Supplementary Results. Metabotropic glutamate receptor 4 impacts adaptive immunity restraining neuroinflammation

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1 Supplementary Results Metabotropic glutamate receptor 4 impacts adaptive immunity restraining neuroinflammation Francesca Fallarino, Claudia Volpi, Francesco Fazio, Serena Notartomaso, Carmine Vacca, Carla Busceti, Silvio Bicciato, Giuseppe Battaglia, Valeria Bruno, Paolo Puccetti, Maria C. Fioretti, Ferdinando Nicoletti, Ursula Grohmann * & Roberto Di Marco * Ursula Grohmann, Ph.D. (ugrohmann@tin.it) University of Perugia Department of Experimental Medicine and Biochemical Sciences Via del Giochetto Perugia 06126, Italy This Supplementary File Includes: Supplementary Figures 1 to 17 with Legends Supplementary Tables 1 to 7 Supplementary Text with References 1

2 Figure S1. Lack of mglur4 increases the CNS invasion by CD4 +, CD8 +, and MHC class II + mononuclear cells in mice undergoing EAE. Spinal cord sections from WT and Grm4 / mice, vaccinated 30 d before with MOG as in Fig. 1b, were immunostained with anti-cd4, -CD8, and anti-mhc class II antibodies. The brown staining specifies cell surface binding of the specific antibodies. Representative positive cells are indicated by arrows. One experiment is shown representative of four. 2

3 Figure S2. Percentages of CD4 +, CD4 + CD25 +, CD4 + Foxp3 +, CD8 +, γδ, B220 +, CD11b + and CD11c + cells are grossly normal in the spleens of Grm4 -/- naïve mice. Total spleen cells of the indicated genotypes were analyzed for surface (or intracellular for Foxp3) expression of the indicated markers by flow cytometry. Numbers show percentages of positive cells in the indicated gate. Data are representative of three independent experiments with three mice per genotype. 3

4 Figure S3. Higher percentages of CD4 +, CD8 +, CD11b + and CD11c + cells infiltrates CNS in Grm4 / mice with EAE. Brain-infiltrating leukocytes were purified from WT and Grm4 / mice at day 30 post-vaccination with MOG. Numbers show percentages of positive cells in the indicated gate. Data are representative of three independent experiments with three mice per genotype. 4

5 Figure S4. Grm4 +/ and Grm4 /, but not Grm4 +/+, littermates from heterozygote breeding are highly susceptible to MOG-induced EAE. Grm4 +/+ (n = 9), Grm4 +/ (n = 12), and Grm4 / (n = 7) littermates from heterozygote breeding were vaccinated with MOG peptide on day 0. Clinical EAE scores (means ± s.d.) over time are shown. Results are compiled data from two experiments each consisting of two sets of littermates. P values (indicated) were determined by linear regression analysis (not shown in the figure). 5

6 Figure S5. CD4 + T cells polarized from naïve Grm4 / mice produce a normal cytokine profiles. a, Naïve CD4 + CD25 T cells purified from WT and Grm4 / LNs were activated with anti-cd3 and anti-cd28 in the absence (TH0) or presence of different recombinant cytokine/neutralizing antibody mixtures specific for TH1, TH2, TH17, or inducible Treg (itreg) cell commitment (see Methods). Natural Treg (ntreg) cells (i.e., naïve CD4 + CD25 + cells) were also assayed after activation with anti-cd3 and anti-cd28. No significant difference in the production of prototypical cytokines could be found between the two groups in any condition. Data are means ± s.d. of three experiments, each performed in triplicate. b, Expression of Foxp3 in CD4 + CD25 T cells purified from WT and Grm4 / LNs cultured as above in either neutral (TH0) or Treg-favouring conditions (itreg). One experiment representative of four. 6

7 Figure S6. A higher number of CD4 + T cells is recovered upon coculture with mglur4-deficient DCs. Five 10 5 naïve CD4 + T cells/well, purified from WT LNs, were activated with anti-cd3 and anti-cd28 in the presence of cdcs (ratio of 3:1) or pdcs (1:1), both purified from spleens of either genotype. After 4 d, viable cells were harvested and counted. Data are means ± s.d. of triplicate samples. One experiment representative of four is shown. 7

8 Figure S7. Silencing of the Grm4 gene is both efficient and specific in splenic cdcs. The kinetics of PCR analysis of Grm4 expression in cdcs treated with Grm4 sirna are shown. Control cells were treated with negative control (nc) sirna. Transcriptional expression of the Grm8 gene was also assayed. One experiment is shown representative of four. 8

9 Figure S8. PHCCC does not modulate cytokine production by DCs in response to LPS in the absence of mglur4 or in the presence of 8-Bromo-cAMP. Cytokine contents were measured in supernatants from Grm4 / DCs and WT DCs (both at ), the latter being either untransfected or transfected with Grm4 small interfering RNA, stimulated with 1 µg ml 1 LPS for 24 h in the presence of PHCCC (40 µm). Untransfected DCs were stimulated in the presence of 100 µm 8-Bromo-cAMP (8-BrcAMP). Data are means ± s.d. of three experiments, each performed in triplicate. 9

10 Figure S9. Treatment with the mglur4 enhancer, PHCCC, protects from EAE. Linear regression analysis of the experiment in Fig. 6a is shown. 10

11 Figure S10. PHCCC prevents but does not reverse EAE. WT mice were vaccinated with the MOG peptide on day 0 and PHCCC (3 mg/kg; n = 12) or vehicle (n = 15) was administered daily s.c. starting one day after the appearance of neurologic signs (16-19 d in this experiment). Clinical EAE scores (means ± s.d.) over time are shown. Compiled data are shown from two experiments. 11

12 Figure S11. PHCCC treatment reduces CNS invasion by CD4 +, CD8 +, and MHC class II + mononuclear cells in WT but not Grm4 / mice undergoing EAE. Spinal cord sections from WT and Grm4 / mice, vaccinated 30 d earlier with MOG as in Fig. 6a and treated with vehicle or PHCCC, were immunostained with anti-cd4, anti- CD8, and anti-mhc class II antibodies. The brown staining specifies cell surface binding of the indicated antibodies. Representative positive cells are indicated by arrows. One experiment representative of three is shown. 12

