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 and Matthew C. Cook
J Immunol June 1, 2012, 188 (11) 5478-5488; DOI: https://doi.org/10.4049/jimmunol.1102994

Abstract

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 demonstrate 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 vivo and that requires T cell 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.

Activated Th cells orchestrate lymphocyte responses by influencing both B and T cells: they drive B cells to produce Abs and create positive feedback activation loops that reinforce particular types of T cell response (1). In the case of the Th2 response, IL-4–producing T cells promote their own production by means of a molecular circuit mediated by the transcription factors STAT-6 and GATA-3 and the cytokine IL-4 (2–5). IL-4 also acts to augment immunogenic Ag presentation to other T cells by potently inducing MHC class II and CD86 expression on APCs (6–8). These polarizing and amplifying effects facilitate rapid and coherent T cell mobilization against a pathogen insult; however, they also carry the risk of immunopathology, especially in the case of a persistent Ag such as a parasite or allergen. Hence, an important unanswered question is whether Th2 cells can also initiate a counterbalancing negative feedback mechanism to inhibit further recruitment of helper cells into the response.

A large body of work demonstrates the critical function of Foxp3+ regulatory T cells (Tregs) that form before the response initiates (9–12). Foxp3+ Tregs develop from appropriately stimulated T cells in the thymus (natural Tregs) (13, 14) or periphery (induced Tregs) (15), and their deficiency results in widespread inflammation, including unrestrained Th2 responses (16, 17). Tregs regulate Th cell responses in various ways using a suite of immunosuppressive proteins, including the CD86/CD80 counter-receptor CTLA-4 (18, 19) and the cytokine IL-10 (20–22). These molecules reduce the immunogenicity of dendritic cells and macrophages for CD4 T cells by downregulating MHC class II and costimulatory molecules and the production of proinflammatory cytokines such as IL-12 (8, 23).

Although IL-10 production by Foxp3+ natural Tregs is part of a dedicated intrathymic differentiation program that creates “anticipatory” regulatory cells (20–22), the cytokine is also expressed by activated effector T cells (24–28). There is compelling evidence that effector T cell-derived IL-10 limits inflammation in the setting of chronic Th1-inducing infections (29–33). However, whereas IL-10 was first described as a product of Th2 clones (34), it remains unknown how the balance of immunostimulatory and suppressive actions of IL-10 and IL-4 are coordinated during Th2 differentiation.

In this study, we describe how IL-10 is synthesized by a discrete Th2 subset that forms from Th2 effector cells during chronic inflammation in vivo. Remarkably, these IL-10+ Th2 cells are also distinguished by their upregulation of a broader program of trans-acting T cell inhibitory molecules including CTLA-4 and granzyme B and by their ability to inhibit naive CD4+ T cell proliferation and IL-4 production in vitro. We show that these IL-10+ Th2 inhibitory (Th2i) cells can arise directly from nonsuppressive Th2 effector precursors under the influence of effector cytokines including IL-2 and IL-21. These results clarify IL-10 production by Th2 cells in vivo and illuminate a cellular mechanism by which Th2 responses may exert feedback to prevent further recruitment of helper cells and immunopathological responses.

Materials and Methods

Mice

All mice were on the C57BL/6 background. Foxp3null males were analyzed at 20–30 d of age, and Foxp3+/null carrier females were used for breeding (35). Card11unm mice were generated by ENU mutagenesis on the C57BL/6 background and have been described (36, 37). Ndfip10/0 mice (on the C57BL/6 background) carry an ENU-induced point mutation in a splice donor site of the Ndfip1 gene that leads to a severely truncated Ndfip1 transcript and the development of disease resembling that seen in Ndfip1null mice (38). All animal procedures were approved by the Australian National University Animal Ethics and Experimentation Committee.

