The importance of regulatory T cells (Tregs) for immune tolerance is well recognized, yet the signaling molecules influencing their suppressive activity are relatively poorly understood. In this article, through in vivo studies and complementary ex vivo studies, we make several important observations. First, we identify the cytoplasmic tyrosine phosphatase Src homology region 2 domain-containing phosphatase 1 (SHP-1) as an endogenous brake and modifier of the suppressive ability of Tregs; consistent with this notion, loss of SHP-1 expression strongly augments the ability of Tregs to suppress inflammation in a mouse model. Second, specific pharmacological inhibition of SHP-1 enzymatic activity via the cancer drug sodium stibogluconate potently augmented Treg suppressor activity both in vivo and ex vivo. Finally, through a quantitative imaging approach, we directly demonstrate that Tregs prevent the activation of conventional T cells and that SHP-1–deficient Tregs are more efficient suppressors. Collectively, our data reveal SHP-1 as a critical modifier of Treg function and a potential therapeutic target for augmenting Treg-mediated suppression in certain disease states.

Regulatory T cells (Tregs) have been recently shown by a number of laboratories as important players in maintaining immune tolerance. However, the mechanism of Treg-mediated suppression is still poorly understood. Although there are unanswered questions at many levels about Treg-mediated immune suppression (reviewed in Refs. 1, 2), our knowledge is particularly limited with respect to intracellular signaling molecules that regulate/modify Treg function. Previously, several molecules essential for Treg development and/or function such as STAT5 (3), Foxp3 (4, 5), or CTLA-4 (6, 7) have been identified. Other molecules have been linked to some of the proposed mechanisms of Treg-mediated suppression either in vitro or in vivo, including cAMP (8), galectin-1 (9), CD39/CD73 (10), LAG-3 (11), IL-35 (12), IL-10, and TGF-β (reviewed in Ref. 1). However, our knowledge of intracellular pathways that can modulate the suppressive activity of Tregs is rather limited. To date, sphingosine 1-phosphate receptor type 1 is the only molecule that has been identified as being important for both the development and regulatory function of Tregs via activation of the Akt-mTOR pathway (13).

Src homology region 2 domain-containing phosphatase 1 (SHP-1) is a nontransmembrane protein tyrosine phosphatase expressed in hematopoietic cells of all lineages including conventional T cells (Tcons) and Tregs. SHP-1 is now widely accepted as an important negative regulator of TCR-mediated signaling in Tcons (reviewed in Refs. 14, 15). Because Tregs have been shown to require stimulation via the TCR for their full function (1619), we asked whether SHP-1 might regulate Treg activity through its effects on TCR-mediated signaling. The existence of a murine genetic model for SHP-1 deficiency has significantly aided our understanding of the biological function of SHP-1. A splicing mutation within the Ptpn6 (Shp1) locus leads to no detectable SHP-1 protein and causes the motheaten (me/me) mouse phenotype (20, 21). We have previously shown that me/me mice have increased numbers of naturally occurring Tregs and that these Tregs can mediate suppression of Tcon responses (22). However, whether SHP-1 also influences the suppressive potential of Tregs (i.e., magnitude of suppression) and how loss of SHP-1 expression might affect Treg function are not known.

In this study, we have attempted to address how stimulation via the TCR affects Treg function using a combination of complementary genetic and pharmacological approaches, specifically targeting the function of the tyrosine phosphatase SHP-1. Our data presented in this study suggest that the strength of TCR-initiated signaling within the Tregs directly affects their level of suppressive activity and that SHP-1 functions downstream of the TCR in Tregs and thereby directly modulates their suppressive potential. Our data using sodium stibogluconate (SSG), a specific inhibitor targeting SHP-1 activity (2325), further support an important role for SHP-1 as modifier of the strength of suppression. Interestingly, SSG has been previously approved for treatment of leishmaniasis (26) and is currently tested in three phase I clinical trials for patients with advanced solid tumors, lymphoma, or myeloma (2729). We also addressed mechanistically how Tregs from me/me mice are more capable of suppression. Using a quantitative single cell-based imaging approach, we show that Tregs can suppress the activation of Tcons via at least two levels, both of which are regulated by SHP-1. During these studies, we also propose a mechanism by which Tregs, by being part of the same complex with Tcons and APCs, can directly suppress the activation of Tcons. Thus, the data presented in this work linking SHP-1 to the strength of Treg-mediated immune suppression may also have clinical relevance in providing a therapeutic target to enhance Treg function in certain disease states.

Mice used for this study were bred in our colony. All mice are on the BALB/c background. TCR-transgenic (Tg) +/+: DO11.10 and me/+: DO11.10 and non–TCR-Tg me/+ were used to generate +/+, me/+, and me/me mice on TCR-Tg and non-Tg backgrounds (30). TS1-HA TCR-Tg mice (31) were generously provided by Dr. Kenneth Tung (University of Virginia, Charlottesville, VA). Genotyping of all mice was done by PCR as described previously for the me allele and DO11.10 TCR (30), and for TS1-HA TCR (32). Unless mentioned otherwise, 17–19-d-old mice were used for this study. All mice were bred and maintained in accordance with the policies of the Institutional Animal Care and Use Committee at the University of Virginia. All experiments involving mice were conducted with the approval of Institutional Animal Care and Use Committee.

CD4+CD25 and CD4+CD25+ T cells were isolated from lymph nodes (combined inguinal, axillary, brachial, cervical, lumbar, sacral, renal, and pancreatic nodes unless otherwise indicated) using the Regulatory T cell Isolation kit (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s protocol.

Bone marrow-derived dendritic cells (BMDCs) were generated as previously described (33). Briefly, bone marrow cells isolated from mouse femurs and tibias were plated at 3 × 106 cells/well in six-well plates and cultured for 5 or 6 d in RPMI 1640 medium (with 10% FCS, 5 × 10−5 M 2-ME, 2 mM l-glutamine, and antibiotics) supplemented with GM-CSF (1000 U/ml) and IL-4 (100 U/ml) (PeproTech, Rocky Hill, NJ) followed by positive selection using CD11c-microbeads (N418) on a MACS column (Miltenyi Biotec). Obtained BMDCs were >95% positive for CD11c as assessed by flow cytometric analysis.

Cells were stained with Abs recognizing the indicated surface markers in PBS supplemented with 1% BSA and 0.1% sodium azide. Intracellular staining was done following fixation and permeabilization of the cells using the Fixation/ Permeabilization kit from eBioscience (San Diego, CA). CD4-PE/PerCP, CD25-allophycocyanin/PE, CD62L-PE, CD38-PE, CTLA-4–PE, CD103-PE/FITC, ICAM-1–PE/FITC, LAG-3–PE, CD62P-FITC, CD3-PerCP, CD279 (PD-1)-PE, CD45-RB-PE, IgG1-PE, IgG-allophycocyanin, CD95-PE.Cy7, CD127 (IL-7Rα)-PE, CD102 (ICAM-2)-FITC, CD184 (CXCR4)-FITC, CD28-PE, FR4-PE, ICOS-PE, and CD122 (IL-2Rβ)-PE were purchased from BD Pharmingen (San Jose, CA). CD29-PE.Cy7, CD49c (integrin α3)-FITC, CD49d (integrin α4)-FITC, CD49e (integrin α5)-PE, CD44-Alexa 488, CD40L-PE, LFA-1–Alexa 488, GITR-Alexa 488, and IgG-Alexa 488 were purchased from Biolegend (San Diego, CA). TLR4-PE, CD80-PE, CD86-PE, 4-1BB-PE, CD69-PE.Cy5, CD4-PE.Cy7, IFN-γ–PE, IgG-FITC, CD11c-FITC/allophycocyanin, OX40-PE, CCR7-allophycocyanin, and FoxP3-PE/FITC Ab and staining kit were from eBioscience (San Diego, CA). KJ1-26-PE and KJ1-26-FITC were purchased from Caltag Laboratories (Burlingame, CA). CD104-PE, CD-2 (LFA-2)-FITC, CD27 (TNFR)-FITC, and CD49f (integrin α6)-FITC were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Stained cells were collected on an FACSCalibur instrument using CellQuest software (BD Biosciences, San Jose, CA), and subsequent analyses were done using FlowJo software (Tree Star, Ashland, OR). Analyses were conducted on live cells (>95%) as defined by forward and side-angle scatter. Gates were set using isotype-matched control Abs.