13 Figure S12. Protection against EAE by PHCCC is dose-dependent. Groups of WT mice (n = 8 10), vaccinated with MOG on day 0, received daily doses of PHCCC on days At the end of the experiment, frequencies of responders were recorded for each dose, response being defined as a highest clinical score never exceeding 1.0 at any time during the experiment. A fitting sigmoid dose-response curve (allowing for calculation of the mean effective dose, ED50) was obtained by the use of Prism (GraphPad Software). In parallel, groups of nonvaccinated WT mice (n = 5) were assayed for acute toxicity by PHCCC at 30 3,000 mg kg 1 as a single bolus. No deaths occurred over a 2-wk observation period, indicating a mean lethal dose of the drug exceeding 3,000 mg kg 1. 13

14 Figure S13. WT mice vaccinated with MOG and treated with PHCCC develop CD4 + CD25 + T cells that impede adoptive transfer of EAE. Linear regression analysis of the experiment in Fig. 6e is shown. 14

15 Figure S14. The in vivo effect of PHCCC in EAE is partly reversible. WT mice were vaccinated with MOG peptide on day 0. PHCCC (3 mg kg 1 ) was administered daily for 20 d starting from the day of vaccination (n = 6) or 40 d (n = 6). Control mice were injected with vehicle for 20 d (n = 8). Clinical EAE scores (means ± s.d.) over time are shown. One experiment of two is shown. 15

16 Figure S15. EAE in BM chimeras reveals that protective mglur4 signaling occurs in the periphery. EAE was induced with MOG in BM chimeras, which consisted of WT mice engrafted with either WT (WT WT) or Grm4 / (Grm4 / WT) BM cells. Clinical scores were evaluated daily and data are shown as means ± s.d. (n = 5 per group). P was assessed by linear regression analysis (not shown in the figure). 16

17 Figure S16. PHCCC inhibits IL-6 production in vitro by LPS-stimulated human DCs. Three 10 5 /ml human DCs, obtained as described in Methods, were incubated for 1 h with different concentrations of PHCCC prior to stimulation with 1 µg ml 1 LPS. After 24 h, IL-6 was measured in culture supernatants. Data are means ± s.d. of three experiments, each performed in triplicate. *P < 0.01 and **P <

18 Figure S17. Meta-analysis of DC gene expression data. a, GRM4, GRM6, GRM7, and GRM8 absolute expression levels in a panel of 60 untreated DC samples derived from public-available gene expression datasets. b, Heat-map depicts the relative changes of GRM4, GRM6, GRM7, and GRM8 scaled expression values in 60 untreated DC samples derived from eight different GEO series. 18

19 Supplementary Table 1 EAE in WT and Grm4 -/- mice. Exp No. Mice (n) Incidence (%) Mortality (%) Day of onset (mean ± s.d) Maximum score (mean ± s.d) 1 WT ± ± Grm4 / ± ± 0.8 P = P = P = WT ± ± Grm4 / ± ± 0.9 P = P < P = WT ± ± Grm4 / ± ± 0.8 P = P < P = EAE was induced in WT and Grm4 / mice by immunization with the MOG peptide on day 0. Only animals developing disease were included in the analysis. 19

20 Supplementary Table 2 Quantitative analysis of cell populations in total cells from spleens, lymph nodes (LNs) and brain-infiltrating leukocytes (BILs) in WT and Grm4 / mice under basal conditions or at 30 d of vaccination with MOG. Spleens LNs BILs Mice strain WT Grm4 / WT Grm4 / WT Grm4 / MOG Cell phenotype % of total cells CD a 4.3* * * nd 2.9 nd 9.6 CD * * nd 2.3 nd 6.8 γδ nd 0.3 nd 0.5 B * * nd 7.5 nd 10.1 CD11b nd nd nd nd nd 4.6 nd 6.5 CD11c nd 2.9 nd 7.9 % of gated CD4 + cells CD nd nd nd nd Foxp nd nd nd nd nd, not determined a, Data are mean percentages of positive cells from three experiments, each consisting of three mice per group. s.d. (not included in the Table) never exceeded 15% of the mean value. *P < 0.01 (EAE vs. respective control); P < 0.05 (EAE vs. respective control); P < 0.05 (Grm4 / vs. WT mice). 20

21 Supplementary Table 3 EAE in WT and Grm4 / mice treated with PHCCC. Mice Treatment (n) Incidence (%) Mortality (%) Day of onset (mean ± s.d) Maximum score (mean ± s.d) WT vehicle ± ± 0.8 WT PHCCC ± ± 0.8 P = P = P < Grm4 / vehicle ± ± 1.0 Grm4 / - PHCCC ± ± 1.3 P = P = P = EAE was induced in WT and Grm4 / mice by immunization with MOG peptide on day 0. PHCCC was administered daily s.c. at the dose of 3 mg kg 1 starting from day 1 of MOG vaccination. Control mice received vehicle (sesame oil) alone. Only animals developing disease were included in the analysis. 21

22 Supplementary Table 4 Quantitative analysis of cell populations in total cells from spleens, lymph nodes (LNs) and brain infiltrating leukocytes (BILs) in WT mice vaccinated with MOG (30 d) and treated daily with PHCCC or vehicle from day 1. Spleens LNs BILs PHCCC Cell phenotype % of total cells CD CD γδ B CD11b nd nd CD11c % of gated CD4 + cells CD * nd nd Foxp * nd nd nd, not determined a, Data are mean percentages of positive cells of three experiments, each consisting of three mice per group. s.d. (not included in the Table) never exceeded 15% of the mean value. *P < 0.05 (PHCCC vs. respective control). 22

23 Supplementary Table 5 Effect of PHCCC on relapsing-remitting EAE (RR- EAE). Mice Treatment n Incidence (%) Mortalit y (%) Day of onset (mean ± s.d) Maximum score (mean ± s.d) Cumulative disease score First relapse disease score Second relapse disease score SJL vehicle SJL PHCCC ± ± ± ± ± ± 2.6 ± 35.9 ± 15 ± ± P = P = P = <0.001 P = P <0.001 RR-EAE was induced in SJL/j mice by immunization with the PLP peptide on day 0. PHCCC was administered daily s.c. at the dose of 3 mg kg 1 starting from onset of the first clinical RR-EAE attack. Control mice received vehicle (sesame oil) alone. 23