Ex vivo stimulation and intracellular staining

Lymphocytes from pooled lymph nodes (cervical, inguinal, axillary, brachial) were suspended in RPMI 1640 media containing 10% FCS (Life Technologies). Cells were plated at 1 × 105 per well in 96-well tissue culture plates in 200 μl of media containing PMA (50 ng/ml; Sigma), ionomycin (500 ng/ml; Sigma), and GolgiStop (1/1000; BD) and left to accumulate cytokines for 3–4 h at 37°C. After stimulation, cells were harvested and surface stained for 20 min at 4°C with allophycocyanin–Cy7–conjugated anti-mouse CD4 (clone GK1.5; BD), Pacific blue-conjugated anti-mouse CD44 (clone IM7; BioLegend), Alexa Fluor 700-conjugated anti-mouse CD45.1 (clone A20; BioLegend), PerCP–Cy5.5–conjugated anti-mouse CD45.2 (clone 104; BD), FITC-conjugated anti-mouse ST2 (clone D.8; MD Bioproducts), and/or allophycocyanin-conjugated anti-mouse CTLA-4 (clone UC10-4B9; eBioscience). Cells were fixed and permeabilized using the eBioscience intracellular staining kit and then stained for 20 min at 4°C with PE–Cy7–conjugated anti-mouse IL-4 (clone BVD6-24G2; eBioscience), PE- or allophycocyanin-conjugated anti-mouse IL-10 (clone JES5-16E3; BD), allophycocyanin-conjugated anti-mouse IFN-γ (clone XMG1.2; eBioscience), FITC-conjugated anti-mouse IL-17A (clone TC11-18H10.1; BioLegend), FITC-conjugated anti-mouse Foxp3 (clone FJK-16a; eBioscience), Alexa Fluor 488-conjugated anti-mouse GATA3 (clone L50-823; BD), allophycocyanin-conjugated anti-mouse CTLA-4 (clone UC10-4B9; eBioscience), allophycocyanin- or PE-conjugated anti-mouse IL-5 (clone TRFK5; BD), Dylight 488-conjugated anti-mouse Blimp-1 (clone 3H2-E8; Novus Biologicals), and/or allophycocyanin- or PE-conjugated anti-human granzyme B (clone GB12; Invitrogen). After staining, cells were washed and then read on an LSR II flow cytometer (BD). FlowJo software (Tree Star) was used for analysis.

Secreted cytokine surface capture

Lymphocytes from pooled lymph nodes were suspended at 4 × 107/ml in PBS (2 mM EDTA and 0.5% FCS) containing 20% of a premixed 1:1 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 RPMI 1640 media containing 10% FCS (Life Technologies), PMA (50 ng/ml; Sigma), and ionomycin (500 ng/ml; Sigma) and then incubated for 2 h at 37°C with constant slow rotation. Cells were then pelleted and resuspended in PBS (2 mM EDTA and 0.5% FCS) containing the mouse IL-10 allophycocyanin detection reagent (Miltenyi Biotec), Alexa 488-conjugated anti-mouse IL-4 (clone 11B11; BD Biosciences), allophycocyanin–Cy7–conjugated anti-mouse CD4 (clone GK1.5; BD), and Pacific blue-conjugated anti-mouse CD44 (clone IM7; BioLegend). IL-4+IL-10–CD44hiCD4+ and/or IL-4+IL-10+CD44hiCD4+ cells were then sorted using a FACSAria II (BD).

Microarray analysis

For the discovery experiment, IL-4+IL-10–CD44hiCD4+ and IL-4+IL-10+CD44hiCD4+ cells were sorted from Foxp3null mice using secreted cytokine surface capture. Sorted cell pellets were snap-frozen and shipped on dry ice to the Miltenyi Biotec Genomic Service Department for Agilent Whole Genome Microarray Service. For the verification experiment, samples from different mice were sorted in an independent experiment and the RNA extracted using the TRIzol reagent (Invitrogen). RNA quality was verified using a Bioanalyzer (Agilent) and pellets then submitted to the Biomolecular Resource Facility at the John Curtin School of Medical Research for reverse transcription, amplification, and hybridization to Affymetrix GeneChip Mouse Gene 1.0 ST Arrays. The complete microarray data has been deposited in ArrayExpress (http://ebi.ac.uk/arrayexpress/) under accession number E-MTAB-948.

Stimulation with HEL protein in vivo

Suspensions containing an equal mixture of 106 3A9-expressing CD4+ T cells each from the spleens of CD451/2 wild-type 3A9 TCR transgenic (Tg) and CD452/2 Ndfip10/0 3A9 TCR Tg mice were transferred intravenously into CD451/1 wild-type recipients. Recipients subsequently received i.p. injection of 100 μg hen egg lysozyme (HEL) protein in PBS or were left uninjected, and their spleens were collected on day 6 for flow cytometric analysis.