Assessment via [3H]thymidine incorporation.

Proliferation and suppression assays were performed as described previously (22). Briefly, to assess proliferation, 2.5 × 104 CD4+CD25 T cells were plated in triplicate in 200 μl RPMI 1640 medium (supplemented with 10% FCS, 5 × 10−5 M 2-ME, 2 mM l-glutamine, and antibiotics) in round bottom 96-well plates. To measure suppression, CD4+CD25+ T cells were added at indicated ratios to the CD4+CD25 T cells. Irradiated (2000 rad) total (after RBC lysis) or T cell-depleted splenocytes were added at 5 × 104 cells/well together with either anti-CD3 Ab (145-2c11; Cedarlane Laboratories, Burlington, NC) at 6 μg/ml or OVA 323–339 peptide (ISQAVHAAHAEINEAGR) at 125 ng/ml unless otherwise indicated. TS1-HA-TCR-Tg+ cells were stimulated using influenza hemagglutinin 110–119 peptide (SFERFEIFPK) at 500 ng/ml. Peptides were synthesized at the Biomolecular Research Facility, University of Virginia. Cells were cultured for 72 h before they were pulsed with 1 μCi [3H]thymidine for 18 h. [3H]Thymidine incorporation was measured using a Tomec cell harvester and Betaplate counter (PerkinElmer, Waltham, MA).

Assessment via CFSE dilution.

For measuring proliferation and suppression, CD4+CD25 T cells were stained with CFSE (CellTrace CFSE cell proliferation kit, Molecular Probes, Invitrogen, Carlsbad, CA) at a final concentration of 10 μM for 15 min at 37°C (34). The cells were washed, and 2.5 × 104 cells were plated onto 96-well plates either alone or with different ratios of CD4+CD25+ Tregs. Irradiated (2000 rad) T cell-depleted splenocytes (5 × 104 cells/well) or day 5 BMDCs (5 × 103 cells/well) were added along with OVA peptide at 125 ng/ml. Cells were cultured for 4 d followed by flow cytometric analyses.

SHP-1 protein levels were assessed as described previously (35). Briefly, SHP-1 was immunoprecipitated from lysates of each 2.5 × 105 purified CD4+CD25+ Tregs using 2 μg rabbit anti–SHP-1 Abs (Santa Cruz Biotechnology) followed by immunoblotting for SHP-1 with monoclonal anti–SHP-1 (clone 1SH01, NeoMarkers, Fremont, CA) at 1.35 μg/ml. Relative levels of SHP-1 were calculated based on densitometry measurements of immunoblots at linear range using the ImageQuant TL 2005 program.

Preparation of Th1 cells.

Th1 cells were generated and adoptively transferred as previously described (36) with the following modifications. CD4+CD25 T cells from BALB/c: +/+: DO11.10 mice (1× 106cells/ml) were cultured with T cell-depleted irradiated splenocytes (2 × 106cells/ml), 1 μg/ml OVA peptide, 10 μg/ml anti-mouse IL-4 neutralization Ab (R&D Systems, Minneapolis, MN), and 10 ng/ml recombinant murine IL-12 (R&D Systems) in 24-well plates. The cells were supplemented with fresh media on days 3 and 5 and were harvested on day 7. A total of 75–80% of the cells, which were adoptively transferred, expressed IFN-γ as assessed by flow cytometric analysis.

CD4+CD25+ Tregs.

CD4+CD25+ cells were isolated from BALB/c: DO11: +/+ and me/+ mice as described above. For adoptive transfer, purified cells were resuspended in sterile PBS (4 × 105 cells in a maximum volume of 25 μl).

Adoptive transfer.

BALB/c mice (2–3 mo old) were divided into three experimental groups: group I, Th1 only (left footpad, Th1 + APC + peptide; right footpad, Th1 + APC); group II, Th1 plus +/+ Tregs (left footpad, Th1 + APC + peptide + Treg [+/+]; right footpad, Th1 + APC); and group III, Th1 plus me/+ (left footpad, Th1 + APC + peptide + Treg [me/+]; right footpad, Th1 + APC). For group I, equal numbers of Th1 cells and APCs (1.6 × 106) were mixed and injected s.c. in the left footpad along with 10 μg OVA peptide in a volume of 25 μl. For the two groups (II and III) that received Tregs, mice were injected with either +/+ or me/+ Tregs (at a Th1/Treg ratio of 4:1) along with Th1 cells, APCs, and OVA peptide in the left footpad in a volume of 25 μl. For all mice, the right footpad received the same number of Th1 cells and APCs without any peptide and served as the control. Footpad measurements were taken before and 24 h after the injections using a micrometer (Mitutoyo USA, Aurora, IL). All footpad measurements were taken in a manner blinded to the experimental conditions. Footpad swelling is expressed as the percentage increase of the left footpad over the control right footpad.

In vitro treatment with SSG during suppression assays.

Cells were purified and plated into the wells of a 96-well plate as described above for the standard suppression assay using CFSE dilution as readout for proliferation. SSG (10 μg/ml; Calbiochem, San Diego, CA) was added to the culture. A total of 10 μg/ml SSG has been determined to inhibit 99% of SHP-1 activity while minimally affecting the phosphatase activity of SHP-2 or PTP 1B (23).

In vivo treatment with SSG.

+/+ DO11.10 mice (2 mo old) were injected with SSG s.c. at a concentration of 10 mg/mouse on days 1 and 5. Control mice of the same age were treated with sterile PBS. On day 7, Tregs were isolated from the lymph nodes of these mice and used for proliferation/suppression assays.

Conjugation assays were performed as described previously (37) with the following modifications. CD4+CD25 and CD4+CD25+ T cells were isolated, resuspended in PBS to a concentration of 2 × 107 cells/ml, and labeled with an equal volume of succinimidyl esters Alexa 488 (diluted 1:100) or Alexa 633 (diluted 1:50) (Molecular Probes, Invitrogen) for 15 min at room temperature. The cells were washed three times and resuspended in complete RPMI 1640 media. BMDCs, which had been pulsed with the indicated concentrations of OVA peptide for 1 h, were used as APCs and added to T cells (CD4+CD25 or CD4+CD25+ or a combination of both) in a final volume of 200 μl at a T cell/APC ratio of 1:1, 1:2, or 1:4 in a 96-well round bottom plate along with respective concentrations of OVA peptide. Settling of the cells were initiated by spinning the plate at 500 rpm for 30 s followed by incubation at 37°C for indicated time periods indicated. To analyze cell conjugates, cell mixtures were vortexed to disrupt the nonspecific conjugates, washed with PBS (1% BSA), and fixed with 4% paraformaldehyde for 15 min at room temperature. Cells were then stained with anti-CD4 and anti-CD11c for additional identification of CD4 T cells and CD11c+ BMDCs and collected on an FACSCalibur (BD Biosciences) and analyzed using FlowJo software (Tree Star). For analysis of conjugates on an ImageStream100 instrument (Amnis Corporation, Seattle, WA), cells were prestained (1:100 dilution) with succinimidyl esters as follows: Tcon, Alexa 647; Treg, Alexa 488; and BMDC, Alexa 405 followed by incubation for 4 h at a ratio of 1:1:1. Samples were collected on an ImageStream100 instrument using the Extended Depth of Field Technology (EDF) program and analyzed by IDEAS analytical software.

To detect the upregulation of CD25 by Tcons when conjugated to BMDCs, experiments were set up as described above for conjugate assays. Prestained cells were incubated for 8 h, stained for the surface expression of CD25, and acquired using the EDF program on an ImageStream100 instrument. The data were analyzed using the IDEAS analytical software. To quantitate CD25 expression on the surface of Tcons in a triple conjugate with Tregs and BMDCs, CD25 expression by Tregs was masked using the masking function provided by the IDEAS applications. CD25 expression of Tcons was quantitated using the similarity feature, which is the log transformed Pearson’s correlation coefficient and a measure of the degree to which two images are linearly correlated within a masked region (per manufacturer’s instructions). Similarity is expressed as the median. For the detection of intracellular IL-2 production by Tcons, a conjugation assay was performed, and cells were fixed and permeabilized with the reagents from the Fixation-permeabilization kit from BD Biosciences following the manufacturer’s protocol. The cells were not treated with any protein transport inhibitor. Anti–IL-2–PE Ab from eBioscience was used for staining.