24 Supplementary Table 6 Oligonucleotide sequences. Gene Tbx21 Gata3 Rorc Foxp3 Grm4 Grm6 Grm7 Grm8 Gapdh Sense and antisense oligonucleotides for rrt-pcr S, 5 -GGACGATCATCTGGGTCACATTGT AS, 5 -GCCAGGGAACCGCTTATATG-3 S, 5 -TCTGGAGGAGGAACGCTAATG-3 AS, 5 -GGCTGGAGTGGCTGAAGG-3 S, 5 -AGCAGTGTAATGTGGCCTAC-3 AS, 5 -GCACTTCTGCATGTAGACTG-3 S, 5 -AGAGCCCTCACAACCAGCTA-3 AS, 5 -CCAGATGTTGTGGGTGAGTG-3 S, 5 -GGCCCTCAAGTGGAACTATG-3 AS, 5 -CTCGTTGGCAAAGATGATGA-3 S, 5 -AAGTGATCCGGAGGCTCAT-3 AS, 5 -AGGAAGTGGCCAGTCAGGT-3 S, 5 -CCTGGTTATCGTCTCATTGG-3 AS, 5 -CACACAGAGGGTGGGATCT-3 S, 5 -CCACCCATATTCACCAAGC-3 AS, 5 -CTGGGGCTGTAGATGCATAG-3 S, 5 -GCCTTCCGTGTTCCTACCC-3 AS, 5 -CAGTGGGCCCTCAGATGC-3 24

25 Supplementary Table 7 Complete list of the datasets used in this study and their sources. Genome-wide expression levels were quantified using A- MADMAN on a total of 83 samples. A subset of 60 nonstimulated DC samples was used to assess GRM4, GRM6, GMR7, and GRM8 expression profiles. GEO series Platform Total samples in series Unstimulated samples in series GSE8658 HG-U133 Plus GEO accession of used samples GSM GSM GSM GSM GSM GSM GSM GSM GSM GSM GSM GSM GSM GSM GSM GSM GSM GSM Reference GSM GSE4984 HG-U133 Plus2 6 3 GSM GSE13762 HG-U133 Plus GSE5547 HG-U133 Plus2 6 6 GSM GSM GSM GSM GSM GSM GSM GSM GSM GSM GSM GSM GSM

26 GSM GSM GSM GSM GSM GSM GSM GSM GSE7509 HG-U133 Plus GSM GSM GSM GSM GSM GSM GSM GSM GSM GSM GSE9946 HG-U133A 12 3 GSM GSM GSM GSM GSE12773 HG-U133 Plus2 5 5 GSM GSE6965 HG-U133 Plus2 4 2 References to Supplementary Table 7 GSM GSM GSM GSM Szatmari, I. et al. PPARγ regulates the function of human dendritic cells primarily by altering lipid metabolism. Blood 110: (2007). 2.Fulcher, J.A. et al. Galectin-1-matured human monocyte-derived dendritic cells have enhanced migration through extracellular matrix. J. Immunol. 177: (2006). 3.Széles, L. et al. 1,25-dihydroxyvitamin D3 is an autonomous regulator of the transcriptional changes leading to a tolerogenic dendritic cell phenotype. J. Immunol. 182: (2009). 4.Humphreys, T.L et al. Dysregulated immune profiles for skin and dendritic cells are associated with increased host susceptibility to Haemophilus ducreyi infection in human volunteers. Infect. Immun. 75: (2007) 5.Dhodapkar, K.M. et al. Selective blockade of the inhibitory Fcγ receptor (FcγRIIB) in human dendritic cells and monocytes induces a type I interferon response program. J. Exp. Med. 204: (2007). 6.Popov, A. et al. Infection of myeloid dendritic cells with Listeria monocytogenes leads to the suppression of T cell function by multiple inhibitory mechanisms. J. Immunol. 181: (2008). 7.Rate, A., Upham, J.W., Bosco, A., McKenna, K.L., & Holt, P.G. Airway epithelial cells regulate the functional phenotype of locally differentiating dendritic cells: implications for the pathogenesis of infectious and allergic airway disease. J. Immunol. 182:72-83 (2009). 8.Mezger, M. et al. Impact of mycophenolic acid on the functionality of human polymorphonuclear neutrophils and dendritic cells during interaction with Aspergillus fumigatus. Antimicrob. Agents Chemother. 52: (2008). 8 26

27 SUPPLEMENTARY TEXT Features of immune cells in naïve Grm4 / mice. Because Grm4 / mice have never been evaluated in terms of immunologic phenotype, we analyzed (by flow cytometry) the percentages of CD4 +, CD8 +, γδ, B220 +, CD11b +, and CD11c + cells in spleens and pooled lymph nodes (LNs). CD25 and Foxp3 staining was performed on CD4 + lymphocytes gated from spleens and LNs. Under basal conditions (i.e., nonimmunized mice), percentages of immune cells in Grm4 / mice were generally similar to WT animals, with the exception of CD4 + T cells in LNs, which were significantly higher as compared to WT animals (Supplementary Table 2). No gender-related differences were noted in our study, and female and male Grm4 / mice were equally more susceptible to EAE than control mice (data not shown). TH cytokine profiles in WT and Grm4 / mice undergoing EAE. In our experimental system of MOG-induced EAE, we evaluated the cytokine profile in cells harvested at different times after MOG vaccination from LNs, spleens, and braininfiltrating lymphocytes (BILs). Nonvaccinated animals were used as a control. We found that in vitro MOG restimulation of LN CD4 + T cells harvested at 10 d of MOG vaccination induced a higher increase in IL-17A, but not IFN-γ, levels when cells were from Grm4 / than WT mice. TGF-β showed the opposite pattern, whereas IL-10 increased at a similar extent in both Grm4 / and WT mice. Splenic CD11c + DCs from vaccinated animals of both genotypes produced higher, albeit comparable, levels of IL-10, IL-12, IL-23, and TGF-β at 10 d post-vaccination relative to nonvaccinated controls (Fig. 2b). In contrast, IL-6 production was significantly higher and IL-27 lower in supernatants from mglur4-deficient cells. The cytokine pattern of BILs at 30 d resembled the early pattern in peripheral lymphoid organs (Fig. 2c). Therefore, the cytokine profiles in Grm4 / mice with EAE suggested that lack of mglur4 favored the emergence of TH17 over Treg cells in neuroinflammation. DCs produce Glu that can activate mglur4 signaling in the presence of PHCCC. PHCCC (40 µm) was able to inhibit camp formation in cultured DCs expressing mglur4, but not in DCs lacking mglur4, although we used a Glu-free medium (Fig. 4a). However, Glu was measurable (6.0 ± 0.7 µm and 6.6 ± 0.8 µm, mean ± s.d. of quadruplicate samples under basal conditions in mglur4 + and mglur4 DC cultures, respectively) at the end of the incubation, which may reflect the ability of DCs to release Glu into the medium 1. References 1. Franco, R., Pacheco, R., Lluis, C., Ahern, G.P. & O'Connell, P.J. The emergence of neurotransmitters as immune modulators. Trends Immunol. 28, (2007). 27