Suppression assays

CFSE-labeled, sorted CD4+CD44loCD62LhiCD25– lymphocytes from Ly5a/a mice (responders) were cocultured in 96-well plates with or without sorted CD3–CD4–CD8– splenocytes from Ly5a/b mice (APCs) and secreted cytokine surface capture-sorted IL-4+IL-10–CD44hiCD4+ or IL-4+IL-10+CD44hiCD4+ lymphocytes from Ly5b/b Foxp3null mice (regulators) in the 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 presentation). Total numbers of APCs per well was within the range of 1.5 × 105 to 3.75 × 105, being a constant value within a given experiment. In the IL-10 neutralization experiment, IL-10 neutralizing Ab (clone JES5-2A5; BD Biosciences) or isotype control (clone MG1-45; BioLegend) was added at 10 μg/ml. On day 3, cells were restimulated with PMA and ionomycin and stained with Abs to detect Ly5a, Ly5b, IL-4, IL-10, and CTLA-4 using the method described earlier.

Cell culture

Sorted IL-4+IL-10–CD44hiCD4+ cells (1 × 104) from Foxp3null mice were cultured in 96-well plates in 200 μl RPMI 1640 media containing 10% FCS (Life Technologies) with anti-mouse IL-2 (10 μg/ml) or the following recombinant cytokines: IL-2 (5 ng/ml), IL-4 (10 ng/ml), IL-5 (20 ng/ml), IL-6 (20 ng/ml), IL-7 (10 ng/ml), IL-10 (10 ng/ml), IL-12 (10 ng/ml), IL-15 (10 ng/ml), IL-21 (10 ng/ml), or TGF-β (2 ng/ml). After 0, 20, or 44 h culture, cells were restimulated with PMA and ionomycin and stained with Abs to detect IL-4, IL-10, and CTLA-4 using the method described earlier.

Statistical analysis

Unpaired or paired t tests were used to compare two groups of data. For situations where more than two groups were compared, ANOVA followed by pairwise posttests were used. The α value applied for significance was 0.05. GraphPad Prism (GraphPad Software) was used for statistical analysis.

Results

Identification of an IL-10–producing Th2 cell subset during chronic inflammation in vivo

In Foxp3-deficient mice, IL-4–producing CD4 T (Th2) cells form spontaneously, typically composing >20% of the CD44hiCD4+ activated effector/memory T cell pool present in lymph nodes (Fig. 1A, 1B). We took advantage of this robustly detectable Th2 population to test whether discrete subsets of Th2 cells exist in the context of chronic inflammation in vivo. Single-cell analysis revealed two prominent Th2 subsets: one producing IL-4 without IL-10, and the other producing both IL-4 and IL-10 (Fig. 1A). Both IL-10– and IL-10+ populations of IL-4+ cells expressed high levels of the master Th2 transcription factor GATA-3. Double-producing IL-4+IL-10+ cells composed an average of ∼6% of the total CD44hiCD4+ population and ∼15% of the total IL-4+ CD44hiCD4+ population (Fig. 1B). Foxp3null mice also contained expanded subsets of CD44hiCD4+ T cells producing IFN-γ (Th1) or IL-17 (Th17), but these subsets contained few IL-10-producers (Fig. 1C).

FIGURE 1.
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 and Foxp3null mice. (C) Production of IFN-γ, IL-17, and IL-10 by gated CD44hiCD4+ lymph node T cells from wild-type and Foxp3null 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.

To determine whether formation of the identified IL-10+ Th2 subset depends on Foxp3+ Treg deficiency or is a more general feature of chronic Th2 inflammation in vivo, we examined a second mouse model of chronic Th2 inflammation. Ndfip1 encodes a protein that binds to the E3 ubiquitin ligase Itch, and deficiency of Ndfip1 or Itch causes chronic inflammatory disease with accumulation of the Th2 transcription factor JunB in T cells (38–40). Analysis of CD44hiCD4+ T cells from Ndfip1-deficient mice revealed an expanded IL-4–producing population in vivo despite normal frequencies of Foxp3+ Tregs (Fig. 1D, 1E). Moreover, the Th2 population arising in Ndfip1-deficient mice was similar to that observed in Foxp3null mice as it included distinct populations of IL-4+IL-10– and IL-4+IL-10+ CD44hiCD4+ T cells, both expressing high levels of GATA-3, but not Foxp3 (Fig. 1E). Thus, a distinct IL-10+ Th2 population arises during chronic Th2 inflammation in vivo, and this occurs within Foxp3–IL-4+ cells in the presence or absence of the Foxp3 gene and co-residing Foxp3+ Tregs.