The p values were calculated with unpaired Student t test; p values <0.05 were considered significant.

To study the role of SHP-1 in Treg function, we used a murine model for SHP-1 deficiency, the so-called me/me mouse, that in previous studies has significantly aided the understanding of the biological function of SHP-1. We have previously demonstrated that me/me mice have increased percentages of functionally active CD4+CD25+ Tregs (22); however, whether and if so, how the loss of SHP-1 influences Treg suppressive activity is not known. To specifically assess Ag-specific signaling downstream of the TCR, mice carrying the me/me allele were crossed with mice Tg for the DO11.10 TCR that recognizes the OVA323–339 peptide (30). Although there are multiple subsets of T cells exhibiting suppressive activity, in this study, we focused on CD4+CD25+Foxp3+ Tregs. Tregs purified from control and mutant SHP-1 mice were assessed for in vitro suppression using varying ratios of Treg versus Tcons. Tregs from homozygous me/me and heterozygous me/+ mice were more suppressive than control +/+ littermates in response to OVA peptide stimulation (Fig. 1A). Monitoring the proliferative response of Tcons (by CFSE labeling and assessing the dilution of the CFSE signal using flow cytometry) confirmed that the Treg effect seen is specifically due to decreased proliferation of Tcons under these assay conditions (Fig. 1B). In addition to the OVA peptide-induced responses, the increased suppressive potential of Tregs lacking SHP-1 was also observed after more generic anti-CD3–mediated TCR stimulation (Fig. 1C). Notably, SHP-1–deficient Tregs showed a strong suppressive activity even at a Treg/Tcon ratio of 1:32, whereas control Tregs had no suppressive activity at this ratio (Fig. 1A–C). It should also be noted that the purified Treg populations from me/+ and me/me cells displayed almost identical profiles for CD4, CD25, Foxp3, CD3, and the Tg TCR DO11.10 compared with Tregs from +/+ mice (Fig. 1D), ruling out another not previously identified population of Tregs as being responsible for the observed effects.

FIGURE 1.

me/me and me/+ Tregs are more potent suppressors than +/+ Tregs. A, CD4+CD25+ (Treg) and CD4+CD25 (Tcon) cells were purified from lymph nodes of +/+, me/+, and me/me DO11.10 TCR-Tg mice. Tregs at the indicated ratios were added to a constant number of +/+ Tcons (2.5 × 104). Irradiated total splenocytes were used as APCs, and proliferation was measured by [3H]thymidine incorporation in response to 125 ng/ml OVA peptide. Proliferation of Tcons in the absence of any Tregs was set at 100%. Results shown are averages of six to seven independent experiments with multiple mice for each genotype per experiment. Error bars indicate ± SEM. B, Suppression assays were set up as described for A, but Tcons were prestained with CFSE and proliferation was measured by dilution of the CFSE fluorescence intensity. Irradiated, T cell-depleted splenocytes were added along with OVA peptide at 125 ng/ml, and flow cytometric analyses were performed after 4 d of culture. The data are representative of three independent experiments. C, Suppression assays were set up as described for A, but proliferation was measured in response to 6 μg/ml anti-CD3 Ab. Results shown are averages of three independent experiments with one to three mice for each genotype per experiment. Error bars indicate ± SEM. D, SHP-1 was immunoprecipitated from lysates of Tregs isolated from +/+ and me/+ mice and analyzed by anti–SHP-1 immunoblotting. Relative levels of SHP-1 were calculated based on densitometry of immunoblots at linear range using the ImageQuant program. Histograms depict surface expression levels of indicated molecules by Tregs isolated from lymph nodes of +/+, me/+, and me/me mice.

FIGURE 1.

me/me and me/+ Tregs are more potent suppressors than +/+ Tregs. A, CD4+CD25+ (Treg) and CD4+CD25 (Tcon) cells were purified from lymph nodes of +/+, me/+, and me/me DO11.10 TCR-Tg mice. Tregs at the indicated ratios were added to a constant number of +/+ Tcons (2.5 × 104). Irradiated total splenocytes were used as APCs, and proliferation was measured by [3H]thymidine incorporation in response to 125 ng/ml OVA peptide. Proliferation of Tcons in the absence of any Tregs was set at 100%. Results shown are averages of six to seven independent experiments with multiple mice for each genotype per experiment. Error bars indicate ± SEM. B, Suppression assays were set up as described for A, but Tcons were prestained with CFSE and proliferation was measured by dilution of the CFSE fluorescence intensity. Irradiated, T cell-depleted splenocytes were added along with OVA peptide at 125 ng/ml, and flow cytometric analyses were performed after 4 d of culture. The data are representative of three independent experiments. C, Suppression assays were set up as described for A, but proliferation was measured in response to 6 μg/ml anti-CD3 Ab. Results shown are averages of three independent experiments with one to three mice for each genotype per experiment. Error bars indicate ± SEM. D, SHP-1 was immunoprecipitated from lysates of Tregs isolated from +/+ and me/+ mice and analyzed by anti–SHP-1 immunoblotting. Relative levels of SHP-1 were calculated based on densitometry of immunoblots at linear range using the ImageQuant program. Histograms depict surface expression levels of indicated molecules by Tregs isolated from lymph nodes of +/+, me/+, and me/me mice.

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Notably, Tregs with a heterozygous me/+ genotype displayed an intermediate phenotype, suggesting that ∼50% reduction in SHP-1 protein can still have a detectable effect on the suppressive activity of Tregs (Fig. 1D). The me/me mice have a short life span and die at ∼3 wk of age, requiring that the above set of experiments be performed using mice <20 d old. To rule out the possibility that the observed phenotype might have arisen due to the young mice used, we repeated the suppression assays comparing +/+ and me/+ Tregs from adult mice. These assays using me/+ Tregs confirmed that the increased suppressive activity of Tregs from the me/me background is independent of their age (Supplemental Fig. 1).

To better understand the mechanism by which SHP-1 may influence Treg activity, we asked whether SHP-1 affects the strength of TCR-mediated signaling. We designed suppression assays such that the Tregs can be stimulated with increasing concentrations of antigenic stimulation, whereas the Tcons are stimulated at a constant dose of Ag peptide. This was achieved through the use of two different TCR-Tg mice as sources for Tcon and Tregs, respectively; HA-specific TCR-expressing Tcons were used as responders, whereas the OVA-specific TCR-expressing Tregs were used as suppressors. Both TCRs recognize their respective peptides presented by class II MHC H-2d, allowing peptide presentation to both Tregs and Tcons by the same APCs. These mixed suppression assays revealed several important observations (Fig. 2). First, the Treg-mediated suppression was absolutely dependent on TCR-dependent activation of Tregs, because no suppression was detectable in the absence of OVA peptide. Moreover, Tregs were able to suppress proliferation of Tcons even if the Tcons and Tregs recognize different MHC–peptide complexes [as it has been previously reported (16)]. Second, me/me Tregs were more potent suppressors than +/+ Tregs under these mixed suppression conditions. Third, upon increased peptide concentrations, both +/+ and me/+ Tregs showed enhanced suppressive activity, indicating a direct correlation between the strength of the TCR-mediated signaling and the suppressive activity of Tregs. In contrast, me/me Tregs lacking SHP-1 were insensitive to increased concentrations of OVA peptide; this indicated that SHP-1 is a critical, nonredundant, and significant brake on TCR signal strength in Tregs such that in the absence of SHP-1, Treg-mediated suppression reaches a maximum even at lower peptide concentrations. Lastly, even though increased TCR stimulation on Tregs (via increasing OVA peptide concentrations) caused a more potent suppression by +/+ Tregs, their suppressive activities never reached the level observed by me/me Tregs, suggesting that the SHP-1–mediated regulation of suppression may involve events beyond the recognition of peptide + MHC by TCR on Tregs (further addressed below).

FIGURE 2.

Strength of TCR signal affects the suppressive efficiency of Tregs. Tregs from +/+, me/+, and me/me DO11.10 TCR-Tg mice were cultured with 2.5 × 104 Tcons from HA-TCR-Tg mice at 1:16 ratio of Treg/Tcons. Tregs were stimulated with increasing concentrations of OVA peptide (no peptide, 125–1000 ng/ml), whereas Tcons were activated with a constant dose of 500 ng/ml HA peptide. Irradiated, T cell-depleted splenocytes were used as APCs. The proliferation of Tcons in the presence and absence of Tregs was measured by [3H]thymidine incorporation. Data shown are the average of three independent experiments (± SEM) with multiple mice for each genotype per experiment.

