD28 itself also reduced the frequency of IL-10 producers among In view of the severe effect of IL-2 deficiency on the formation of Th2 cells formed in vivo: IL-10+ cells were about half as frequent Th2 cells generally, we next tested the effects of partial reduc- among IL-4+ cells in CD28null 3 Foxp3null mice as in CD28- tions in the efficiency of T cell activation and signaling to IL-2 sufficient controls. Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 6. Effect of loss-of-function IL-2, Card11, or CD28 on formation of IL-10+ Th2 cells. (A) Frequency of IL-4+IL-10– and IL-4+IL-10+ cells among gated CD44hiCD4+ lymph node T cells from Il2+/+Foxp3null and Il2null/nullFoxp3null mice. Data are collated from two independent experiments. (B) Plots (top) and quantitation (bottom) of IL-4 and IL-10 production by gated CD44hiCD4+ lymph node T cells from Foxp3null mice that are also bearing a hypomorphic allele of Card11 (Card11unm) or a CD28 null-allele (CD28null). (C) Expression of total CTLA-4 (icCTLA-4) and granzyme B on gated IL-4+ CD44hiCD4+ cells from Foxp3null (white histogram) and Card11unm 3 Foxp3null (gray histogram) mice detected by ex vivo stimulation and intracellular or cell surface staining. Plots show the corresponding mean fluorescence intensities (MFIs) from three individual mice of each genotype expressed as a percentage of the MFI of IL-4–CD44hiCD4+ cells (non-Th2 effector cells) in Foxp3null mice. (D) Viable IL-4+IL-10–CD44hiCD4+ cells were sorted from Card11unm 3 Foxp3null mice using secreted cytokine surface capture technology and analyzed by intracellular staining for IL-4, IL-10, and CTLA-4 after 20 or 116 h of culture with IL-2, followed by 3-h stimulation with PMA and ionomycin. The left plots show all cells, and the right plots show gated IL-4+ cells. The Journal of Immunology 9

The observed dependence on fully efficient signaling for T cell flammation and disease that is more severe than when the gene is activation extended beyond IL-10 production to the suppressive ablated selectively within Foxp3-expressing cells (18) indicates Th2 program more broadly: expression of CTLA-4 and granzyme that CTLA-4 can also mediate suppression independently of B by Th2 cells were also markedly reduced in mice homozygous Foxp3. Because IL-4+ cells account for a majority of the cells that for Card11unm (Fig. 6C). We conclude that formation of the IL-10+ express surface CTLA-4 in the lymph nodes of Foxp3null mice Th2 subset in vivo requires instructive signals mediated by opti- (Supplemental Fig. 1), and mice bearing Foxp3Cre-driven Ctla4 mal T cell activation through TCR/CD28–NF-kB signaling that ablation also develop a robust Th2 response (18), it is plausible are dispensable for formation of the IL-10– Th2 subset. that Th2i cells mediate some of the Foxp3-independent suppres- Finally, we tested whether supplementation with IL-2 could sion by CTLA-4 evident in these mice. overcome the deficit of IL-10+ Th2 cells when Card11unm was The external signals that orchestrate the conversion of Th2 cells homozygous. IL-4+IL-10– cells were sorted from Card11unm 3 into Th2i cells during chronic inflammation in vivo are likely to be Foxp3null donors and cultured in the presence of IL-2 for various multifaceted. In contrast to previous work showing that repeated periods. Although few IL-4+ cells had become IL-10+CTLA-4hi stimulation of Th2 cells in vitro with IL-4 results in accessibility of after 20-h culture, 116 h of culture led to the emergence of the IL-10 locus for transcription (60), the results here show no a prominent IL-10+CTLA-4hi Th2 population (Fig. 6D). Thus, activity of ex vivo IL-4 treatment in inducing the Th2i phenotype despite its deficiency in Card11unm 3 Foxp3null mice, the IL-10+ (Fig. 5). Rather, there are a number of indications that IL-2 may be Th2 subset can form from Card11unm IL-10– Th2 precursors when an important mediator of Th2i formation. First, some of the genes they are supplemented with exogenous IL-2 for a prolonged period. identified by microarray as increased in IL-10+ Th2 cells are

known to be induced by IL-2, including Ctla4 (61, 62), Gzmb (63), Downloaded from Discussion Prdm1 (63), and Il10 itself (63–65). Second, both IL-2 and its The findings here clarify the production of IL-10 during Th2 high-affinity receptor chain CD25 are known to be potently in- responses in vivo. They demonstrate that IL-10 is produced by duced by NF-kB signaling resulting from antigenic stimulation of a Th2 cell subset characterized by its upregulation of a suite of T cells in a CD28- and Card11-dependent manner, which makes suppressive molecules including CTLA-4 and granzyme B and by hypoinduction of autocrine or paracrine IL-2 signaling a plausible unm its ability to suppress, rather than promote, the differentiation of contributor to the failure to form Th2i cells in Card11 and http://www.jimmunol.org/ naive T cells into Th2 cells. These IL-10+ Th2i cells represent CD28null mice. Third, ex vivo culture of IL-10– Th2 cells indicates a form of feedback