Distinct gene expression profile of IL-10+ Th2 cells

We next investigated whether the in vivo-formed Th2 subsets were distinguished other than by their IL-10–producing capacity. Using secreted cytokine surface capture technology (41), we isolated in vivo-formed IL-4+IL-10– and IL-4+IL-10+ cells from three individual Foxp3null mice without the need for fixation or permeabilization (Fig. 2A), extracted RNA, and compared global gene expression on microarrays. One hundred thirty-six genes had probes whose intensity was above the array background (median of the normalized intensities >3 for the six samples) and showed a mean fold change (FC) >2.5 (IL-4+IL-10+ versus IL-4+IL-10–), with p value <0.1 (comparing biological replicates) (Fig. 2B).

FIGURE 2.
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 CD44hiCD4+ 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.

This list of candidate genes that distinguish IL-10+ from IL-10– Th2 cells was evaluated by repeating the same comparison of sorted IL-4+IL-10+ versus IL-4+IL-10– cells in an independent experiment using a different microarray platform. Of the 136 genes short-listed in the first experiment, 28 showed an FC of >2.5 in the second experiment (comparing two samples of each cell type) and so are considered with high confidence to be genes upregulated in the IL-4+IL-10+ subset (Fig. 3A). Of these, the most highly upregulated was Il10 itself (FC = 88 on array experiment 1, FC = 84 on array experiment 2). Strikingly, 9 of the 28 are genes known to be upregulated in Foxp3+ Tregs and/or to contribute to their suppressive capacity. In order of decreasing FC (indicated in parentheses), these are Il10 (Ref. 22) (88), Ctla4 (Ref. 18) (9.2), Pparg (Ref. 42) (8.7), Fgl2 (Ref. 43) (7.3), Prdm1 (Ref. 44) (3.6), Gzmb (Ref. 45) (3.1), Ikzf2 (Ref. 46) (2.9), Ahr (Ref. 47) (2.6), Ccr8 (Ref. 48) (2.8).

FIGURE 3.
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 histogram) cells from Foxp3null mice detected by ex vivo stimulation and intracellular or cell surface staining. Plots show the corresponding mean fluorescence intensities (MFIs) from three individual Foxp3null mice, expressed as a percentage of the MFI of IL-4–CD44hiCD4+ cells (non-Th2 effector cells).

Using intracellular staining followed by flow cytometry, we confirmed changes in gene expression at the protein level for CTLA-4 (Ctla4), granzyme B (Gzmb), IL-5 (Il5), ST2 (Il1rl1), and Blimp-1 (Prdm1) (Fig. 3B). CTLA-4 was found to be the most sensitive and specific marker of in vivo IL-10+ Th2 cells after IL-10 itself: total CTLA-4 was detected at low levels in IL-4+IL-10– cells and at a high level in most of the IL-4+IL-10+ cells. Surface CTLA-4 was detected on ∼10% of IL-10– cells but on a majority of the IL-10+ cells, and its expression was nearly exclusive to Th2 cells: IL-4+ cells accounted for ∼80% of all cells expressing surface CTLA-4 in Foxp3null lymph nodes (Supplemental Fig. 1).

IL-10+ and IL-10– Th2 subsets from Ndfip10/0 mice were also tested by flow cytometry for expression of Ags encoded by candidate genes identified in the microarray experiments. Consistent with results from the Foxp3null mice, IL-4+IL-10+ T cells in Ndfip10/0 mice showed a markedly elevated expression of CTLA-4 and granzyme B expression relative to their IL-4+IL-10– counterparts (Supplemental Fig. 2). Notably, however, ST2, Blimp-1 and IL-5 were not highly upregulated in IL-10+ Th2 cells from the Ndfip10/0 mice.