FIGURE 2.

Strength of TCR signal affects the suppressive efficiency of Tregs. Tregs from +/+, me/+, and me/me DO11.10 TCR-Tg mice were cultured with 2.5 × 104 Tcons from HA-TCR-Tg mice at 1:16 ratio of Treg/Tcons. Tregs were stimulated with increasing concentrations of OVA peptide (no peptide, 125–1000 ng/ml), whereas Tcons were activated with a constant dose of 500 ng/ml HA peptide. Irradiated, T cell-depleted splenocytes were used as APCs. The proliferation of Tcons in the presence and absence of Tregs was measured by [3H]thymidine incorporation. Data shown are the average of three independent experiments (± SEM) with multiple mice for each genotype per experiment.

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To assess whether the increased suppressive activity of SHP-1–deficient Tregs seen ex vivo would be relevant under in vivo conditions, we tested Treg-mediated suppression in a mouse model of delayed-type hypersensitivity (36). Limiting numbers of Tregs were injected, and the efficiency of suppression was assessed. Under conditions when +/+ Tregs failed to show a statistically significant suppression, the me/+ Tregs were able to potently suppress OVA-induced footpad swelling (Fig. 3). Thus, SHP-1 deficiency causes Tregs to become more potent suppressor cells in the context of a whole animal. Collectively, these data identified SHP-1 as a key regulator of Treg suppressive activity in vitro and in vivo.

FIGURE 3.

me/+ Tregs are more efficient in suppressing a DTH response than +/+ Tregs. Total of 1.6 × 106 ex vivo skewed Th1 (DO11.10 TCR Tg) cells were injected into the footpads of BALB/c mice along with equal numbers of irradiated T cell-depleted splenocytes and 10 μg/ml OVA peptide. Tregs (+/+ or me/+) were added at a ratio of 1:4 Treg/Th1 cells. Footpad measurements were taken prior to and 24 h after the injections. Data are the average of three independent experiments. n indicates the number of mice analyzed for each experimental condition. Error bars denote ± SEM.

FIGURE 3.

me/+ Tregs are more efficient in suppressing a DTH response than +/+ Tregs. Total of 1.6 × 106 ex vivo skewed Th1 (DO11.10 TCR Tg) cells were injected into the footpads of BALB/c mice along with equal numbers of irradiated T cell-depleted splenocytes and 10 μg/ml OVA peptide. Tregs (+/+ or me/+) were added at a ratio of 1:4 Treg/Th1 cells. Footpad measurements were taken prior to and 24 h after the injections. Data are the average of three independent experiments. n indicates the number of mice analyzed for each experimental condition. Error bars denote ± SEM.

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As a complementary approach, and to directly correlate the phenotype of the Tregs from me/me mice to the enzymatic activity of SHP-1, we used SSG, a pharmacological inhibitor of SHP-1. SSG has previously been shown to specifically inhibit 99% of SHP-1 activity at low concentrations (10 μg/ml), whereas the closely related phosphatase SHP-2 was fully active under these conditions (23). We next asked how SSG affected Treg activity. To focus on the effect of SSG on Treg activity, suppression assays were performed using peptide concentrations that provided optimal proliferative stimulation for Tcons. It is important to note that SSG treatment alone did not affect Tcon proliferation under these conditions (Fig. 4A, top panel, no Treg). This allowed us to directly address the effect of SSG on Treg-mediated suppression and to make the following key observations. First, SSG treatment of Tregs from +/+ mice potently increased their suppressive activity. (Fig. 4A, bottom left panel). Second, in the context of Tregs from me/+ mice, SSG further enhanced the suppressive activity, likely due to inhibition of the residual SHP-1 activity in the heterozygous mice (Fig. 4A, bottom middle panel). Third, SSG had no effect on the already enhanced suppressive activity of Tregs from me/me mice (i.e., no effect in the absence of SHP-1 expression), which further supports the proposed specificity of SSG (Fig. 4A, bottom right panel). Moreover, the short-term SSG treatment of wild-type Tregs leading to the enhanced suppressive potential, essentially phenocopying the results with Tregs from me/me mice, helped rule out any secondary effects due to the loss of SHP-1 in other tissues within the me/me mice, but indicated a cell autonomous phenotype. Collectively, these data demonstrate that the enzymatic activity of SHP-1 normally functions as a brake to control the suppressive potential of Tregs and that altering the function of SHP-1 could be a tool to modify the functional efficiency of Tregs.

FIGURE 4.

SSG, a specific inhibitor of SHP-1, increases the suppressive potential of Tregs in vitro and in vivo. A, Suppression assays (as described in Fig. 1B) were set up with or without addition of 10 μg/ml SSG. Cells were cultured for 4 d followed by flow cytometric analysis. The data are representative of three independent experiments. B, Tregs (+/+) were mixed with CFSE-labeled Tcons (+/+) at a ratio of 1:4 with or without addition of SSG (10 μg/ml). Irradiated, T cell-depleted splenocytes were added along with indicated concentrations of OVA peptide followed by flow cytometric analysis after 4 d of culture. The data are representative of three independent experiments. C, +/+ DO11.10 mice (2 mo old) were injected with SSG (10 mg/mouse) or vehicle (PBS) s.c. on days 1 and 5. On day 7, Tregs were purified from lymph nodes and tested for suppression with CFSE-labeled Tcons at a ratio of 1:8. Irradiated, T cell-depleted splenocytes were added along with indicated concentrations of OVA peptide followed by flow cytometric analyses after 5 d of culture. The data are representative of three independent experiments.

FIGURE 4.

SSG, a specific inhibitor of SHP-1, increases the suppressive potential of Tregs in vitro and in vivo. A, Suppression assays (as described in Fig. 1B) were set up with or without addition of 10 μg/ml SSG. Cells were cultured for 4 d followed by flow cytometric analysis. The data are representative of three independent experiments. B, Tregs (+/+) were mixed with CFSE-labeled Tcons (+/+) at a ratio of 1:4 with or without addition of SSG (10 μg/ml). Irradiated, T cell-depleted splenocytes were added along with indicated concentrations of OVA peptide followed by flow cytometric analysis after 4 d of culture. The data are representative of three independent experiments. C, +/+ DO11.10 mice (2 mo old) were injected with SSG (10 mg/mouse) or vehicle (PBS) s.c. on days 1 and 5. On day 7, Tregs were purified from lymph nodes and tested for suppression with CFSE-labeled Tcons at a ratio of 1:8. Irradiated, T cell-depleted splenocytes were added along with indicated concentrations of OVA peptide followed by flow cytometric analyses after 5 d of culture. The data are representative of three independent experiments.

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Two additional observations supported and extended our conclusion that SSG enhances the suppressive activity of Tregs in vitro and in vivo. First, SSG, due to its ability to inhibit SHP-1, has been shown to enhance Tcon effector function (24, 38). When we tested the effect of SSG under suboptimal peptide concentrations in which SSG augments Tcon proliferation (Fig. 4B, compare rows 1 and 3), addition of Tregs suppressed Tcon proliferation to the same extent or even further than in the absence of SSG (Fig. 4B, compare rows 2 and 4). Thus, SSG-mediated enhancement of Treg suppressive activity appears more dominant than the SSG effect on Tcon proliferation. Secondly, when mice were injected with SSG, we found that the suppressive activity of Tregs from SSG-treated mice was enhanced compared with the activity of PBS-injected control mice (Fig. 4C). Collectively, these observations suggested that SSG enhances Treg suppressive activity in vivo and that the effect of SSG on Tregs is greater than the effect on Tcons.

To mechanistically understand how SHP-1 could influence the suppressive activity of Tregs, we compared the gene expression and protein profile of Tregs from control and SHP-1–deficient mice. Using microarray analyses, the mRNA profiles of three genetically different Treg populations (+/+, me/+, and me/me) were compared. Based on our observation that me/+ Tregs display an intermediate phenotype in the functional assays, a special emphasis was also placed on detecting mRNAs that show a consistent change from +/+ to me/+ and me/me. However, no obvious changes in among between the +/+, me/+, and me/me Treg gene expression profiles were noted, and no statistically significant differences were observable among Treg signature genes (Supplemental Fig. 2).

