B6/COLODR/SPL/11C/83/LAP/#2.006 B6/COLODR/SPL/11C/86/LAP/#2.016 CD11C B6/COLODR/SPL/11C/80/LAP/#2.011 CD11C

Transcription

1 CD3-specific antibody-induced immune tolerance and suppression of autoimmune encephalomyelitis involves TGF-β production through phagocytes digesting apoptotic T cells Sylvain Perruche 1,3, Pin Zhang 1, Yongzhong Liu 1, Jeffrey A. Bluestone 2, and WanJun Chen 1 * a b CD86 CD83 Clodronate B6//SPL/11C/83/LAP/#1.3 B6/COLODR/SPL/11C/83/LAP/# CD11C CD11c B6//SPL/11C/86/LAP/# CD11C CD11c R1 R1 R2 R CD11C B6/COLODR/SPL/11C/86/LAP/#2.16 R1 R1 R2 R CD11C Cell # (%) Cell # (%) Clodro P =.12 CD11c + CD83 + (R1) + + Clodro P =.97 CD11c + CD86 +hi (R1) P =.3 CD11c + CD83 (R2) P =.2 CD11c + CD86 /low (R2) c CD8 B6//SPL/11C/8/LAP/#1.8 B6/COLODR/SPL/11C/8/LAP/# CD11C CD11c R1 R1 R2 R CD11C Cell # (%) Clodro P =.27 CD11c + CD8 +hi (R1) P =.5 CD11c + CD8 /low (R2) Supplementary Fig. S1 Clodronate-loaded liposomes selectively deplete idcs. C57BL/6 mice were injected i.v. with - or clodronate-loaded liposomes. Spleen cells were then harvested 24 h later, stained with CD11c-FITC and CD83- (a), CD86- (b), or CD8-PE (c) antibodies and analyzed with FACS calibur. Data are shown as representative FACS profiles of CD11c vs. indicated surface markers in each group of mice (left panel). Data are shown as mean ± s.d. of indicated cell numbers of individual mice (n = 2-3 in each group, right panel). The experiment was repeated twice with similar results.

2 a F4/8 + MΦ:Red; Apo-T cells: Green. b 4 c F4/8 + MΦ: Red; Apo CD4 + T cells: Green. TGF-β1 (pg/ml) d Relative cell number MΦ+Med MΦ+Apo anti-cd FL4-LAP-TGFb LAP-TGF-β M1 e LAP-TGF-β + cells (%) (within F4/8 + ) P =.2 anti-cd3 in vivo treatment Supplementary Fig. S2 Macrophages engulf and digest apoptotic T cells and produce TGF-β. (a) Macrophages stained with PE-F4/8 antibody (red) were co-cultured with apoptotic thymocytes pre-labeled with CFSE for 12 h and examined under immuno-fluorescence microscopy. White arrows: apoptotic cells that are engulfed and ingested by macrophages; yellow arrows: apoptotic contacting macrophages. (b) The supernatants from macrophages cultured alone and plus apoptotic thymocytes were tested for total TGF-β1 by ELISA. The data are shown as mean ± s.d. of the duplicate wells and representative of three independent experiments. (c) Macrophages take up and digest apoptotic CD4 + T cells in vivo. The experiment was done as described in Fig. 2e. Green color: apoptotic T cells, red color: F4/8 + macrophages in spleen. (d,e) Macrophages increase LAP-TGF-β after CD3- antibody treatment in vivo. C57BL/6 mice were injected with or CD3-antibody. Spleen cells were stained for F4/8 and LAP-TGF-β 24 h later. (d) A representative FACS profile of LAP-TGF-β expression on macrophages. Dashed line indicates the histogram of isotype control antibody. (e) Data are shown as mean ± s.d. of LAP-TGF-β + cells within F4/8 + macrophages in individual mice (n = 7 mice in each group).

3 25 anti-cd3-day 1 anti-cd3-day 4 P =.3 P =.4 CD4 + CD25 + Foxp3 + Tregs (%) P =.2 P =.3 P =.2 P =.3 P =.5 5 P =.3 P =.4 nd Blood PLN MLN Spleen Lungs Supplementary Fig. S3 CD3-antibody treatment induced increase in CD4 + Foxp3 + Treg cells in vivo. C57BL/6 mice were injected with - or clodronate-loaded liposomes one day before CD3-antibody treatment. On days one and four post-antibody injection, T cells from blood, spleen, peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN) and lungs were isolated and stained with CD4-, CD8-, CD25- and Foxp3-antibodies and analyzed by FACS calibur. CD4 + T cells were gated and the frequency of CD4 + CD25 + Foxp3 + Treg cells is shown. The data are shown as mean ± s.d. of individual mice in each group. ( vs. anti- CD3-day 4: blood: n = 7 mice; spleen: n = 11 mice, PLN: n = 9-11 mice; MLN: n = 4 mice; lungs: n = 5 mice. anti- CD3-day 1: n = 2 mice per group). nd: not done.

4 a Total # of DLN cells per mouse (*1 6 ) Apo cell + Necro cell b 14, 12, P =.3 P =.8 3 H uptake (CPM) 1, 8, 6, 4, 2, + Apo cell + Necro cell in vivo treatment Supplementary Fig. S4 Necrotic T cells fail to induce immune tolerance in vivo. C57BL/6 mice were injected with apoptotic (+ Apo cell) or necrotic T cells (+ Necro cell) followed by immunization with OVA peptide in CFA. (a) One week later, the draining lymph nodes were harvested and pooled for each group (n = 3 mice) and total cell numbers were calculated. (b) The cells were then cultured with OVA protein (5 µg/ml) for 3 d and antigen-specific T cell proliferation was determined by 3 H thymidine incorporation assay. The data are shown as mean ± s.d. of the triplicate wells of the plate.

