Indoleamine 2,3-dioxygenase controls conversion of Foxp3+ Tregs to TH17-like cells in tumor-draining lymph nodes
Blood Sharma et al.
113: 6102
Supplemental materials for: Sharma et al
Reagents, 1MT, and cell lines
1-methyl-D-tryptophan (catalog #452483) and 1-methyl-L-tryptophan (catalog #447439) were purchased from Sigma (St. Louis MO) or supplied by NewLink Genetics Inc. (Ames, IA) and dissolved as described.1 For in vivo use, D 1MT in drinking water at 2 mg/ml (or vehicle control) was administered as described.2
Conjugated antibodies against mouse CD4, CD8, IL-10, Thy1.1, TNF, and CD11c were from BD-Pharmingen; antibodies against IL 17A, granzyme B, IL 2, and IL 6 were from eBioscience; antibodies against IL 22 and CXCR3 were from R&D Systems. Recombinant mouse IL 6 (R&D Systems) was used at 100 ng/ml. Polyclonal anti-mouse IL 6 antibody (cat. # AF-406-NA, R&D Systems) was used at 100 ug/ml. Recombinant mouse CD28/Fc chimeric protein (#483-CD, R&D Systems) was used at 20 ug/ml.
Cell lines used were B16F103 (obtained from ATCC, Manassas, VA) and B16 OVA (B16F10 transfected with full-length chicken ovalbumin, clone MO44, obtained from Dr. Alan Houghton, Memorial Sloan Kettering).
Mouse strains and radiation chimeras
TCR-transgenic OT I mice (CD8+, B6 background, recognizing the SIINFEKL peptide of ovalbumin on H2Kb) were purchased from Jackson Laboratories (Bar Harbor, ME). GCN2 KO mice1 (B6 background) were a generous gift from the laboratory of David Ron, New York University School of Medicine. Foxp3GFP mice5 were the generous gift of Alexander Rudensky and were inbred >5 generations onto the B6 background. Rorγtgfp/gfp mice6 were the generous gift of Dan Littman, New York University. A1 mice (CBA background, recognizing an H Y peptide presented on IEk) and IDO1 KO mice (B6 background) were as described.2
For Treg adoptive transfer, Tregs were isolated from spleens of TCR-tg OT II mice bred onto the Foxp3GFP background.5 OT II mice were obtained from Jackson and crossed to Foxp3GFP mice on a Thy1.1-congenic background. OT II mice possess Tregs which can respond to cognate OVA antigen7 and to B16 OVA tumors8; naive OT II T cells are capable of differentiation into both Tregs and TH17 cells under appropriate conditions.9
For radiation chimeras, wt B6 recipients were irradiated (9.5 Gy) and transplanted with 1 × 107 nucleated bone-marrow cells from either RORγtgfp/gfp mice or wt B6 controls. Chimeras were used 8 weeks after transplant. IAb-KO mice lack an IAb promoter-exon 1 region due to Cre/loxP mediated deletion and were generated as described10 by crossing mice carrying a floxed IAb allele with TIE2-Cre mice (both on C57BL/6J background). The resultant heterozygous mice were interbred to establish an IAb-KO breeding colony.
Vaccines
Recombinant lentivector expressing truncated cytoplasmic chicken ovalbumin (OVA Lv) was prepared by using transient co-transfection method as described.11 Plasmid DNA containing the mutant TRP1 gene (muTRP1) has been previously described12 and was the generous gift of Jose Guevara-Patino. Lentivector vaccines were delivered by footpad injection 5 days after tumor implantation, timed such that maximal production of antigen would coincide with the OT I injection on day 7. CpG vaccines were prepared by emulsifying 100 ug OVA protein (Sigma F-5506) and 50 ug CpG 1826 (gift of Coley Pharmaceuticals) in incomplete Freund’s adjuvant (Sigma) as described13 and administered in the footpad on day 7 at the time of OT I transfer.
Treg activation co-cultures and readout assays
The Treg culture system has been described in detail.2 Pre-activation cultures contained 2 × 103 pDCs, isolated by sorting for the CD11c+B220+ fraction, which contains the IDO-expressing subset of CD19+ pDCs, as previously described.14 To these were added 1 × 105 sorted CD8+ OT I cells, 100 nM SIINFEKL peptide, and 5 × 103 sorted CD4+CD25+ Tregs from spleens of non–tumor-bearing B6 mice, or CD4+GFP+ Tregs from Foxp3GFP mice. All cultures received a feeder layer of 1 × 105 T-cell–depleted B6 spleen cells (CD4NEGCD8NEG), as described,2 in order to maintain viability of the sorted cell populations. We have previously shown that these feeder cells are entirely nonspecific, and can be MHC-mismatched or IDO-deficient.2 For αCD3-induced activation, identical cultures received 200 uM 1MT plus 0.1 ug/ml αCD3 mAb and 10 ng/ml IL-2. Cultures were harvested after 2 days, and either stained for FACS analysis, or the Tregs re-stained for CD4 expression and sorted for functional suppression assays (comprising 1 × 105 A1 cells, 2 × 103 CD11c+ DCs from CBA spleen, and H Y peptide). Proliferation of readout assays was measured after 72 hrs by thymidine incorporation. An allogeneic readout was used to prevent any activation of the Tregs during the readout assay, so that suppression of the readout T cells was strictly dependent on activation of the Tregs in the pre-activation cultures.