We next explored the possibility that protein expression may vary between the different genotypes and focused on proteins associated with Treg activity and/or function. Many surface molecules previously linked to Treg development/function were unchanged (including Foxp3, CD25, CD122, LAG-3, ICAM-2, CD28, CD86, CD69, TLR-4, CD95, CD38, CCR7, CXCR4, CD49c, CD49d, CD49e, CD49f, and CD31) or even downmodulated (CD62L, FR4, and CD40L). However, a few surface molecules showed notable differences in expression among +/+, me/+, and me/me Tregs (Fig. 5A, Supplemental Fig. 3). The difference was manifested at two levels. First, at steady state, a higher fraction of me/me Tregs expressed the following markers: CD103, 4-1BB, intracellular CTLA-4, CD80, CD44, CD27, CD62P, LFA-1, ICOS, and CD29; however, the expression level of the individual markers on a per-cell basis was not changed. Second, Tregs from SHP-1–deficient mice displayed an increase in surface expression (on a per-cell basis) of specific molecules, such as ICAM-1, IL-7Rα, CD104, OX40, GITR, and LFA-2. This difference in the surface profile of Tregs in the me background was not due to the source of the lymph node from which the cells were derived, because Tregs from inguinal, axillary, brachial, cervical, lumbar, sacral, renal, and pancreatic nodes as well as spleen were comparable (Supplemental Fig. 4A, 4B). Moreover, the difference seen in Tregs in the me background was independent of whether the Tregs were derived from non–TCR-Tg or the DO11.10 TCR-Tg mice (Supplemental Fig. 4C). Taken together, these data showed that SHP-1 expression (or the lack thereof) affects the expression of a subset of proteins on Tregs.

FIGURE 5.

SHP-1 deficiency causes a more activated phenotype of Tregs. A, CD4+ T cells from lymph nodes of +/+, me/+, and me/me DO11.10 TCR-Tg mice were analyzed for the expression of indicated surface or intracellular molecules. Shaded histograms represent CD4+CD25 T cells (Tcon) derived from +/+ mice. B, Increased suppressive activity of me/+ Tregs directly correlates with increased percentages of CD103+ cells. Tregs were isolated from individual me/+ mice (marked as A, B, C, and D) and their suppressive activities compared with +/+ Tregs. Surface expressions of CD103 and CD54 (ICAM-1) on the respective mice are also shown. Tregs were added to 2.5 × 104 of +/+ Tcons at a Treg/Tcon ratio of 1:8 along with 5 × 104 APCs and OVA peptide (125 ng/ml). Percentages of maximal proliferation were calculated based on proliferation of Tcons in the absence of Tregs. Error bars denote ± SEM. Each data point represents an individual mouse.

FIGURE 5.

SHP-1 deficiency causes a more activated phenotype of Tregs. A, CD4+ T cells from lymph nodes of +/+, me/+, and me/me DO11.10 TCR-Tg mice were analyzed for the expression of indicated surface or intracellular molecules. Shaded histograms represent CD4+CD25 T cells (Tcon) derived from +/+ mice. B, Increased suppressive activity of me/+ Tregs directly correlates with increased percentages of CD103+ cells. Tregs were isolated from individual me/+ mice (marked as A, B, C, and D) and their suppressive activities compared with +/+ Tregs. Surface expressions of CD103 and CD54 (ICAM-1) on the respective mice are also shown. Tregs were added to 2.5 × 104 of +/+ Tcons at a Treg/Tcon ratio of 1:8 along with 5 × 104 APCs and OVA peptide (125 ng/ml). Percentages of maximal proliferation were calculated based on proliferation of Tcons in the absence of Tregs. Error bars denote ± SEM. Each data point represents an individual mouse.

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One of the main differences among +/+, me/+, and me/me Tregs is the fraction of cells positive for CD103 (αEβ7-integrin), and CD103 is thought to mark the in vivo activated effector/memory subpopulation of CD4+Foxp3+ Tregs (39, 40). When we gated on this CD103+ population, many other markers linked to Treg-mediated suppression were also altered, including ICAM-1, CD80, 4-1BB (CD137), LAG-3 (CD223), and CD62L (Supplemental Fig. 5A). To correlate the altered marker expression to Treg function, we took advantage of the variability we see in the suppressive ability of Tregs from me/+ mice. Whereas heterozygous me/+ Tregs consistently show an intermediate phenotype between +/+ and me/me mice, there are significant variations between individual me/+ mice. Some more closely resembled the me/me phenotype, whereas others are closer to a wild type phenotype. Interestingly, when we compared four randomly chosen me/+ littermates (denoted mouse A, B, C, and D in Fig. 5B) for CD103 and ICAM-1 expression, we observed a close correlation between suppressive potential and the fraction of Tregs that were CD103 positive (Fig. 5B). When we tested the hypothesis that the increase in the fraction of CD103-positive cells in the me background might be due to the heightened TCR response in the absence of SHP-1, this was not found to be the case (Supplemental Fig. 5B). This further supports our previous findings (Fig. 2) that in Tregs, SHP-1 affects additional signaling pathways besides TCR-mediated signaling.

The phenotypic and functional differences among me/me, me/+, and +/+ Tregs indicate that SHP-1–deficient Tregs are in a state of heightened activation. One of the functional requirements for successful suppression is the formation of a conjugate between the Tregs and the APCs, and many of the surface molecules upregulated on me/+ and me/me Tregs are adhesion molecules. This prompted us to test whether me/+ and me/me Tregs might form conjugates with APCs more efficiently. To have a more homogenous Ag-presenting population for these assays, we used primary BMDCs from control mice (reviewed in Ref. 41). We confirmed that differences in suppressive activities between me/+ and +/+ Tregs can still be observed using BMDCs as APCs (data not shown). Using a flow cytometric approach, we examined the conjugate formation between Tregs and BMDCs (Fig. 6A). Even in the absence of cognate peptide, 7–9% of the Tregs formed conjugates with BMDCs, with me/me-derived Tregs forming slightly more conjugates. Upon addition of peptide, an increased percentage of Tregs (16%) from control mice formed conjugates, whereas Tregs from me/me mice formed many more conjugates (25%). To better define the composition of the conjugates, such as the number of cells that form the conjugates, we used the Imagestream100 (Amnis Corporation) instrument, which combines flow cytometry with fluorescence microscopy; this allowed imaging large numbers of individual conjugates while the cells are being analyzed by traditional parameters used in flow cytometry. We scored the individual BMDC–Treg conjugates as BMDC conjugated to 1, 2, or ≥3 Tregs (Fig. 6B). Whereas the overall fraction of Tregs found in conjugate with APCs increases upon addition of peptide, the me/+ and me/me Tregs consistently formed more conjugates than +/+ Tregs (Fig. 6C, Supplemental Fig. 6). Taken together, these data support our hypothesis that SHP-1–deficient Tregs are more efficient in forming conjugates with APCs, which may contribute to their increased suppressive activity.

FIGURE 6.

me/me and me/+ Tregs are more efficient than +/+ Tregs in forming conjugates with BMDCs and downmodulating CD80 on BMDCs. A, Schematic depiction of experimental setup; CD4+CD25+ T cells were isolated and labeled with succinimidyl ester Alexa 488. Following washing, cells were resuspended and mixed with BMDCs (without peptide or prepulsed with 100 ng/ml OVA peptide) at 1:1 ratio for 4 h at 37°C. Cells were harvested, stained with anti-CD4 and anti-CD11c, and analyzed by flow cytometry. Conjugates were identified based on CD4 and Alexa 488 staining for Tregs and CD11c staining for BMDCs. Numbers indicate percentage of Tregs conjugated to BMDCs. Experiment shown is representative of three independent experiments. B, Tregs (prestained with Alexa 488) and BMDCs (prestained with Alexa 405) loaded with no peptide or 100 or 500 ng/ml OVA peptide were incubated at a 1:1 ratio for 4 h at 37°C followed by analysis on an ImageStream100 instrument (Amnis Corporation) using the EDF program for sample acquisition followed by data analysis with IDEAS analytical software. Representative images of individual conjugates are shown. Examples for 1, 2 or ≥3 Tregs per BMDC are depicted. C, Quantitative analysis of data obtained in B. Graphs depict percentages of Tregs (+/+, me/+, and me/me) conjugated to BMDCs pulsed with 100 ng/ml or 500 ng/ml OVA peptide. Percentages were calculated based on the number of conjugates formed and the number of individual Tregs conjugated to BMDC. Primary data and detailed calculations are provided in Supplemental Fig. 6.