To test whether cells of the IL-10+ Th2 phenotype arise during a synchronized immune response to a specific Ag, we bred Ndfip10/0 mice with mice bearing the 3A9 TCR transgene that recognizes HEL peptide in the context of H2-IAk. Equal numbers of naive HEL-specific CD4+ T cells from wild-type 3A9 Tg and Ndfip10/0 3A9 Tg donors were then transferred into congenic CD451/1 hosts, some of which were injected i.p. with 100 μg HEL protein (Supplemental Fig. 3). Under these immunization conditions, no IL-4+IL-10+ cells were detected among the wild-type donor cells, whether they were naive (in uninjected hosts) or Ag-activated (in HEL-injected hosts). The naive Ndfip1-deficient T cells were also uniformly IL-4–IL-10–, however a prominent IL-4+IL-10+ population emerged among the Ag-activated Ndfip1-deficient T cells. Further phenotypic analysis revealed that like the IL-4+IL-10+ cells that arise spontaneously in Foxp3-deficient and Ndfip1-deficient mice, the foreign Ag-driven IL-4+IL-10+ cells expressed high levels of CTLA-4 and granzyme B.

IL-10+ Th2 cells suppress proliferation

The analyses described earlier revealed that IL-10+ Th2 cells upregulate a range of additional molecules that are known to inhibit the activation of other T cells, including CTLA-4 (49, 50), Gzmb (45), and Fgl2 (43, 51). To test whether IL-4+IL-10+ cells can modify the proliferation and differentiation of naive T cells, IL-4+IL-10+ or IL-4+IL-10– cells were isolated from Foxp3null mice and added as a third population to cultures of Ly5a/a (CD45.1) congenically marked and CFSE-labeled naive CD4+ responder cells and Ly5a/b marked APCs, together with anti-CD3 Ab (Fig. 4A, 4B). Whereas none of the naive CD4 responder cells cultured in the absence of APCs had divided after 3 d, 50% had divided ≥2 times in cultures where APCs were present, although few produced IL-4. When sorted IL-4+IL-10– cells were added as the third population, proliferation of the responder cells was modestly increased, and they now exhibited a division-linked differentiation to IL-4 secretion, so that ∼90% of the naive responder cells were IL-4+.

FIGURE 4.
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 (“regulators”). 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.

By contrast, when IL-4+IL-10+ cells were added as the third population, division of the naive CD4 responder cells was markedly suppressed so that 25% had divided ≥2 times. IL-4 expression by the naive responder cells was induced by the IL-4+IL-10+ cells but to a lower level than that induced by IL-4+IL-10– cells, gauged either by overall frequency or assessed in relation to cell division number (Fig. 4A, 4B). A similar effect was observed when the assay was modified by doubling the frequency of APCs: this led to more robust proliferation among responders, but proliferation and IL-4 production were still markedly reduced when IL-4+IL-10+ cells were present (Supplemental Fig. 4). We conclude that IL-4+IL-10+ cells are functionally distinct from IL-4+IL-10– cells because they suppress the proliferation and differentiation of naive T cells. This suppression is partially dependent on IL-10 because neutralization of this cytokine led to a significant increase in the frequency of responders that divided in the presence of IL-4+IL-10+ cells (Fig. 4C).

IL-10+ Th2 cells form from IL-10– Th2 cells

In the suppression cocultures described earlier, analysis of the regulator populations at the endpoint of the assay revealed that many of the sorted IL-4+IL-10– cells had adopted an IL-4+IL-10+ phenotype by the end of the culture period, but that the converse was not apparent (Fig. 4A), raising the possibility of a precursor–progeny relation between IL-10– and IL-10+ Th2 cells. To test this possibility, sorted IL-4+IL-10– cells from Foxp3null donors were labeled with CFSE and cultured alone or with exogenous cytokines before being briefly restimulated with PMA and ionomycin and stained intracellularly to test their cytokine-producing potential (Fig. 5A, 5B).

FIGURE 5.
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–CD44hiCD4+ 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+ 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 suppression 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 but not IL-10, cells that were restimulated immediately after sorting (without a period in culture) included a large fraction that produced IL-4, of which <3% coproduced IL-10. In contrast, after 20-h culture in media alone, a clear subset of IL-4+IL-10+ cells had developed in the absence of any cell division. These accounted for 15% of IL-4+ cells and were enriched for CTLA-4hi cells. The formation of this subset was inhibited by addition of neutralizing Ab to IL-2 (anti–IL-2) and markedly augmented by addition of exogenous IL-2, both effects being observed in the absence of any cell division (Fig. 5A).