because they develop directly from non- that exposure to IL-2, including autocrine IL-2, provides a stimu- suppressive Th2 effector cells in a manner that can be promoted lus for division-independent conversion of IL-10– Th2 cells into by cytokines including IL-2, IL-10, and IL-21 and that depends on Th2i cells (Fig. 5). Fourth, IL-2–deficient Foxp3null mice show full T cell activation through CD28, Card11, and IL-2. Thus, our a more severe deficiency in Th2i cells than in their Th2 precursors observations illuminate a mechanistic basis by which the opposing (Fig. 6A). Such a model would be consistent with work that functions of IL-4 and IL-10 can be coordinated during Th2 establishes IL-2 as a key differentiation factor for Th2 cells in vivo responses in vivo. (66) and would extend this concept to the terminal differentiation The gene expression profile of Th2i cells is reminiscent of of Th2 cells from helper cells into cells that inhibit proliferation of by guest on September 26, 2021 Foxp3+ Tregs in that the most upregulated transcripts include other CD4 cells. It would also be consistent with the action of IL-2 molecules known to suppress the activation of other T cells. In to optimize expression of CTLA-4 and confer regulatory compe- addition to IL-10, these include CTLA-4, FGL2, and granzyme B. tence on Foxp3+ Tregs (62). That some of the hallmark suppressive arms of Foxp3+ Tregs Despite the evidence that IL-2 promotes and is required for should become coordinately expressed in cells of an effector lin- Th2i formation, CTLA-4loIL-10– Th2 cells sorted from animals eage, independently of Foxp3, is intriguing. Moreover, the fact with chronic inflammation can also become suppressive Th2i cells that Th2 cells are the predominant Th cell source of these sup- in response to IL-10 or IL-21 within 20 h and in a division- pressive molecules in Foxp3null mice (Supplemental Fig. 1) sug- independent fashion (Fig. 5A). This observation is consistent gests that Th2 cells are best equipped to fill a “suppressor” niche with a model where the concerted activity of several factors normally occupied by Foxp3+ Tregs. While it is known that (rather than a single “master” factor) coordinates the switch to Foxp3-deficient mice contain distinct populations of peripheral a suppressive state. For instance, it is likely that some of the T cells, including IL-4+ cells, that have activated the Foxp3 locus necessary transcriptional basis for IL-10 production, such as chro- [detected by a targeted reporter Foxp3gfpko allele (55)], several matin remodeling by GATA-3 (67), is set in place in IL-10– Th2 lines of evidence indicate that the Th2i subset identified in this cells, whereas other components are provided by more transient study is not an intermediate on its way to differentiating into cytokine-induced signals. STAT3 may be one such component, a Foxp3+ Treg. These are as follows: 1) a similar IL-4+IL-10+ as activation of this transcription factor is common to IL-2, IL-10, subset forms in Foxp3wt Ndfip10/0 animals and does not express and IL-21 (68–71). Notably, STAT3 has been shown to bind to Foxp3 (Fig. 1 and Supplemental Fig. 2); 2) many of the IL-10+ a motif in the human IL-10 promoter and thereby promote the cells in Foxp3gfpko mice are not actively transcribing the Foxp3 gene’s expression (72, 73). Other transcription factors that are locus (because they are GFP–) (55); and 3) the Th2i subset has known to promote IL-10 production by CD4 T cells and whose suppressive function, in contrast to GFP+ cells from Foxp3gfpko/wt message is highly upregulated in the IL-10+ Th2 subset include mice, which do not (55). PPARg (74), AHR (75), and Blimp-1 (76). Ctla4 encodes an inhibitory receptor that competes with CD28 Th2i cells have potent suppressive activity in vitro, raising the for engagement of, and acts to downregulate, CD80/CD86 co- possibility that they mediate essential negative feedback during the stimulatory molecules on APCs (56–59). Like IL-10, CTLA-4 acts in vivo Th2 responses in which they form. Moreover, as it is known to reduce the immunogenicity of Ag presentation to T cells and so that immunosuppression characterizes chronic Th2 responses, such serves an essential function in the maintenance of Th cell toler- as those that affect the large proportion of the human population ance and homeostasis (19, 49, 57). Although this function is bearing chronic parasite infection, it will be interesting to see largely attributable to its constitutive expression on the surface of whether Th2i cells also arise in the context of such infection. Foxp3+ Tregs, the fact that global deletion of Ctla4 leads to in- Conversely, failure to form Th2i cells, for example due to relative 10 Th2 EFFECTORS FORM Th2 INHIBITORY CELLS DURING INFLAMMATION deficiencies of the cytokines critical for their formation, might 2011. CD83 increases MHC II and CD86 on dendritic cells by opposing IL-10- driven MARCH1-mediated ubiquitination and degradation. J. Exp. 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