Flow cytometric analysis and cell sorting

T_conv (CD4⁺CD25⁻CD45RB^hi) and T_reg (CD4⁺CD25⁺CD45RB^lo) cells from the spleens and lymph nodes of C57BL/6 or knockout age-matched mice were positively sorted by FACS. Purity of sorted T_conv and T_reg was verified by Foxp3 staining and FACS analysis. Where indicated, T_conv (CD4⁺Foxp3⁻CD45RB^hi) and T_reg(CD4⁺ Foxp3⁺CD45RB^lo) cells from the spleens and lymph nodes of Foxp3^gfp mice were positively sorted by FACS. Following red blood cell lysis with Gey's solution, cells were stained with antibodies against CD4, CD25, and CD45RB (eBioscience, San Diego, CA) for T cell isolation and sorted on a MoFlo (Dako, Fort Collins, CO) or Reflection (i-Cyt, Champaign, IL). Flow cytometric analysis was performed using a FACSCalibur (Becton Dickinson, San Jose, CA).

Anti-CD3/CD28-coated latex beads

4µM sulfate latex beads (Molecular Probes) were incubated overnight at room temperature with rotation in a 1;4 dilution of anti-CD3 + anti-CD28 antibody mix (13.3 µg/ml anti-CD3 Ab (eBioscience) and 26.6 µg/ml anti-CD28 (eBioscience). Beads were washed 3 times with 5mM phosphate buffer pH 6.5 and resuspended at 5×10⁷/ml in sterile phosphate buffer with 2mM BSA.

Polarized cell generation

Murine T cells were purified by MACS separation using biotinylated CD8, CD11b, CD11c, CD25, Ter119, B220, panNK, Mac-1 and Gr-1 antibodies to deplete non-T cells. MACS purified T cells were seeded at 2×10⁶/ml in a 24 well plate coated with anti mCD3ε (4µg/ml) and soluble anti-CD28 (2µg/ml). Recombinant cytokines and neutralizing antibodies were added as indicated for polarization. Th1 conditions: 35ng/ml recombinant IL-12, 10 µg /ml αIL-4; Th2 conditions: 50ng/ml recombinant IL-4, 10 µg /ml αIFNγ. Cells were split on day 3 and expanded in media containing IL-2 (10 Units/ ml). On day 6, cells were collected, washed and used in functional assays. Polarization was verified by intracellular cytokine staining, cytokine secretion using Luminex™ technology, and Tbet (Th1)/Gata3 (Th2) qPCR.

RNA, cDNA and quantitative real-time PCR

Purified T_conv and T_reg were cultured with anti-CD3/CD28-coated latex beads or in the absence of stimuli. Where indicated, T_reg were activated in the presence of T_conv at a 4:1 (T_conv : T_reg) ratio. T_conv were used at 2×10⁶ cells/ml and T_reg at 5 ×10⁵ cells/ml in 100µl used in a total volume of 1 ml, with or without anti-CD3/CD28-coated latex beads. After 48 h, T_conv and T_reg were re-sorted based on Foxp3 expression or based on the expression of congenic markers Thy1.1 for T_conv and Thy1.2 for T_reg for analysis. T cell RNA was isolated from purified cells using the Qiagen micro RNA extraction kit (Valencia, CA) or by using the Trizol reagent. RNA was quantitated spectrophotometrically and cDNA generated using the Applied Biosystems (Foster City, CA) high capacity cDNA reverse transcription kit. The cDNA samples were subjected to 40 cycles of amplification in an ABI Prism 7900 Sequence Detection System instrument using TaqMan or Sybr green PCR master mix (ABI) (primer sequences listed in Supplementary Table I). Quantitation of relative mRNA expression was determined by the comparative CT method (ABI User Bulletin #2, pg. 11 - http://docs.appliedbiosystems.com/pebiodocs/04303859.pdf) whereby the amount of target mRNA, normalized to endogenous β actin or cyclophillin expression was determined by the formula: 2^−ΔΔCT.