We tested the ability of various other factors known to influence T cell survival and/or differentiation (IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15, IL-21, TGF-β) to drive the conversion of IL-4+IL-10– cells into IL-4+IL-10+ cells (Fig. 5B). Supplementation with IL-4, IL-5, IL-6, IL-7, IL-12, or IL-15 did not lead to an altered proportion of IL-10+ cells relative to the “media only” condition after 20 h of culture. In contrast, IL-10 and IL-21 each robustly increased the proportion of IL-10+ cells to 30–40% of IL-4+ cells, and this was accompanied by striking increases in CTLA-4 expression on the IL-10+ cells. Addition of TGF-β led to a small, but statistically significant, decrease in the proportion of IL-10+ cells. Because CFSE remained undiluted in all treatments at the 20-h time point, the emergence of IL-4+IL-10+CTLA-4hi cells at this time point could only be accounted for by division-independent conversion of IL-4+IL-10–CTLAlo precursors. Nevertheless, at the 44-h time point, the cultured cells underwent some division, by which time 35, 40, and 60% of the IL-2–, IL-10–, and IL-21–treated cells, respectively, had become IL-10–producing, including many that had upregulated CTLA-4 considerably relative to IL-4+IL-10– cells in the same cultures.

We tested whether conversion of the Th2 cells into IL-10+CTLA-4hi cells was accompanied by acquisition of suppressive activity against naive T cell proliferation. Sorted IL-4+IL-10– CD4 cells from Foxp3null donors were cultured as above with IL-2 for 0, 6, 24, or 48 h prior to being tested for their ability to suppress the proliferation of naive T cells (Fig. 5C). Over these time points, the fraction of cultured cells that had adopted an IL-10+CTLA-4hi phenotype increased steadily and, depending on the time point, this was either associated (48 h) or not associated (0, 6, 24 h) with cell proliferation (data not shown). In parallel, the cultured IL-4+IL-10– cells gained the capacity to suppress proliferation of naive responder T cells.

Formation of the IL-10+ Th2 subset depends on IL-2, Card11, and CD28 in vivo

We next investigated whether formation of the suppressive IL-10+ Th2 subset requires a distinctly regulated event in vivo. A stochastic model would predict that the IL-10+ subset forms during any Th2 response. A regulated conversion model, by contrast, predicts that there exist stimuli necessary for the formation of the IL-10+ subset from IL-10– Th2 effectors and that removal of these stimuli allows inflammatory states where Th2 effectors accumulate but their IL-10+ Th2 progeny are selectively reduced or absent.

In view of its ability to promote IL-4+IL-10– cell formation ex vivo, we began by testing whether IL-2 was required for the subset’s formation in vivo. We generated mice that were doubly deficient in Foxp3 and Il2. Consistent with an important role for IL-2 in Th2 cell differentiation generally, the frequency of IL-4+IL-10– cells was ∼10-fold lower in these mice than in Foxp3null controls (Fig. 6A). However, there was an ∼100-fold decrease in the frequency of IL-4+IL-10+ cells in the IL-2–deficient Foxp3null mice, indicating that formation of this subset is even more dependent on IL-2 than other Th2 cells.

FIGURE 6.
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 × 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 × 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.

In view of the severe effect of IL-2 deficiency on the formation of Th2 cells generally, we next tested the effects of partial reductions in the efficiency of T cell activation and signaling to IL-2 on formation of Th2 subsets. To this end, we introduced loss-of-function alleles of Card11 or CD28 onto the Foxp3null background. Card11 is a critical intracellular scaffolding protein that couples TCR and CD28 receptor signals to activation of canonical NF-κB transcription factors required for T cell activation and IL-2 production (36, 52–54). Homozygosity for the hypomorphic allele Card11unm (36, 37) abolished selectively the formation of the IL-4+IL-10+ cells while preserving the high frequency of IL-4+IL-10– cells and severe inflammatory disease in Foxp3-deficient mice: whereas 10–18% of IL-4+ cells coproduce IL-10 in Foxp3null mice, this was reduced to 1–2% of IL-4 producers in Card11unm × Foxp3null mice (Fig. 6B). Mice heterozygous for Card11unm showed an intermediate decrease, with 7% of IL-4+ cells producing IL-10 on average. Although not as striking as Card11unm, which interferes with both TCR and CD28 signaling, deficiency of CD28 itself also reduced the frequency of IL-10 producers among Th2 cells formed in vivo: IL-10+ cells were about half as frequent among IL-4+ cells in CD28null × Foxp3null mice as in CD28-sufficient controls.