FIGURE 6.

me/me and me/+ Tregs are more efficient than +/+ Tregs in forming conjugates with BMDCs and downmodulating CD80 on BMDCs. A, Schematic depiction of experimental setup; CD4+CD25+ T cells were isolated and labeled with succinimidyl ester Alexa 488. Following washing, cells were resuspended and mixed with BMDCs (without peptide or prepulsed with 100 ng/ml OVA peptide) at 1:1 ratio for 4 h at 37°C. Cells were harvested, stained with anti-CD4 and anti-CD11c, and analyzed by flow cytometry. Conjugates were identified based on CD4 and Alexa 488 staining for Tregs and CD11c staining for BMDCs. Numbers indicate percentage of Tregs conjugated to BMDCs. Experiment shown is representative of three independent experiments. B, Tregs (prestained with Alexa 488) and BMDCs (prestained with Alexa 405) loaded with no peptide or 100 or 500 ng/ml OVA peptide were incubated at a 1:1 ratio for 4 h at 37°C followed by analysis on an ImageStream100 instrument (Amnis Corporation) using the EDF program for sample acquisition followed by data analysis with IDEAS analytical software. Representative images of individual conjugates are shown. Examples for 1, 2 or ≥3 Tregs per BMDC are depicted. C, Quantitative analysis of data obtained in B. Graphs depict percentages of Tregs (+/+, me/+, and me/me) conjugated to BMDCs pulsed with 100 ng/ml or 500 ng/ml OVA peptide. Percentages were calculated based on the number of conjugates formed and the number of individual Tregs conjugated to BMDC. Primary data and detailed calculations are provided in Supplemental Fig. 6.

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We next asked whether the increased conjugate formation due to SHP-1 deficiency correlates with a Treg-mediated inhibitory effect on the APCs. Tregs have been shown to decrease the surface expression of the coreceptors CD80 and CD86 (18, 42) on APCs, and this is considered one of the mechanisms by which Tregs mediate their suppressive function. We noted that day 5 BMDCs have very low levels of surface CD80/CD86, whereas their expression is clearly detectable at day 6. The addition of Tregs for 24 h caused a downmodulation of CD80 surface expression (Fig. 7A, 7B). Incubation with me/+ or me/me Tregs resulted in an additional decrease of CD80 expression, which was small but consistent, when compared with BMDCs incubated with +/+ Tregs. This effect of Tregs requires Ag-mediated interaction with BMDCs, as there was no effect in the absence of OVA peptide. In comparison, Tcons showed no inhibitory effect on CD80, but rather an increase in CD80 expression (Fig. 7A, 7B).

FIGURE 7.

Tregs cause decrease of CD80 surface expression on BMDCs. BMDCs were harvested on day 6, and an aliquot of cells was assessed for CD80 surface expression by flow cytometry (denoted as day 6 BMDC). Tregs or Tcons were added to day 6 BMDCs at ratios of 1:1 (A) or 1:2 (B) and cultured with or without 100 or 1000 ng/ml OVA peptide. C, Tregs (+/+, me/+, and me/me) and Tcons (+/+) were added at a 1:2:4 ratio and cultured with or without the indicated concentrations of peptide. AC, After 24 h of incubation, cells were collected and stained for the surface expression of CD80, CD11c, and CD4 followed by flow cytometry. BMDCs still conjugated to CD4+ T cells were excluded from final analyses. Graphs depict mean fluorescence intensity (MFI) of CD80 on BMDC (CD11c+CD4). The graphs are representative for two to four independent experiments.

FIGURE 7.

Tregs cause decrease of CD80 surface expression on BMDCs. BMDCs were harvested on day 6, and an aliquot of cells was assessed for CD80 surface expression by flow cytometry (denoted as day 6 BMDC). Tregs or Tcons were added to day 6 BMDCs at ratios of 1:1 (A) or 1:2 (B) and cultured with or without 100 or 1000 ng/ml OVA peptide. C, Tregs (+/+, me/+, and me/me) and Tcons (+/+) were added at a 1:2:4 ratio and cultured with or without the indicated concentrations of peptide. AC, After 24 h of incubation, cells were collected and stained for the surface expression of CD80, CD11c, and CD4 followed by flow cytometry. BMDCs still conjugated to CD4+ T cells were excluded from final analyses. Graphs depict mean fluorescence intensity (MFI) of CD80 on BMDC (CD11c+CD4). The graphs are representative for two to four independent experiments.

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As under physiological conditions, APCs can form conjugates containing both Tcon and Tregs, we next asked how CD80 expression is influenced when both Tregs and Tcons are added to BMDCs. Whereas Tcons cause an increase in CD80 expression, addition of Tregs still decreases CD80 expression (Fig. 7C). Under these conditions, Tregs from me/+ and me/me mice were again slightly more effective in inhibiting CD80 expression. In fact, at high peptide concentrations (1 μg) in which Tregs have been shown to be less efficient (11), Tregs from the me/me background were still capable of substantially decreasing CD80 expression (whereas the +/+ Tregs showed a diminished effect) (Fig. 7C). These data further suggest that Tregs with loss of SHP-1 expression are more effective suppressor cells and are capable of actively downmodulating CD80 expression to levels lower than initially expressed on the APCs.

We then tested whether Tregs would affect the conjugate formation between Tcons and APCs and whether Tregs with or without SHP-1 differed in regulating Tcon–APC interaction and/or Tcon activation (Fig. 8A). The addition of Tregs decreased the number of conjugates formed between Tcon and APCs, with Tregs from me/me mice being slightly more efficient (Fig. 8B). We next assessed whether the decrease in Tcon–BMDC conjugate formation was due to a competition for binding on the BMDC or due to other mechanisms, such as the change in surface molecule expression on BMDCs induced by Tregs (Fig. 7). To test the competition model, we added excess Tcons (with a second fluorescent label) to assess whether it would alter the Tcon–BMDC conjugate formation. Addition of extra Tcons caused only a minor decrease in the Tcon–BMDC conjugate formation; under the same conditions, addition of Tregs caused a significant decrease in Tcon–BMDC conjugates (Fig. 8B). This suggests that inhibition likely occurrs due to direct effects of Tregs on BMDCs, which in turn results in lower conjugate formation between Tcon and BMDCs. In contrast, under our experimental conditions (BMDC/Tcon/Treg ratio 1:1:1), direct competition for BMDC binding between Treg and Tcons contributes only minimally to the inhibitory effects.

FIGURE 8.

me/me and me/+ Tregs are more efficient in preventing productive Tcon–APC conjugate formation than +/+ Tregs. A, Schematic depiction of experimental setup. B, Tcon (prestained with Alexa 633), Tregs (prestained with Alexa 488), and BMDCs loaded with the indicated concentrations of OVA peptide were incubated for 6 h at 37°C at a ratio of 2:1:1 (BMDC/Tcon/Treg). Cells were harvested, stained with anti-CD4 and anti-CD11c, and collected on an FACSCalibur (BD Biosciences). Conjugates were defined based on CD4 and Alexa 633 staining for Tcons and CD11c staining for BMDCs. Numbers indicate percentages of Tcons conjugated to BMDCs. To assess the specificity of Treg-mediated effects, no Tregs or Alexa 488-stained Tcons were added. Experiment shown is representative of four independent experiments. C, BMDC (Alexa 405)–Tcon (Alexa 647)–Treg (Alexa 488) conjugates formed after incubating for 4 h at 37°C at 1:1:1 ratio. Analysis of conjugates was performed on an ImageStream100 as in Fig. 6. Conjugates were defined based on CD4 and Alexa 647 staining for Tcons, Alexa 405 staining for BMDCs, and CD4 and Alexa 488 staining for Tregs. Representative images of individual conjugates are shown. Left panel, BMDC–Tcon conjugates from control culture of BMDC + Tcon + OVA peptide without Tregs. Right panel, Tcon–BMDC–Treg conjugates formed in culture of BMDC + Tcon + Treg + OVA peptide. A quantitative summary of these data are provided in the 1Materials and Methods under the heading Adoptive transfer, in a list of the percentages of Tcon–BMDC conjugates that also contain Tregs. A more detailed record of the conjugate composition is presented in Supplemental Fig. 8.