Immunoprecipitation and Western Blotting

Immunoprecipitation and immunoblotting were performed as previously described (6–8). Cellular supernatants were diluted in lysis buffer containing 0.1% Tween 20, 50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, and 1 complete protease inhibitor tablet (Roche, Indianapolis, IN) per 50 ml lysis buffer. Supernatants were immunoprecipitated with anti-mouse Il12a (p35) antibody (clone C18.2; eBioscience, San Diego, CA) [pre-coupled to Protein G-sepharose beads]. Immunoprecipitates were resolved by SDS-PAGE (Invitrogen Life Technologies, Carlsbad, CA), and blots were probed with a biotinylated monoclonal anti-mouse Ebi3 antibody (30A1, eBioscience). Blots were developed using ECL (Amersham Biosciences, Piscataway, NJ) and bands quantitated by densitometry using a Bio-Rad Gel Documentation System (Hercules, CA).

Cytokine quantitation

Cytokine secretion in cell culture supernatants was measured by Luminex™ technology. T_conv and T_reg were used at 2×10⁶ cells/ml and 5 ×10⁵ cells/ml, respectively, in a 100µl volume in a 96 well round bottom plate with or without anti-CD3/CD28-coated latex beads. After 72 hours, the supernatants from these in vitro co-culture assays and Transwell™ experiments were collected and analyzed for IL-10, IL-2, and TGFβ using Millipore multiplex kits from LINCO Research (St. Charles, MO). Cytokine concentrations were determined, in duplicate, using standard curves of reference proteins supplied by the manufacturer.

Proliferation and T_reg suppression assays

For standard proliferation assays, 5 × 10⁴ T_conv were activated with anti-CD3/anti-CD28 or anti-Vβ8 coated latex beads. Cultures were pulsed with 1 µCi [³H]-thymidine for the final 8 h of the 72 h assay and harvested with a Packard harvester. Counts per minute were determined using a Packard Matrix 96 direct counter (Packard Biosciences, Meriden, CT). In vitro T_reg function was measured by culturing 5 × 10⁴ T_conv with anti-CD3/anti-CD28 coated latex beads and titrations of T_reg. T_conv were activated with anti-CD3/anti-CD28 coated latex beads for 72 h. For direct comparison of T_reg suppressive capacity with and without T_conv cell contact, T_reg were also cultured in direct contact with responder T_conv in the bottom chamber of the Transwell™ plate. In indicated assays, recombinant IL-10 (eBioscience, San Diego, CA) or neutralizing anti-IL-10 antibody (clone JES5-2A5, BD Bioscience, San Diego, CA) were added to standard T_reg assays and Transwell™ experiments. Cultures were pulsed and harvested as described for proliferation assays.

T_conv-potentiated T_reg suppress across a permeable membrane in an IL-35- and IL-10-dependent manner