28 Supplementary Methods Metabotropic glutamate receptor 4 impacts adaptive immunity restraining neuroinflammation Francesca Fallarino, Claudia Volpi, Francesco Fazio, Serena Notartomaso, Carmine Vacca, Carla Busceti, Silvio Bicciato, Giuseppe Battaglia, Valeria Bruno, Paolo Puccetti, Maria C. Fioretti, Ferdinando Nicoletti, Ursula Grohmann * & Roberto Di Marco * Ursula Grohmann, Ph.D. (ugrohmann@tin.it) University of Perugia Department of Experimental Medicine and Biochemical Sciences Via del Giochetto Perugia 06126, Italy This Supplementary File Includes: Supplementary Text with References 1

29 EAE induction and treatment with PHCCC in vivo. Progressive EAE was induced in WT and Grm4 / mice by s.c. immunization with 200 µg of the murine myelinoligodendrocyte glycoprotein (MOG) peptide (MEVGWYRSPFSRVVHLYRNGK) emulsified in 100 µl of complete Freund s Adjuvant. Two hundreds ng of pertussis toxin in 100 µl of phosphate-buffered saline (PBS) were injected i.p. on the day of immunization (day 0) and two days later 1. Neurologic signs (ascending paralysis from the tail to the forelimbs) lasted for at least 2 months (WT mice) and eventually led to death (Supplementary Table 1). N- phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC) was dissolved in sesame oil and administered daily s.c. at the dose of 3 mg kg 1 starting from day 1 (prophylactic regimen). To evaluate any dose-dependency of the effect, PHCCC was also administered at the dose of 0.03, 0.3, and 300 mg/kg. Control mice received vehicle (sesame oil) alone. In the therapeutic regimen, the drug was administered daily s.c. at the dose indicated above but commencing one day after the appearance of initial signs of overt neuropathology (days and in WT and Grm4 / mice, respectively). For adoptive transfer of EAE, mice were immunized as above and used as donors of pathogenic CD4 + T lymphocytes, which were purified from spleens and LNs and incubated for 48 h with anti-cd3ε (145-2C11; 1 µg/ml) and anti-cd28 (PV-1; 1 µg/ml) in the presence of MOG peptide (25 µg/ml). A total of CD4 + cells/mouse was transferred i.p into WT recipients that were sublethally irradiated (450 rad) 18 h before adoptive transfer (day 0). On days 0 and 2 animals were also injected i.p. with 200 ng of pertussis toxin 2. In selected groups, CD4 + T cells were depleted of the CD25 + subset by means of mouse CD25 Microbeads (Miltenyi Biotec). Onset and progression of neurologic signs were monitored as above. Relapsing-remitting EAE (RR-EAE) was induced as described 3. Briefly, SJL/j mice were immunized with 75 µg of proteolipid protein peptide (PLP ; HSLGKWLGHPDKF) emulsified in CFA with 6 mg/ml Mycobacterium tuberculosis (Difco). Each mouse received s.c. injections of 200 µl emulsion divided among four sites draining axillary and inguinal LNs. Pertussis toxin (200 ng/mouse) was administered i.p. on the day of immunization and 48 h later. Clinical assessment of EAE was performed according to the same criteria as above. After the first clinical attack (12-15 d), mice were randomized into two groups to be treated with either 2

30 PHCCC or vehicle. PHCCC (3 mg kg 1 ) or vehicle was administered s.c. daily for 15 d and mice were monitored up to 80 d. A relapse was defined as a sustained (> 2 d) increase in clinical score by at least 1 full grade after the animal had improved previously by at least 1 full grade and stabilized for at least 2 days. EAE induction in bone marrow chimeras. Bone marrow (BM) chimeras were generated as described 4. Briefly, BM cells were isolated from either WT or Grm4 / mice by flushing femur and tibia bones with HBSS. BM was filtered through a 100- µm cell strainer and cells were washed with HBSS. Four to 6 wk-old WT recipient mice were lethally irradiated with 950 cgy and injected i.v. with 10 7 gender matched Grm4 / or WT BM cells, respectively. At 6 wk after engraftment, engrafted animals were immunized with MOG and monitored for clinical signs as above. Acute toxicity. Acute toxicity of PHCCC was studied in C57BL/6 mice receiving i.p. a single dose of the drug (30, 300, 900, 1500, or 3000 mg kg 1 ) dissolved in dimethyl sulfoxide (DMSO). No mortality could be observed up to 3000 mg kg 1 during the 2 wk observation period, indicating that the LD 50 pf PHCCC administered i.p. is > 3 g kg 1. Histology and immunohistochemistry. At day 30 after MOG immunization, mice were anesthetized by i.p administration of Avertin (125 mg/kg) and perfused transcardially with 4% paraformaldehyde in PBS. Spinal cords were removed, fixed for 24 h in ethyl alcohol (60%), acetic acid (10%), and chloroform (30%), and embedded in paraffin. Sections were cut at 5 µm and stained by haematoxylin and eosin (H&E) to reveal CNS inflammatory infiltrates. For immunohistochemistry, 5 µm deparaffinized sections were first soaked in 3% hydrogen peroxide to block endogenous peroxidase activity and then stained with rabbit polyclonal anti-myelin binding protein (MBP; NM_002385, Millipore), mouse monoclonal anti-class II MHC (NM_ , Millipore), rat monoclonal anti-cd4 (sc-58930, Santa Cruz Biotechnology), or rabbit polyclonal anti-cd8 (sc-7188, Santa Cruz Biotechnology) followed by appropriate biotinylated secondary antibodies (Vector Laboratories) and streptavidin-hrp (Zymed). Controls were performed with isotype-matched antibodies (data not shown). 3