The observed dependence on fully efficient signaling for T cell activation extended beyond IL-10 production to the suppressive Th2 program more broadly: expression of CTLA-4 and granzyme B by Th2 cells were also markedly reduced in mice homozygous for Card11unm (Fig. 6C). We conclude that formation of the IL-10+ Th2 subset in vivo requires instructive signals mediated by optimal T cell activation through TCR/CD28–NF-κB signaling that are dispensable for formation of the IL-10– Th2 subset.

Finally, we tested whether supplementation with IL-2 could overcome the deficit of IL-10+ Th2 cells when Card11unm was homozygous. IL-4+IL-10– cells were sorted from Card11unm × Foxp3null donors and cultured in the presence of IL-2 for various periods. Although few IL-4+ cells had become IL-10+CTLA-4hi after 20-h culture, 116 h of culture led to the emergence of a prominent IL-10+CTLA-4hi Th2 population (Fig. 6D). Thus, despite its deficiency in Card11unm × Foxp3null mice, the IL-10+ Th2 subset can form from Card11unm IL-10– Th2 precursors when they are supplemented with exogenous IL-2 for a prolonged period.

Discussion

The findings here clarify the production of IL-10 during Th2 responses in vivo. They demonstrate that IL-10 is produced by a Th2 cell subset characterized by its upregulation of a suite of suppressive molecules including CTLA-4 and granzyme B and by its ability to suppress, rather than promote, the differentiation of naive T cells into Th2 cells. These IL-10+ Th2i cells represent a form of feedback because they develop directly from nonsuppressive Th2 effector cells in a manner that can be promoted by cytokines including IL-2, IL-10, and IL-21 and that depends on full T cell activation through CD28, Card11, and IL-2. Thus, our observations illuminate a mechanistic basis by which the opposing functions of IL-4 and IL-10 can be coordinated during Th2 responses in vivo.

The gene expression profile of Th2i cells is reminiscent of Foxp3+ Tregs in that the most upregulated transcripts include molecules known to suppress the activation of other T cells. In addition to IL-10, these include CTLA-4, FGL2, and granzyme B. That some of the hallmark suppressive arms of Foxp3+ Tregs should become coordinately expressed in cells of an effector lineage, independently of Foxp3, is intriguing. Moreover, the fact that Th2 cells are the predominant Th cell source of these suppressive molecules in Foxp3null mice (Supplemental Fig. 1) suggests that Th2 cells are best equipped to fill a “suppressor” niche normally occupied by Foxp3+ Tregs. While it is known that Foxp3-deficient mice contain distinct populations of peripheral T cells, including IL-4+ cells, that have activated the Foxp3 locus [detected by a targeted reporter Foxp3gfpko allele (55)], several lines of evidence indicate that the Th2i subset identified in this study is not an intermediate on its way to differentiating into a Foxp3+ Treg. These are as follows: 1) a similar IL-4+IL-10+ subset forms in Foxp3wt Ndfip10/0 animals and does not express Foxp3 (Fig. 1 and Supplemental Fig. 2); 2) many of the IL-10+ cells in Foxp3gfpko mice are not actively transcribing the Foxp3 locus (because they are GFP–) (55); and 3) the Th2i subset has suppressive function, in contrast to GFP+ cells from Foxp3gfpko/wt mice, which do not (55).

Ctla4 encodes an inhibitory receptor that competes with CD28 for engagement of, and acts to downregulate, CD80/CD86 costimulatory molecules on APCs (56–59). Like IL-10, CTLA-4 acts to reduce the immunogenicity of Ag presentation to T cells and so serves an essential function in the maintenance of Th cell tolerance and homeostasis (19, 49, 57). Although this function is largely attributable to its constitutive expression on the surface of Foxp3+ Tregs, the fact that global deletion of Ctla4 leads to inflammation and disease that is more severe than when the gene is ablated selectively within Foxp3-expressing cells (18) indicates that CTLA-4 can also mediate suppression independently of Foxp3. Because IL-4+ cells account for a majority of the cells that express surface CTLA-4 in the lymph nodes of Foxp3null mice (Supplemental Fig. 1), and mice bearing Foxp3Cre-driven Ctla4 ablation also develop a robust Th2 response (18), it is plausible that Th2i cells mediate some of the Foxp3-independent suppression by CTLA-4 evident in these mice.