Previous studies using Transwell™ culture plates suggested that T_reg were unable to suppress responder T_conv proliferation when the two cell populations were separated by a permeable membrane (1, 3, 7, 8). It is important to note that in these studies, resting or activated T_reg (in the absence of T_conv) were cultured in the top chamber of a Transwell™ plate and their ability to suppress activated T_conv in the bottom chamber was determined by ³H-thymidine incorporation. However, our data indicate that T_reg required direct contact with T_conv for maximal secretion of IL-35, suggesting that cytokine dependence or independence of in vitro T_reg function should be re-examined under these conditions. Thus, different cell populations in the upper chamber of a Transwell™ plate were assayed for their ability to suppress proliferation of purified “responder” T_conv that were cultured with anti-CD3 (clone 145-2C11)/anti-CD28 (clone 37.51)-coated latex beads in the bottom chamber. As other groups have shown, minimal suppression of responder T_conv proliferation in the lower chamber was observed with resting or activated T_conv or T_reg in the upper chamber (Fig. 2A). Remarkably, wild type T_reg in the presence of T_conv (at a 4:1 T_conv:T_reg ratio in the upper chamber) suppressed responder T_conv proliferation (in the lower chamber) by 40% in the absence of cell contact (Fig. 2A). Control experiments where the total number of cells in the top chamber of the Transwell™ plate were kept the same, demonstrate that this was not merely a result of combined suppression of individual T_conv and T_reg, but rather T_conv potentiation of T_reg suppression (Supplementary Fig. 1). As this suppression occurred across a permeable membrane, this inferred a role for soluble factors. Thus, we next assessed whether the inhibitory cytokines IL-35 and IL-10 contributed to the T_conv-potentiated T_reg suppression. Strikingly, IL-35-deficient T_reg from either Ebi3^−/− or Il12a^−/− mice fail to increase suppression across the permeable membrane in the presence of T_conv cells in the upper chamber (Fig. 2A). This suggests that IL-35 is required for this cell-contact independent suppression by T_regin vitro, which correlates with the 40-fold increase in IL-35 secretion observed upon contact with T_conv cells (Fig. 1C). Previous studies have suggested that IL-10 does not contribute to T_reg function in vitro, even though there is substantial data to support its importance in vivo. To our surprise, in the Transwell™ system, Il10^−/− T_reg in the presence of T_conv have reduced suppressive capacity when compared to wild-type T_reg, however this was greater than in the absence of T_conv cells in the upper chamber (Fig. 2A). This raises the possibility that IL-10 and IL-35 act together or synergize for maximal suppression mediated by T_reg and that their function is potentiated by T_conv cells.

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Figure 2

T_reg mediated suppression is potentiated by T_conv cell contact in an IL-10- and IL-35-dependent, but not IL-2, manner

T_conv or T_reg cells from the spleens and lymph nodes of C57BL/6, Ebi3^−/−, Il12a^−/− and Il10^−/− mice were purified by FACS. (A) Cells assayed for regulatory capacity (T_conv or T_reg alone or in combination at a 4:1 T_conv: T_reg ratio) were cultured in the top chambers of a Transwell™ culture plate as indicated. Freshly purified wild-type “responder” T_conv were cultured in the bottom chamber of the 96-well flat bottom plates in medium containing anti-CD3/anti-CD28-coated latex beads. After 64 h in culture, top chambers were removed and [³H]-thymidine was added directly to the responder T_conv cells in the bottom chambers of the original Transwell™ plate for the final 8 h of the 72 h assay. Cultures were harvested and cpm determined. (B) Purified T_reg cells from wild-type, Ebi3^−/− and Il10^−/− mice were cultured at the indicated ratios with purified T_conv and assayed for regulatory capacity in the top wells of the Transwell™ plate. In parallel, T_reg were assayed for regulatory capacity when T_reg were in direct contact with responder T_conv in the bottom chamber of the Transwell™ plate. Suppression of purified responder T_conv cells was measured by [³H]-thymidine incorporation. (C) RNA was extracted from cells in the bottom chamber of the Transwell™ plate. Groups analyzed were those cultured with no cells in the top chamber or WT T_conv co-cultured with T_reg (WT, Il10^−/−). IL-2 expression was determined by real-time quantitative PCR. Data represent the mean ± SEM of (A) 4, (B) 2, (C) 2 and independent experiments. Statistical analysis: *p< 0.05. Counts per minute of T_conv cells activated alone, in the absence of any suppressors, were 25,000 – 60,000.