31 Leukocyte isolation and stimulation. For in vitro differentiation, purified naïve CD4 + CD25 T cells were activated for 5 d with 1 µg ml 1 of plate-bound anti-cd3 and anti-cd28 in the presence of various combinations of recombinant cytokines and blocking antibodies as follows: IFN-γ (5 ng ml 1 ) and anti-il-4 (11B11; 10 µg ml 1 ) for differentiating T H 1 cells; IL-4 (10 ng ml 1 ) and anti-ifn-γ (XMG1.2; 10 µg ml 1 ) for T H 2 cells; TGF-β (5 ng ml 1 ), IL-6 (20 ng ml 1 ), anti-ifn-γ (10 µg ml 1 ), and anti- IL-4 (10 µg ml 1 ) for T H 17 cells 5 ; and TGF-β (10 ng ml 1 ) for itreg cells. In the T reg suppression assay, CD4 + CD25 cells were cocultured with irradiated T cell-depleted splenocyte samples and CD4 + CD25 + cells for 3 d in the presence of soluble anti-cd3. Proliferation was measured by incorporation of [ 3 H]thymidine according to standard procedures. Splenic DCs were purified by magnetic-activated cell sorting using CD11c MicroBeads and MidiMacs (Miltenyi Biotec), in the presence of EDTA to disrupt DC-T cell complexes, as described 6. For the purification of conventional (cdcs) and plasmacytoid DCs (pdcs), CD11c + cells were further fractionated using CD11b and mpdca-1 MicroBeads (Miltenyi Biotec) 7, respectively. Cytokine production from total or fractionated DC subpopulations was measured in culture supernatants harvested after 24 h cell incubation with medium alone, 1 µg ml 1 lipopolysaccharide (LPS; for cdcs), or 10 µg ml 1 CpG-ODN '-TCCATGACGTTCCTGACGTT- 3' (custom, phosphorothioate from Invitrogen Life Technologies; for pdcs). Splenic CD11b + and B220 + cells and lymph node γδ T cells were purified by magneticactivated sorting according to manufacturer s indicated procedure using specific Microbeads, namely CD11b, CD45R and, for γδ cells, fluorescein isothiocyanate (FITC)-labelled GL3 (hamster anti-γδ T-cell receptor, BD Pharmingen) followed by avidin-conjugated MicroBeads 8. Purification of brain-infiltrating leukocytes (BILs) was performed as described 9,10. Briefly, brains were recovered from anesthetized mice perfused with cold PBS. After centrifugation of brain homogenates, infiltrating leukocytes were separated on a discontinous percoll gradient (Sigma). Cytokine production from BILs was measured in culture supernatants harvested after 24 h cell incubation with medium alone. The expression of Tbx21, Gata3, Rorc, and Foxp3 was evaluated on CD4 + cells sorted from BILs and LN cells. 4

32 Human blood CD11c + DCs were obtained as described 11. Briefly, positively selected CD14 + monocytes (Miltenyi Biotec) were cultured in the presence of 200 ng ml 1 human GM-CSF and 50 ng ml 1 human IL-4 (both from Peprotech). After 6 d, the resultant monocyte-derived DCs were washed and incubated with 2.5, 10, or 40 µm PHCCC prior to the addition of 1 µg ml 1 LPS. After 24 h, IL-6 was measured in culture supernatants. Flow cytometry. In all FACS analyses, cells were treated with rat anti-cd16/32 (2.4G2) for 30 min at 4 C for the blockade of Fc receptors before assaying on an EPICS flow cytometer using EXPO 32 ADC software (Beckman Coulter). CD4 and CD25 expression was analyzed as described 7. Hamster anti-tcr γ/δ PE-conjugated antibody (clone GL3) was from Abcam. For intracellular Foxp3, cells were stained with anti-cd4 (GK1.5)- PE (BD Pharmingen), fixed, permeabilized and stained with Alexa Fluor 488 antimouse Foxp3 (MF-14; Biolegend) or isotype control Alexa Fluor 488 rat IgG2b, as described 12. FITC-labeled rat anti-mouse CD45R/B220 (clone RA3-6B2; BD Pharmingen), hamster anti-mouse CD11c (clone N418, ebioscience), and rat antimouse CD11b (clone M1/70; BD Pharmingen) were also used. In T cell-dc cocultures, the intracellular expression of IL-17A and IFN-γ was evaluated on gated CD4 + cells after staining using Fixation/Permeabilization solution (ebioscience). rrt-pcr, sirna synthesis and transfection Real-time PCR (for Tbx21, Gata3, Rorc, Foxp3, Grm4, Grm6, Grm7, Grm8, and Gapdh) analyses were carried out as described 8, using primers listed in Supplementary Table 6. Unfractionated brain homogenate was used as positive control for the expression of mglurs. For all panels, bars represent the ratio of gene to Gapdh expression as determined by the relative quantification method (ΔΔCT) (mean ± s.d. of triplicate determination). The cdna clone (Thermo Scientific) was used as a positive control for Grm6. For silencing Grm4 mrna, ON- TARGETplus sirna was predesigned on the basis of gene ID sequence and synthesized by Thermo Scientific (Dharmacon RNAi Technologies). ON- TARGETplus Non-Targeting sirnas were used as negative control. Transfection was carried out as described 7. 5