The external signals that orchestrate the conversion of Th2 cells into Th2i cells during chronic inflammation in vivo are likely to be multifaceted. In contrast to previous work showing that repeated stimulation of Th2 cells in vitro with IL-4 results in accessibility of the IL-10 locus for transcription (60), the results here show no activity of ex vivo IL-4 treatment in inducing the Th2i phenotype (Fig. 5). Rather, there are a number of indications that IL-2 may be an important mediator of Th2i formation. First, some of the genes identified by microarray as increased in IL-10+ Th2 cells are known to be induced by IL-2, including Ctla4 (61, 62), Gzmb (63), Prdm1 (63), and Il10 itself (63–65). Second, both IL-2 and its high-affinity receptor chain CD25 are known to be potently induced by NF-κB signaling resulting from antigenic stimulation of T cells in a CD28- and Card11-dependent manner, which makes hypoinduction of autocrine or paracrine IL-2 signaling a plausible contributor to the failure to form Th2i cells in Card11unm and CD28null mice. Third, ex vivo culture of IL-10– Th2 cells indicates that exposure to IL-2, including autocrine IL-2, provides a stimulus for division-independent conversion of IL-10– Th2 cells into Th2i cells (Fig. 5). Fourth, IL-2–deficient Foxp3null mice show a more severe deficiency in Th2i cells than in their Th2 precursors (Fig. 6A). Such a model would be consistent with work that establishes IL-2 as a key differentiation factor for Th2 cells in vivo (66) and would extend this concept to the terminal differentiation of Th2 cells from helper cells into cells that inhibit proliferation of other CD4 cells. It would also be consistent with the action of IL-2 to optimize expression of CTLA-4 and confer regulatory competence on Foxp3+ Tregs (62).

Despite the evidence that IL-2 promotes and is required for Th2i formation, CTLA-4loIL-10– Th2 cells sorted from animals with chronic inflammation can also become suppressive Th2i cells in response to IL-10 or IL-21 within 20 h and in a division-independent fashion (Fig. 5A). This observation is consistent with a model where the concerted activity of several factors (rather than a single “master” factor) coordinates the switch to a suppressive state. For instance, it is likely that some of the necessary transcriptional basis for IL-10 production, such as chromatin remodeling by GATA-3 (67), is set in place in IL-10– Th2 cells, whereas other components are provided by more transient cytokine-induced signals. STAT3 may be one such component, as activation of this transcription factor is common to IL-2, IL-10, and IL-21 (68–71). Notably, STAT3 has been shown to bind to a motif in the human IL-10 promoter and thereby promote the gene’s expression (72, 73). Other transcription factors that are known to promote IL-10 production by CD4 T cells and whose message is highly upregulated in the IL-10+ Th2 subset include PPARγ (74), AHR (75), and Blimp-1 (76).

Th2i cells have potent suppressive activity in vitro, raising the possibility that they mediate essential negative feedback during the in vivo Th2 responses in which they form. Moreover, as it is known that immunosuppression characterizes chronic Th2 responses, such as those that affect the large proportion of the human population bearing chronic parasite infection, it will be interesting to see whether Th2i cells also arise in the context of such infection. Conversely, failure to form Th2i cells, for example due to relative deficiencies of the cytokines critical for their formation, might promote aberrant Th2 responses and allergy. Interventions that drive the conversion of pathogenic Th2 cells into Th2i cells may thus provide a novel and specific form of therapy against such diseases.

Disclosures

The authors have no financial conflicts of interest.

Footnotes

  • This work was supported by National Health and Medical Research Council Program Grant 427620 (to C.C.G. and M.C.C.).

  • The sequences presented in this article have been submitted to ArrayExpress (http://ebi.ac.uk/arrayexpress/) under accession number E-MTAB-948.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    FC
    mean fold change
    HEL
    hen egg lysozyme
    Tg
    transgenic
    Th2i
    Th2 inhibitory
    Treg
    regulatory T cell.

  • Received October 10, 2011.
  • Accepted April 3, 2012.

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