T_conv potentiation of T_reg suppression is robust and not controlled by strength of activation

The ability of T_conv to potentiate T_reg -mediated suppression across the Transwell™ was not dependent upon the strength of proliferation of the responder T_conv in the bottom chamber of the Transwell™. Transwell™ T_reg suppression was seen when the proliferation of responder T_conv alone ranged from 5,000 to 75,000 counts per minute (data not shown). It should be noted that all cell populations were sorted based on cell surface marker expression, T_conv(CD4⁺CD45RB^hiCD25⁻) and T_reg (CD4⁺CD45RB^loCD25⁺) and were both pure and expressed comparable levels of Foxp3, as determined by intracellular staining (Supplementary Fig. 2). Thus, any differences in their suppressive capacities was not due to differences in purity or Foxp3 expression. To illustrate the robustness of the suppression seen across the Transwell™, parallel suppression assays were performed in which T_reg were are placed in contact with T_conv in the lower chamber. Our results show that Transwell™ suppression mediated by T_conv:T_reg co-culture was approximately half what was seen in conventional T_reg assays where T_conv and T_reg are in direct contact [2:1 T_conv: T_reg ratio - 91% vs. 59%; 4:1 T_conv: T_reg ratio - 78% vs. 39%] (Fig. 2B). This is surprisingly robust given that the IL-35 and IL-10 secreted by the T_reg in the upper chamber has to diffuse across the entire well to achieve suppressive concentrations. This contrasts with the substantially higher concentration of cytokine that would be present in close proximity to targets with which T_reg are in contact (22)

Reduced suppression in the absence of IL-10 and IL-35 is not due to differential consumption of IL-2

Recent data suggests that the suppressive effects of T_reg are mediated in part by consumption of IL-2 (23). To determine whether differences seen in the ability of wild-type and cytokine deficient T_reg to suppress across the Transwell™ was due to differential consumption of IL-2, we measured IL-2 concentration in the Transwell™ T_reg assay. Total IL-2 secretion was not reduced in the Transwell™ culture when responder cell proliferation was suppressed (Supplementary Fig. 3a). These data suggest that the Transwell™ suppression observed was not due to IL-2 deprivation-mediated apoptosis.

As there are dual sources of IL-2 in this assay (from T_conv in the top and bottom chambers of the Transwell™), IL-2 secretion may in fact be reduced on a per cell basis. Indeed, if one adjusts for T_conv cell number, IL-2 secretion per 10⁴ cells in the assay was reduced (Supplementary Fig. 3b). To directly assess if IL-2 expression was reduced by Transwell™ suppression, we measured IL-2 mRNA expression in the target responder T_conv in the bottom chamber following suppression by T_conv:T_reg co-cultured in the top chamber. We found that the responder T_conv IL-2 expression was reduced by 30–50%, depending upon the genotype of the T_reg in the top chamber (Fig 2C). This suggests that IL-2 expression is reduced by Transwell™ suppression in the absence of direct contact with target T_conv in the bottom chamber.

The role of pre-activation in mediating Transwell suppression

Recent studies using pre-activated and fixed T_reg have contributed to our understanding of the characteristics and conditions required for T_reg to suppress T_conv cell proliferation (1, 24). Reports indicate that previously activated T_reg do not require re-stimulation through their TCR to suppress T_conv proliferation. Moreover, once pre-activated, T_reg can be fixed and still retain their suppressive capacity. To determine whether Il10^−/− and Ebi3^−/− T_reg were able to suppress under these conditions, wild-type, Il10^−/− and Ebi3^−/− T_reg were assayed for proliferative capacity following pre-activation, with and without fixation, in both conventional and Transwell™ T_reg assays. Like wild-type T_reg, freshly isolated, fixed Il10^−/− and Ebi3^−/− T_reg were unable to suppress T_conv proliferation in a conventional T_reg assay. Not surprisingly, if T_reg were pre-activated prior to fixation, all T_reg regardless of genotype were able to mediate suppression, albeit to a slightly lesser degree that freshly isolated T_reg (Fig. 3A). In a Transwell™ assay, pre-activation did not enhance the suppressive capacity of the T_reg, and pre-activated and subsequently fixed T_reg, regardless of genotype, were unable to suppress responder T_conv proliferation in the bottom well (Fig. 3B)