FIGURE 8.

me/me and me/+ Tregs are more efficient in preventing productive Tcon–APC conjugate formation than +/+ Tregs. A, Schematic depiction of experimental setup. B, Tcon (prestained with Alexa 633), Tregs (prestained with Alexa 488), and BMDCs loaded with the indicated concentrations of OVA peptide were incubated for 6 h at 37°C at a ratio of 2:1:1 (BMDC/Tcon/Treg). Cells were harvested, stained with anti-CD4 and anti-CD11c, and collected on an FACSCalibur (BD Biosciences). Conjugates were defined based on CD4 and Alexa 633 staining for Tcons and CD11c staining for BMDCs. Numbers indicate percentages of Tcons conjugated to BMDCs. To assess the specificity of Treg-mediated effects, no Tregs or Alexa 488-stained Tcons were added. Experiment shown is representative of four independent experiments. C, BMDC (Alexa 405)–Tcon (Alexa 647)–Treg (Alexa 488) conjugates formed after incubating for 4 h at 37°C at 1:1:1 ratio. Analysis of conjugates was performed on an ImageStream100 as in Fig. 6. Conjugates were defined based on CD4 and Alexa 647 staining for Tcons, Alexa 405 staining for BMDCs, and CD4 and Alexa 488 staining for Tregs. Representative images of individual conjugates are shown. Left panel, BMDC–Tcon conjugates from control culture of BMDC + Tcon + OVA peptide without Tregs. Right panel, Tcon–BMDC–Treg conjugates formed in culture of BMDC + Tcon + Treg + OVA peptide. A quantitative summary of these data are provided in the 1Materials and Methods under the heading Adoptive transfer, in a list of the percentages of Tcon–BMDC conjugates that also contain Tregs. A more detailed record of the conjugate composition is presented in Supplemental Fig. 8.

Close modal

Intriguingly, we still observed many Tcon–BMDC conjugates under conditions in which we did not observe robust proliferation of Tcons. When we separated the Tcon/BMDC conjugates into Treg-containing (Tcon–BMDC–Treg) and no-Treg containing conjugates (Tcon–BMDC), we uncovered that a substantial percentage of Tcon–BMDC conjugates also contained Tregs (Fig. 8C, Supplemental Fig. 8). This tripartite conjugate was more pronounced with me/+ or me/me Tregs (e.g., 52% at 100 ng OVA peptide) compared with +/+ Tregs (30%).

Because activated Tcons upregulate CD25 and produce IL-2 in an Ag-dependent manner (Fig. 9A, 9B, bottom panels, Supplemental Fig. 7A, 7B), we directly addressed how the presence of Tregs would affect these parameters. Tcons that are part of the Tcon–BMDC–Treg conjugate failed to upregulate CD25 or produce IL-2 (Fig. 9A, 9B). Interestingly in the same culture, Tcons in the bipartite Tcon–BMDC conjugates (without Tregs) were able to efficiently upregulate CD25 (Fig. 9A). Previous studies have suggested that Tregs can mediate bystander suppression wherein the presence of Tregs in the same culture with Tcon and APC, even when they are not part of the same complex, is sufficient to induce suppression (reviewed in Refs. 1, 2). However, the level and mechanism of such suppression remains poorly understood. Our system of being able to simultaneously quantitate Tcon responses at the single-cell level in the context of conjugates with or without Tregs allowed us to visualize bystander suppression. Remarkably, we observed that IL-2 production by Tcons was partially inhibited by the simple presence of Tregs in the culture, even when the Tcon–BMDC conjugates did not contain Tregs (Fig. 9B). We were concerned that this inhibition of IL-2 production in the Treg-lacking Tcon–BMDC conjugates represented those that initially were tripartite complexes, from which the Tregs had dissociated during the experimental procedures; however, based on time-course experiments, this was not found to be the case (data not shown). Moreover, full suppression of CD25 upregulation on Tcons did not occur via this bystander mechanism, but was only on those Tcons that were part of the Tcon–BMDC–Treg tripartite conjugate. This suggests that different parameters of Tcon activation are differentially suppressed, perhaps indicating that Tregs may use multiple mechanisms of suppression.

FIGURE 9.

Presence of Tregs in Tcon–APC conjugates inhibits Tcon activation. A and B, Tcon (prestained with Alexa 647), Treg (prestained with Alexa 488), and BMDCs (prestained with Alexa 405) loaded with 500 ng/ml OVA peptide were incubated for 8 h at 37°C at a ratio of 1:1:1. Cells were harvested and stained with anti-CD25 (A) or anti–IL-2 (B) preanalysis on an ImageStream100 instrument (Amnis Corporation) as described above. The punctate staining pattern of IL-2 has been described previously (47). Representative images of individual conjugates are shown. Top panels, Examples of BMDC–Tcon–Treg conjugates (note that CD25 staining colocalizes with Tregs but not with Tcons; open arrows point to representative Tcons, filled arrows point to representative Tregs). Middle panels, Examples of BMDC–Tcon conjugates that lack Tregs in conjugate (note colocalization of CD25 with Tcons). Bottom panels, Examples of BMDC–Tcon conjugates are shown from a control culture of BMDC + Tcon + OVA peptide without added Tregs. The median for similarities between CD25 and Tcon staining in each sample are indicated below the representative pictures in A. In B, the percentages of IL-2–producing Tcons within conjugates are indicated below the representative pictures. C and D, Quantitative analysis of data obtained in the experiment described in Fig. 8C. The data presented were obtained from experiments using BMDCs loaded with 100 ng/ml or 500 ng/ml OVA peptide. Graphs depict the percentages of input Tcons conjugated with BMDCs that do not contain Tregs in the same conjugate. Percentages incorporate number of individual Tcons conjugated to BMDCs. Primary data for one representative experiment and detailed calculations are provided in Supplemental Fig. 8.

FIGURE 9.

Presence of Tregs in Tcon–APC conjugates inhibits Tcon activation. A and B, Tcon (prestained with Alexa 647), Treg (prestained with Alexa 488), and BMDCs (prestained with Alexa 405) loaded with 500 ng/ml OVA peptide were incubated for 8 h at 37°C at a ratio of 1:1:1. Cells were harvested and stained with anti-CD25 (A) or anti–IL-2 (B) preanalysis on an ImageStream100 instrument (Amnis Corporation) as described above. The punctate staining pattern of IL-2 has been described previously (47). Representative images of individual conjugates are shown. Top panels, Examples of BMDC–Tcon–Treg conjugates (note that CD25 staining colocalizes with Tregs but not with Tcons; open arrows point to representative Tcons, filled arrows point to representative Tregs). Middle panels, Examples of BMDC–Tcon conjugates that lack Tregs in conjugate (note colocalization of CD25 with Tcons). Bottom panels, Examples of BMDC–Tcon conjugates are shown from a control culture of BMDC + Tcon + OVA peptide without added Tregs. The median for similarities between CD25 and Tcon staining in each sample are indicated below the representative pictures in A. In B, the percentages of IL-2–producing Tcons within conjugates are indicated below the representative pictures. C and D, Quantitative analysis of data obtained in the experiment described in Fig. 8C. The data presented were obtained from experiments using BMDCs loaded with 100 ng/ml or 500 ng/ml OVA peptide. Graphs depict the percentages of input Tcons conjugated with BMDCs that do not contain Tregs in the same conjugate. Percentages incorporate number of individual Tcons conjugated to BMDCs. Primary data for one representative experiment and detailed calculations are provided in Supplemental Fig. 8.

Close modal

Although there is some inhibition of Tcon activation even in the exclusive Tcon–BMDC conjugates, these conjugates are also the only ones in these cultures that are capable of any activation. This prompted us to focus on the number of bipartite Tcon–BMDC conjugates and in turn ask how this would be influenced by Tregs from control and SHP-1–deficient mice. Calculating the conjugates using IDEAS analytical software (Supplemental Fig. 8), we made two key observations: first, the addition of +/+ Tregs decreased the percentage of exclusive Tcon–BMDC conjugates by 50–65% (Fig. 9C); second, Tregs from the me/me background were more efficient and decreased the percentage of Tcon–BMDC conjugates by 75–80% (me/me) (Fig. 9D). Taken together, in the context of SHP-1 deficiency, the overall decrease in Tcon–BMDC conjugates with a concurrent increased fraction of Treg–BMDC–Tcon conjugates (which are nonproductive for Tcon activation) results in fewer exclusive Tcon–BMDCs conjugates and translates to an enhanced suppressive activity of Tregs from me/me mice readily seen ex vivo and in vivo. These studies identify SHP-1 as an essential and nonredundant intracellular signaling molecule that modulates the potency of Treg-mediated suppression.