Transfection of T-REX cells with IDO and western blot for NF IL6
Western analysis of the LIP isoform of NF-IL6/CEBPß was performed as described15 using equal amounts of protein from lysates derived from T-REX cells stably transfected with an inducible IDO construct (pCDNATO4-IDO, as described15). Cells were seeded into 12 well dishes. IDO was not expressed in uninduced cells, and could be induced by treatment with Doxycycline (20 ng/mL). Replicate cultures of cells induced with Doxycycline were also treated with 50, 25, and 10µM of the IDO enzyme inhibitors L-1MT or MTH-tryptophan.16 To confirm functional IDO expression, cells were harvested 48 hours following treatment and lysed in RIPA buffer. Kynurenine production in cells was analyzed essentially as described.16 Briefly, 200 µL of the media form the treated cells was mixed with 12.5 µL 30% TCA, incubated 30 min at 50˚C, and clarified by 10 min centrifugation at 3–10,000 rpm. Supernatants (100 µL) were removed to a new dish, mixed with 100 µL Ehrlich’s reagent (2% p-dimethylaminobenzaldehyde w/v in glacial acetic acid), and incubated 10–30 min at room temperature. Absorbance was determined at 490 nm.
To confirm IDO induction in transfected cells by western blot, affinity purified rabbit polyclonal anti-IDO was prepared by a commercial supplier (Covance). Antisera was raised against a mixture of murine and human GST-conjugated IDO1. Antisera was screened for reactivity against the immunizing antigen by ELISA and western, and samples with high titer were purified by affinity chromatography. Specifically, antisera was preabsorbed to protein column containing GST and GST-IDO2 (and IDO related protein) The unbound material was then affinity purified on an antigen specific peptide column containing human and mouse His-tagged IDO1. The resulting antibody was analyzed and determine to be IDO1 specific with no cross-reactivity with IDO2. The primary antibody was detected using HRP-conjugated goat anti-rabbit antibody and chemiluminescence.
REFERENCES
1. Munn DH, Sharma MD, Baban B, et al. GCN2 kinase in T cells mediates proliferative arrest and anergy induction in response to indoleamine 2,3-dioxygenase. Immunity. 2005;22:633–642.
2. Sharma MD, Baban B, Chandler P, et al. Plasmacytoid dendritic cells from mouse tumor-draining lymph nodes directly activate mature Tregs via indoleamine 2,3-dioxygenase. J Clin Invest. 2007;117:2570–2582.
3. Nicolson GL, Brunson KW, Fidler IJ. Specificity of arrest, survival, and growth of selected metastatic variant cell lines. Cancer Res. 1978;38:4105–4111.
4. Falo LD, Jr., Kovacsovics-Bankowski M, Thompson K, Rock KL. Targeting antigen into the phagocytic pathway in vivo induces protective tumour immunity. Nat Med. 1995;1:649–653.
5. Fontenot JD, Rasmussen JP, Williams LM, Dooley JL, Farr AG, Rudensky AY. Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity. 2005;22:329–341.
6. Eberl G, Marmon S, Sunshine MJ, Rennert PD, Choi Y, Littman DR. An essential function for the nuclear receptor RORgamma(t) in the generation of fetal lymphoid tissue inducer cells. Nat Immunol. 2004;5:64–73.
7. Sutmuller RP, den Brok MH, Kramer M, et al. Toll-like receptor 2 controls expansion and function of regulatory T cells. J Clin Invest. 2006;116:485–494.
8. Wang L, Pino-Lagos K, de Vries VC, Guleria I, Sayegh MH, Noelle RJ. Programmed death 1 ligand signaling regulates the generation of adaptive Foxp3+CD4+ regulatory T cells. Proc Natl Acad Sci U S A. 2008;105:9331–9336.
9. Mucida D, Park Y, Kim G, et al. Reciprocal TH17 and regulatory T cell differentiation mediated by retinoic acid. Science. 2007;317:256–260.
10. Shimoda M, Mmanywa F, Joshi SK, et al. Conditional ablation of MHC-II suggests an indirect role for MHC-II in regulatory CD4 T cell maintenance. J Immunol. 2006;176:6503–6511.
11. He Y, Zhang J, Donahue C, Falo LD, Jr. Skin-derived dendritic cells induce potent CD8(+) T cell immunity in recombinant lentivector-mediated genetic immunization. Immunity. 2006;24:643–656.
12. Guevara-Patino JA, Engelhorn ME, Turk MJ, et al. Optimization of a self antigen for presentation of multiple epitopes in cancer immunity. J Clin Invest. 2006;116:1382–1390.
13. Miconnet I, Koenig S, Speiser D, et al. CpG are efficient adjuvants for specific CTL induction against tumor antigen-derived peptide. J Immunol. 2002;168:1212–1218.
14. Munn DH, Sharma MD, Hou D, et al. Expression of indoleamine 2,3-dioxygenase by plasmacytoid dendritic cells in tumor-draining lymph nodes. J Clin Invest. 2004;114:280–290.
15. Metz R, Duhadaway JB, Kamasani U, Laury-Kleintop L, Muller AJ, Prendergast GC. Novel tryptophan catabolic enzyme IDO2 is the preferred biochemical target of the antitumor indoleamine 2,3-dioxygenase inhibitory compound D-1-methyl-tryptophan. Cancer Res. 2007;67:7082–7087.
16. Muller AJ, Duhadaway JB, Donover PS, Sutanto-Ward E, Prendergast GC. Inhibition of indoleamine 2,3-dioxygenase, an immunoregulatory target of the cancer suppression gene Bin1, potentiates cancer chemotherapy. Nat Med. 2005;11:312–319.