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Figure 3

Regulatory capacity of fresh and pre-activated fixed T_reg

T_conv or T_reg cells from the spleens and lymph nodes of C57BL/6, Ebi3^−/−, and Il10^−/− mice were purified by FACS. Cells were cultured fresh or pre-activated for 24h prior to culture, with or without fixation with 20% formaldehyde. Cells assayed for regulatory capacity (T_conv or T_reg alone or in combination at a 4:1 T_conv:T_reg ratio) were cultured in (A) direct contact with responder T_conv in a 96-well round bottom plate. Statistical analysis indicates fixation does not affect the ability of pre-activated T_reg to suppress while in direct contact with responder T_conv (B) top chambers of a Transwell™ culture plate as indicated. Freshly purified wild-type “responder” T_conv were activated in the bottom chamber of the 96-well plates. After 64 h in culture, top chambers were removed and [³H]-thymidine was added directly to the responder T_conv cells in the bottom chambers of the original Transwell™ plate for the final 8 h of the 72 h assay. Cultures were harvested and cpm determined. Statistical analysis: *p< 0.05.

The role of IL-10 in T_reg suppression in vitro

It is well known that IL-10 is essential for T_reg suppression in vivo (4, 9–17). However, previous studies using IL-10 neutralizing antibodies suggested that IL-10 is not required for T_reg -mediated suppression in vitro (1, 3, 8). We sought to expand upon these results by using an IL-10 neutralizing antibody in both conventional and Transwell™ T_reg assays. We show that IL-10 neutralization has no effect on suppression in a conventional T_reg assay, as previously reported, however in the Transwell™ system, neutralizing IL-10 reduces the suppression mediated by wild-type T_reg from 40% to 15% and further reduces the suppression by Il10^−/− T_reg from 25% to 5% (Fig. 4). Hence, IL-10 secretion from both the T_reg and suppressed T_conv populations appear important to maintaining the fidelity of T_reg suppression across the Transwell™. This is surprising given that the amount of IL-10 that the suppressed target T cells (T_sup) produce appears small when Il10^−/− T_reg were cultured with T_conv (Fig. 1A/B). However, it is possible that the actual IL-10 contribution is much higher as T_reg-derived IL-10 may potentiate IL-10 production by the T_sup. Importantly, Transwell™ suppression by Il10^−/− T_reg was restored by addition of recombinant IL-10 (Fig. 4). Thus, the differential and potentially synergistic use of IL-10 and IL-35 by T_reg cells may help explain and unify in vitro and in vivo results regarding the necessity of IL-10 for T_reg suppression. In a Transwell™ T_reg assay, approximately 300pg/ml TGFβ was detected in the culture supernatant (data not shown). Given the modest amount of TGFβ present in the Transwell™ T_reg assay, it is unlikely that suppression of T_conv cell proliferation is influenced by TGFβ, however this will need to be formally tested.

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Figure 4

T_reg mediated suppression across the Transwell™ is IL-10 dependent

Purified T_reg were assayed for regulatory capacity in conventional T_reg and Transwell™ T_reg assays in the presence of an IL-10 neutralizing antibody (10µg/ml) or with recombinant IL-10 (100ng/ml) as indicated. Percent suppression was calculated in reference to the activated T_conv control under the conditions indicated. All % suppression to the left of the line was in reference to T_conv without anti-IL-10 or rIL-10 and the reference for % suppression to the right of the line was T_conv with anti-IL-10 or rIL-10, respectively. Data represent the mean ± SEM of 3 independent experiments. Statistical analysis: *p< 0.05. Counts per minute of T_conv cells activated alone, in the absence of any suppressors, were 25,000 – 60,000.

We wish to thank Karen Forbes for colony management, Richard Cross for FACS, Jennifer Smith for cytokine analysis, staff of the St Jude ARC Histology Laboratory and Animal Husbandry Unit, and Peggy Just, Gangzhou Li, and Diana Mitchell of eBioscience for Ebi3 Western Blotting reagents. We also wish to thank Richard Blumberg and Tim Kuo for the Ebi3^−/− mice, Alexander Rudensky for the Foxp3^gfp knockin mice, and Terrence Geiger for the Il10^−/− mice.