Despite the significant knowledge acquired about Tregs over the past few years, very little is known about signaling events within Tregs that can alter the potency of their suppressive activity. In this report, we identify the intracellular signaling molecule SHP-1 as a nonredundant and cell autonomous modulator of Treg function both in vitro and in vivo. Using mice either homozygous or heterozygous for the me/me allele, we found that SHP-1 is an endogenous brake for the potency of Treg-mediated suppression and that loss of SHP-1 expression manifests as increased suppressive activity. Furthermore the SHP-1–specific pharmacological inhibitor SSG complemented the data from me/me mice and demonstrated that SHP-1 modulates Treg function in a cell-autonomous manner. It is interesting to note that SSG via its inhibitory effect on SHP-1 causes an activation of Treg function. SSG is a drug originally marketed to treat Leishmania infections (26). Interestingly, Tregs have been shown to dampen the immune response, thereby allowing low level persistence of the Leishmania parasite in the host, which confers long-term immunity and resistance to reinfection (43). In particular, CD103+ Tregs, which we found to be enriched in SHP-1–deficient mice, have been shown to be the subpopulation accumulating at the site of Leishmania infection (44). In addition, more recent studies have proposed an antitumor effect for SSG via a T cell-dependent mechanism (24, 25). In fact, SSG is currently tested in several phase I clinical trials for patients with advanced solid tumors, lymphoma, or myeloma (2729). Because enhanced Treg suppressor activity could interfere with antitumor immunity, our findings that SSG can potentiate Treg suppressor activity suggests that these treatments should also be evaluated for concurrent effects on Tregs in the patients.

Comparable expression profiles between SHP-1–sufficient and SHP-1–deficient Tregs obtained from the microarray data further confirmed that SHP-1 affects Treg function at the level of signaling and likely not at the developmental level. Interestingly, despite the lack of differences at the mRNA level (Supplemental Fig. 2), we detected significant changes in the surface expression profiles of several molecules associated with Treg function among +/+, me/+, and me/me Tregs, indicating posttranscriptional regulatory mechanisms. This finding emphasizes the need to complement microarray analyses with studies assessing protein levels. At this point, it is unknown at what level the expression of the individual proteins is regulated. Future studies will be required to differentiate whether protein synthesis or degradation is affected by SHP-1 deficiency and whether this is directly linked to SHP-1, or whether it is a secondary effect reflecting a difference in activation status. Whereas earlier studies had supported the notion that TCR-mediated activation is absolutely required for Tregs to mediate their suppression (1618), a recent study by Szymczak-Workman et al. (45) suggested that Tregs may not require activation by the cognate peptide, although activation increased the suppressive activity. In our study, we found that Ag-mediated activation is required for suppression (Fig. 2). However, we used a very low Treg/Tcon ratio of 1:16. At this ratio, Szymczak-Workman et al. (45) also did not detect any suppression in the absence of TCR-mediated activation. Interestingly, we found that me/me Tregs demonstrated some suppression even in the absence of stimulation. Similarly, our conjugate assays showed a basal conjugate formation between Tregs and APCs even in the absence of peptide (Fig. 6), which was not observed when Tcons were conjugated with APCs (data not shown). It is therefore possible that a subpopulation (5–10% of total) of freshly isolated Tregs is already activated, either in the animal or during the isolation procedure, and can confer suppression without further stimulation. Consistent with the data presented by Szymczak-Workman et al. (45), this phenotype will be more obvious at higher ratios of Tregs. Our studies extend previous findings by demonstrating that the strength of signaling downstream of the TCR directly affects the suppressive activity of the Tregs. Moreover, our data indicate that at least one mechanism by which SHP-1 modulates Treg activity is targeting signaling pathways downstream of the TCR.

Although the mRNA expression profiles are similar among +/+, me/+, and me/me Tregs, there are substantial differences at the protein expression levels. Whereas SHP-1–deficient Tregs showed selective upregulation of a number of proteins, the expression of other molecules associated with the status of Treg activation, such as LAG-3 (46) and CD69, was not affected by the presence or absence of SHP-1. Most prominently, SHP-1 deficiency caused an increase in Tregs expressing adhesion molecules associated with an activated phenotype. This, in turn correlated with SHP-1–deficient Tregs being more efficient in conjugate formation than Tregs of control mice. However, conjugate formation of me/me or me/+ Tregs is still Ag-dependent indicating that loss of SHP-1 heightens the activity but does not uncouple activity from other regulatory mechanisms (such as TCR dependency). Correlating with the increased ability to form conjugates, SHP-1–deficient Tregs are more efficient in inhibiting the upregulation of the costimulatory molecules CD80/CD86 on APCs. This further supports the hypothesis that APCs are a target of Treg-mediated suppression, which in turn could affect how well these APCs can activate Tcons.

Whereas several previous studies have documented that Tregs inhibit the overall proliferation and IL-2 production of Tcons in vitro, many of these studies were based on analyses of whole populations and not at the level of single cells. Using a combined flow cytometry/microscopy approach that allows visualization of individual Tcon–APC–Treg conjugates, we demonstrate that Tregs inhibit the activation of Tcons at two levels. First, there is a modest inhibition of conjugate formation between Tcons and APCs. This is more evident at suboptimal levels of Ag (100 ng OVA peptide) than at optimal levels (500 ng OVA peptide), consistent with previous data that Tregs are more efficient suppressors at low Ag dose (11). Second, the Tcon–APC conjugates can be divided into two groups depending on whether Tregs are coconjugated to the complexes. Our data show that Tcons conjugated to APCs with Tregs are not activated as evidenced by an almost complete failure to upregulate CD25 or express IL-2. Remarkably in the same culture, a fraction of Tcons conjugated to APCs without Tregs can be fully activated, whereas partial inhibition is also detectable in other conjugates; this suggests that the most efficient and complete Treg-mediated suppression is limited to the APCs that are in direct contact with the Tregs. Interestingly, any so-called bystander suppression that we observed in our culture was limited to IL-2 production by Tcons with relatively little apparent effect on CD25 upregulation. This suggests that suppression by Tregs may occur via different mechanisms and thereby affect or interfere with Tcon activation at various levels. Furthermore, our dose-response experiments demonstrate that at low Ag concentration (100 ng/ml OVA peptide), direct inhibition of Tcon–APC conjugate formation is a major contributor to Treg-mediated suppression. In contrast, at high concentrations (500 ng/ml OVA peptide), inhibition of conjugate formation plays a relatively minor role, but suppression is instead mediated by the formation of tripartite conjugates (Tcon–APC–Treg). Quite interestingly, SHP-1–deficient Tregs are more effective at both levels of suppression. me/me and me/+ Tregs are more efficient than +/+ Tregs at inhibiting conjugate formation between Tcons and BMDCs. In addition, SHP-1–deficient Tregs are also more effective in forming tripartite conjugates (Tcon/APC/Treg) and thereby limiting the number of bipartite Tcon–APC conjugates. Based on our quantitative analysis of these assays, loss of SHP-1 results in ∼2- to 3-fold increased suppressive activity compared with control +/+ Tregs. Finally, the data presented in this work linking SHP-1 to the strength of Treg-mediated immune suppression, along with the effect of the drug SSG, could prove therapeutically useful in disease states in which enhancing Treg function could be beneficial.

We thank Dr. Kodi Ravichandran for critical reading of the manuscript, comments, and suggestions. We thank Joanne Lannigan and Michael Solga (Flow Cytometry Core Facility at the University of Virginia) for outstanding technical and intellectual help with flow cytometric and Imagestream100 analyses. We also thank Dr. Irene Mullins for help provided with the RNA preparation and amplification for the microarray analyses.

Disclosures The authors have no financial conflicts of interest.

This work was supported by National Institutes of Health Grant RO1 AI48672 (to U.L.).

The online version of this article contains supplemental material.

Abbreviations used in this paper:

BMDC

bone marrow-derived dendritic cell

EDF

Extended Depth of Field Technology

me/me

motheaten

MFI

mean fluorescence intensity

SHP-1

Src homology region 2 domain-containing phosphatase 1

SSG

sodium stibogluconate

Tcon

conventional T cell

Tg

transgenic

Treg

regulatory T cell.

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