33 Western blot analysis and cytokine determination. Expression of mglur4 protein was examined by immunoblotting. Briefly, whole cell lysates were obtained from CD4 + CD25 + T cells activated by means of plate-bound anti-cd3 and anti-cd28 antibodies or from DCs stimulated with LPS, and Western blot analysis was conducted as described 13 using a specific polyclonal antibody recognizing the C-terminus sequence of rodent mglu4r 14. Anti β-actin antibody (Sigma) was used as a normalizer. Mouse cytokines (IL-2, IL-4, IL-6, IL-10, IL-12, IL-17A, IL-22, IL-23, IL-27, IFN-γ, TNF-α and TGF-β1) were measured in culture supernatants by ELISA using specific kits (R&D Systems and Abnova Corporation) or previously described reagents and procedures 7,8. The detection limits (pg ml 1 ) of the assays were 5 for IL-2, 2 for IL-4, 2 for IL-6, 4 for IL-10, 3 for IL-12 p70, 5 for IL-17A, 8 for IL-22, 4 for IL-23, 4 for IL-27 p28, 2 for IFN-γ, 6 for TNF-α and 15 for TGF-β1. Human IL-6 was measured in culture supernatants by ELISA using a high sensitivity (> 0.8 pg ml 1 ) kit (Abcam). Determination of intracellular camp and extracellular Glu levels. Measurements of intracellular camp levels were performed essentially as described 15. Briefly, unfractionated DCs ( cells/ 300µl/ sample) were pre-incubated in Locke s solution buffer (glutamate-free), ph 7.4, containing 0.5 mm isobutylmethylxanthine (a phosphodiesterase inhibitor) for 20 min to block the breakdown of camp. PHCCC (dissolved in DMSO at the initial concentration of 20 mm and diluted in Locke s solution to final concentrations of 2.5, 10, or 40 µm) or L-(+)-2-amino-4- phosphonobutanoate (L-AP4, 5 µm; from Tocris Cookson Ltd) was added 30 s before the addition of 10 µm forskolin (FSK; Sigma-Aldrich) or 1 µg ml 1 LPS and the incubation was continued for additional 10 min. The reaction was stopped by the addition of an equal volume of 0.8 N HClO 4. Samples were then sonicated and centrifuged at low speed (1500 g for 10 min). One hundred eighty microliters of the supernatant were added to 20 µl of K 2 CO 3 (2 M) and, after centrifugation in a microfuge (2 min at maximal speed), 20 µl were used for the camp assay. Intracellular camp levels were measured by Cyclic AMP [ 3 H] Biotrak Assay System (Amersham Biosciences). The same samples were also used for measuring extracellular Glu by HPLC as described 16. 6

34 Meta-analysis of dendritic cell gene expression data. To evaluate the expression of GRM4 and other members in human untreated DCs, different gene expression datasets were downloaded from Gene Expression Omnibus ( using A-MADMAN (Annotation-based MicroArray Data Meta ANalysis tool 17 ). A-MADMAN is a web application to retrieve, annotate and manage gene expression data. The rationale behind this tool is automating the error-prone steps necessary to set-up a working environment for the meta-analyses of expression data. A-MADMAN allows the automatic download and organization of GEO, proprietary raw data, and annotations, the automatic import of metadata from GEO records into a local relational database, the subsequent manual annotation and selection of samples through user-defined tags, and the selection of samples to be analyzed using a complex logical query on tags. The A-MADMAN low-level analysis of expression data uses the R backend powered by Bioconductor packages. Raw expression data (i.e., CEL files) obtained from different platforms are integrated using an approach inspired by the generation of custom Chip Definition Files (CDFs 18 ). In custom CDFs, probes matching the same transcript, but belonging to different probe sets, are aggregated into putative custom-probe sets, each one including only those probes with a unique and exclusive correspondence with a single transcript. Similarly, probes matching the same transcript but located at different coordinates on different type of arrays may be merged in custom-probe sets and arranged in a virtual platform grid. As for any other microarray geometry, this virtual grid may be used as a reference to create the virtual-cdf file containing the probes, shared among the platforms of interest, and their coordinates on the virtual platform, and the virtual-cel files containing the intensity data of the original CEL files properly re-mapped on the virtual grid. Once defined the virtual platform through the creation of its custom-cdf and transformed the CEL files into virtual-cels, raw data, originally obtained from different platforms, are homogeneous in terms of platform and can be preprocessed and normalized adopting standard approaches, as RMA or GCRMA. Specifically, 8 GEO series comprising 83 samples of human DCs, obtained using Affymetrix arrays HG-U133A and HG-U133 Plus2, were downloaded and organized in a proprietary database. Supplementary Table 7 reports the complete list of the datasets used in this study and their sources. After manual reannotation and tagging, all samples were meta-analyzed to derive gene expression 7

35 profiles. Expression values were generated from intensity signals using the combined HG-U133A/HG-U133 Plus2 virtual-cdf file, the custom definition files for human GeneChips based on GeneAnnot (version ), and the transformed virtual-cel files. Intensity values for a total of meta-probe sets have been background adjusted, normalized using quantile normalization, and gene expression levels calculated using median polish summarization (RMA algorithm 19 ). A subset of 60 untreated samples was used to assess the GRM4, GRM6, GMR7, and GRM8 expression profiles reported in Supplementary Fig

36 References 1. Mendel, I., Kerlero de Rosbo, N. & Ben-Nun, A. A myelin oligodendrocyte glycoprotein peptide induces typical chronic experimental autoimmune encephalomyelitis in H-2 b mice: fine specificity and T cell receptor Vβ expression of encephalitogenic T cells. Eur. J. Immunol. 25, (1995). 2. Stromnes, I.M. & Goverman, J.M. Passive induction of experimental allergic encephalomyelitis. Nat. Protoc. 1, (2006). 3. Mangano, K. et al. Variable effects of cyclophosphamide in rodent models of experimental allergic encephalomyelitis. Clin. Exp. Immunol. 159, Liu, J.Q. et al. CD24 on the resident cells of the central nervous system enhances experimental autoimmune encephalomyelitis. J. Immunol. 178, (2007). 5. Schraml, B.U. et al. The AP-1 transcription factor Batf controls T(H)17 differentiation. Nature 460, (2009). 6. Grohmann, U. et al. CTLA-4-Ig regulates tryptophan catabolism in vivo. Nat. Immunol. 3, (2002). 7. Grohmann, U. et al. Reverse signaling through GITR ligand enables dexamethasone to activate IDO in allergy. Nat. Med. 13, (2007). 8. Romani, L. et al. Defective tryptophan catabolism underlies inflammation in mouse chronic granulomatous disease. Nature 451, (2008). 9. Liu, T. & Chambers, T.J. Yellow fever virus encephalitis: properties of the brain-associated T-cell response during virus clearance in normal and γ interferon-deficient mice and requirement for CD4 + lymphocytes. J. Virol. 75, (2001). 10. Fitzgerald, D.C. et al. Suppressive effect of IL-27 on encephalitogenic Th17 cells and the effector phase of experimental autoimmune encephalomyelitis. J. Immunol. 179, (2007). 11. Orabona, C. et al. Toward the identification of a tolerogenic signature in IDOcompetent dendritic cells. Blood 107, (2006). 12. Fallarino, F. et al. Therapy of experimental type 1 diabetes by isolated Sertoli cell xenografts alone. J. Exp. Med. 206, (2009). 13. Ngomba, R.T. et al. Positive allosteric modulation of metabotropic glutamate 4 (mglu4) receptors enhances spontaneous and evoked absence seizures. Neuropharmacology 54, (2008). 14. Corti, C., Aldegheri, L., Somogyi, P. & Ferraguti, F. Distribution and synaptic localisation of the metabotropic glutamate receptor 4 (mglur4) in the rodent CNS. Neuroscience 110, (2002). 15. Matrisciano, F. et al. Defective group-ii metaboropic glutamate receptors in the hippocampus of spontaneously depressed rats. Neuropharmacology 55, (2008). 16. Bruno, V. et al. Selective activation of mglu4 metabotropic glutamate receptors is protective against excitotoxic neuronal death. J. Neurosci. 20, (2000). 17. Bisognin, A. et al. A-MADMAN: annotation-based microarray data metaanalysis tool. BMC Bioinformatics 10, 201 (2009). 18. Ferrari, F. et al. Novel definition files for human GeneChips based on GeneAnnot. BMC Bioinformatics 8, 446 (2007). 19. Irizarry, R.A. et al. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4, (2003). 9

37 Supplementary Note Metabotropic glutamate receptor 4 impacts adaptive immunity restraining neuroinflammation Francesca Fallarino, Claudia Volpi, Francesco Fazio, Serena Notartomaso, Carmine Vacca, Carla Busceti, Silvio Bicciato, Giuseppe Battaglia, Valeria Bruno, Paolo Puccetti, Maria C. Fioretti, Ferdinando Nicoletti, Ursula Grohmann * & Roberto Di Marco * Ursula Grohmann, Ph.D. (ugrohmann@tin.it) University of Perugia Department of Experimental Medicine and Biochemical Sciences Via del Giochetto Perugia 06126, Italy This Supplementary File Includes: Supplementary Text with References 1

38 Signaling and expression of group-iii metabotropic glutamate receptors. Metabotropic glutamate receptors (mglurs) form a family of eight subtypes, subdivided into three groups on the basis of their amino acid sequence and G-protein coupling. Group I includes mglur1 and mglur5, which are coupled to G q protein. Group II includes mglur2 and mglur3, which are coupled to G i /G o proteins; group III includes mglur4, mglur6, mglur7 and mglur8, which are also coupled to G i /G o proteins in heterologous expression systems 1,2. Although data have indicated the presence of mglur1 and mglur5 in human T cells 3 and mouse thymocytes 4, the expression and function of mglur4 or other group-iii mglurs have never been examined in cells of the immune system. In contrast, expression of all mglurs is well known in the central nervous system (CNS), with the exception of mglur6 that appears to be confined to rod bipolar cells of the retina 5. Of particular relevance in the present context is the fact that group-iii mglur4 and mglur8 display a differential expression pattern in distinct types of MS lesions 6. In the rim of chronic active lesions, both mglur4 and mglur8 have been found to be expressed by a population of reactive astrocytes. In contrast, in active lesions, mglur8, but not mglur4 is expressed in cells of the microglia/macrophage lineage, whereas fewer macrophage-like cells express the receptor in chronic active and inactive lesions 6. Interestingly, we found that Grm8 expression is prominent in peripheral CD11b + cells (mainly macrophages and myeloid DCs) and, to a lesser extent, in B220 + cells (mostly B lymphocytes) (Fig. 3a). Thus, taking into consideration the acute nature of MOG-induced EAE, literature data combined with our current results would argue against an immunoregulatory effect of mglur4 signaling at the CNS level. The peripheral effect of PHCCC was also confirmed using experimental autoimmune neuritis, a T-cell mediated autoimmune inflammatory demyelinating disease of the peripheral nervous system (data not shown). T H cytokine profiles and EAE. In adaptive responses, a paradigm of two functionally opposing CD4 + T H cell populations T H 1 and T H 2 cells 7 has long dominated acquired immunity scene. The recent discovery of T H 17 cells as a distinct subset of effector cells has led to a revised model of adaptive immunity 8,9. More plastic than T H 1 and T H 2 cells 10, T H 17 cells develop under strict, bidirectional influence by T reg cells 11. The development of T H 17 and T reg cells is, in turn, conditioned by T H 1-associated cytokines such as interferon-γ (IFN-γ) and IL-27. These cytokines, both strictly linked to the development and function of T H 1 cells, can have profound effects on the commitment of other CD4 + T cell subtypes. Besides representing the functional marker of T H 1 cells and antagonizing the rise of T H 2 cells 7, IFN-γ can induce the generation of T reg cells in vivo either directly, inducing Foxp3 in CD4 + CD25 T cells 12,13, or indirectly, by influencing the function of antigen-presenting cells 14. IFN-γ is also important for the regulatory function of T reg cells in vivo 15. In addition to T reg cells, IFN-γ inhibits the development of T H 17 cells 16 and their production of IL IL-27, produced by macrophages and dendritic cells (DCs) 18, induces the development of T H 1 cells. IL-27 also suppresses the development of T H 17 cells via inhibition of IL-6 signaling 19 and exerts antiinflammatory properties in various models of infectious diseases and autoimmunity 20, including EAE 18,21. More recently, IL-27 has been shown to block Rorc expression directly, thus inhibiting the lineage commitment of T H 17 cells 22. However, IL-27 has little effect on committed T H 17 cells, despite their expression of a functional IL-27 receptor 23. 2

39 Orthosteric and allosteric modulators of mglurs. A positive allosteric modulator or enhancer is, by definition, a drug that amplifies receptor function acting at a site distinct from the recognition site of orthosteric ligands. In the case of mglu4rs, the enhancer N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC) binds to a putative site located in the heptaspanning transmembrane (7-TM) region, whereas the endogenous (Glu) and exogenous (L-AP4) orthosteric agonists bind to a recognition site located in the N-terminus extracellular domain 24,25. The enhancer amplifies receptor function in an activity-dependent manner, i.e., the enhancer recruits only those receptors that are activated by endogenous or exogenous orthosteric agonists. There are several advantages of allosteric modulators over traditional orthosteric agonists/antagonists 26,27. Firstly, specific allosteric modulators (i.e., those with no intrinsic agonist activity) induce effects that are contingent upon copresence of endogenous ligands. Secondly, it is possible to observe greater receptor selectivity due to sequence divergence in allosteric binding sites. In fact, L-AP4 is not selective for mglur4 and can activate also mglur6, mglur7, and mglur8. Thirdly, for targets such as mglurs, orthosteric ligands are mostly found among amino acid analogs which display poor brain penetration and suboptimal pharmacokinetics. 3

40 References 1. Nicoletti, F., Bruno, V., Copani, A., Casabona, G. & Knopfel, T. Metabotropic glutamate receptors: a new target for the therapy of neurodegenerative disorders? Trends Neurosci. 19, (1996). 2. Conn, P.J. & Pin, J.P. Pharmacology and functions of metabotropic glutamate receptors. Annu. Rev. Pharmacol. Toxicol. 37, (1997). 3. Pacheco, R. et al. Glutamate released by dendritic cells as a novel modulator of T cell activation. J. Immunol. 177, (2006). 4. Storto, M. et al. Expression of metabotropic glutamate receptors in murine thymocytes and thymic stromal cells. J. Neuroimmunol. 109, (2000). 5. Nomura, A. et al. Developmentally regulated postsynaptic localization of a metabotropic glutamate receptor in rat rod bipolar cells. Cell 77, (1994). 6. Geurts, J.J. et al. Expression patterns of Group III metabotropic glutamate receptors mglur4 and mglur8 in multiple sclerosis lesions. J. Neuroimmunol. 158, (2005). 7. Mosmann, T.R. & Coffman, R.L. TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu. Rev. Immunol. 7, (1989). 8. Langrish, C.L. et al. IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J. Exp. Med. 201, (2005). 9. Ivanov, II et al. The orphan nuclear receptor RORγt directs the differentiation program of proinflammatory IL-17 + T helper cells. Cell 126, (2006). 10. Zhou, L., Chong, M.M. & Littman, D.R. Plasticity of CD4 + T cell lineage differentiation. Immunity 30, (2009). 11. Bettelli, E. et al. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 441, (2006). 12. Wang, Z. et al. Role of IFN-γ in induction of Foxp3 and conversion of CD4 + CD25 T cells to CD4 + Tregs. J. Clin. Invest. 116, (2006). 13. Feng, G., Wood, K.J. & Bushell, A. Interferon-γ conditioning ex vivo generates CD25 + CD62L + Foxp3 + regulatory T cells that prevent allograft rejection: potential avenues for cellular therapy. Transplantation 86, (2008). 14. Wood, K.J. & Sawitzki, B. Interferon γ: a crucial role in the function of induced regulatory T cells in vivo. Trends Immunol. 27, (2006). 15. Sawitzki, B. et al. IFN-γ production by alloantigen-reactive regulatory T cells is important for their regulatory function in vivo. J. Exp. Med. 201, (2005). 16. Kimura, A., Naka, T. & Kishimoto, T. IL-6-dependent and -independent pathways in the development of interleukin 17-producing T helper cells. Proc. Natl. Acad. Sci. U.S.A. 104, (2007). 17. Park, H. et al. A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat Immunol 6, (2005). 18. Kastelein, R.A., Hunter, C.A. & Cua, D.J. Discovery and biology of IL-23 and IL-27: related but functionally distinct regulators of inflammation. Annu. Rev. Immunol. 25, (2007). 19. Stumhofer, J.S. et al. Interleukin 27 negatively regulates the development of interleukin 17-producing T helper cells during chronic inflammation of the central nervous system. Nat. Immunol. 7, (2006). 4

41 20. Yoshida, H. & Miyazaki, Y. Regulation of immune responses by interleukin- 27. Immunol. Rev. 226, (2008). 21. Fitzgerald, D.C. et al. Suppressive effect of IL-27 on encephalitogenic Th17 cells and the effector phase of experimental autoimmune encephalomyelitis. J. Immunol. 179, (2007). 22. Diveu, C. et al. IL-27 blocks RORc expression to inhibit lineage commitment of Th17 cells. J. Immunol. 182, (2009). 23. El-behi, M. et al. Differential effect of IL-27 on developing versus committed Th17 cells. J. Immunol. 183, (2009). 24. Maj, M. et al. (-)-PHCCC, a positive allosteric modulator of mglur4: characterization, mechanism of action, and neuroprotection. Neuropharmacology 45, (2003). 25. Sarichelou, I. et al. Metabotropic glutamate receptors regulate differentiation of embryonic stem cells into GABAergic neurons. Cell Death Differ. 15, (2008). 26. Marino, M.J. & Conn, P.J. Glutamate-based therapeutic approaches: allosteric modulators of metabotropic glutamate receptors. Curr. Opin. Pharmacol. 6, (2006). 27. Conn, P.J., Christopoulos, A. & Lindsley, C.W. Allosteric modulators of GPCRs: a novel approach for the treatment of CNS disorders. Nat. Rev. Drug Discov. 8, (2009). 5