5 a.35 IL-17 + cells in spleen (%) b.8 Clodro anti- CD3 anti-cd3 + Clodro IFN-γ + cells in DLNs (%) c.14 Clodro anti- CD3 anti-cd3 + Clodro.12 TNF-α in DLNs (Δ O.D. values) Clodro anti- CD3 anti-cd3 + Clodro Supplementary Fig. S5 MOG-specific T cell tolerance in CD3-specific antibody treated mice is reversed by phagocyte depletion. IL-17 expressing cells (a) were detected in spleens and IFN-γ expressing cells in the draining lymph nodes (DLNs) (b) ex vivo 21 d after MOG-immunization in the different groups:, clodronate-loaded liposomes (Clodro), CD3-specific (anti-cd3) or clodronate in addition to CD3-specific antibody (anti-cd3 + Clodro). IL17 and IFN-γ expressing cell percentages are given in the CD4 + T cell population. (c) T cells from draining lymph nodes of the different groups were also cultured for 3 d with MOG-stimulation and the supernatants were quantified for TNF (O.D., mean ± s.e.m.). Representative results from 2 independent experiments.

6 5 4 anti-cd3 anti-cd3 + anti-tgf-β Mean clinical score ns * * Time (d) post immunization Supplementary Fig. S6 Injection of anti-tgf-β antibody reverses intact CD3-antibodymediated therapeutic effects on relapsing-remitting EAE. SJL mice were immunized with PLP peptide and then randomly divided into the following 3 groups: untreated control (, n = 12), treated with CD3-antibody (anti-cd3, n = 6), or with CD3-antibody plus anti- TGF-β 1,2,3 -antibody (anti-cd3 + anti-tgf-β, n = 6). As indicated (blue arrow), CD3-antibody (5 µg/per mouse) was injected i.p. on days *P <.5; ns: no statistical significance (one-way ANOVA test). The data are shown as mean ± s.e.m. of clinical scores of individual mice in two independent experiments.

7 Mice T cell αcd3 injection activation/ proliferation/ apoptosis IL2,IFN-γ TNF MØ idcs T APC activation/ phagocytosis - TNF TGF-β Foxp3 + Treg Tolerance Supplementary Fig. S7 A proposed model of T cell apoptosis/phagocyte mediated immune tolerance induced by intact CD3-specific antibody treatment in mice. Intact antibody induces transient activation and release of T cell-inflammatory cytokines, followed by T cell deletion. Macrophages and idcs then engulf and digest apoptotic T cells to produce TGF-β. TGF-β then in turn not only downregulates consequent activation of immune cells and resolve the inflammation, but also establishes an immunosuppressive microenvironment under which newly generated antigen specific T cells are anergized and/or promoted to differentiate into T regulatory cells. Immune tolerance ensues.

8 Supplementary Table 1 Depletion of phagocytes abrogates CD3-antibody treatment mediated increase in ratios of Foxp3 + to IL-17 + or IFN-γ + in spinal cords of MOG-induced EAE a Treatment Ratio of Foxp3 + /IL-17 + Ratio of Foxp3 + /IFN-γ Clodronate Anti-CD Anti-CD3 + Clodronate a Four days before immunization with MOG peptide, C57BL/6 mice received (n = 3), Clodronate (n = 4), anti-cd3 + (n = 3) or anti-cd3 + Clodronate (n = 3). 21 days post immunization, spinal cords from each group were pooled together and cells were isolated for flow cytometry analysis. The ratios of CD4 + Foxp3 + Treg cells to CD4 + IL-17 + or IFN-γ + cells were calculated as the following formula: the % of Foxp3 + Treg cells/ % of IL-17 + or IFN-γ + cells.

9 Supplementary Table 2 Depletion of phagocytes abrogates CD3-antibody treatment-mediated increase in the ratios of Foxp3 + to IL-17 + or IFNγ + cells in relapsing-remitting EAE a Treatment Tregs (%, SC) Ratio (SC) (Foxp3 + /IL-17 + ) Ratio (SC) (Foxp3 + /IFN-γ + ) Treg / CD25 + Foxp3 cells (%, Spleen) /17 Anti-CD /15 Anti-CD3 + Clodronate /19 3-IgG /5.3 3-IgG3 + Clodronate /5.1 a SJL mice were immunized with PLP peptide in CFA. At the peak of the acute EAE (d 13), the mice were randomly divided into the five following groups: (n = 4), anti-cd3 + (n = 3), anti-cd3 + Clodronate (n = 3), 3-IgG3 + (n = 3) and 3-IgG3 + Clodronate (n = 4). Mice from each group were pooled together. Cells were isolated and then divided into two parts, one for Foxp3 + Treg cell staining and the other to study IL-17 and IFN-γ production in CD4 + T cells, both by flow cytometry. The ratios of CD4 + Foxp3 + Treg cells to CD4 + IL-17 + or IFN-γ + cells were calculated as the following formula: the % of Foxp3 + Treg cells/% of IL-17 + or IFN-γ + cells. Foxp3 + Treg cells to CD25 + Foxp3 effector T cells ratio was also assessed in the pooled spleens.

10 Supplementary methods. Antibodies. The following antibodies were purchased form BD Biosciences: Purified anti-cd16/cd32; FITC-conjugated-CD3-, CD4-, CD11c-, and IFN-γ- antibodies; PEconjugated CD8-, CD11c-, CD8-, CD83-, CD86-, MAC3-, TNF-, IL-4-, and IFN-γantibodies ; PercP-conjugated CD4-, CD8- antibodies; APC-conjugated CD45 LCA-antibody. FITC-conjugated F4/8 antibody was purchased from Caltag (Invitrogen). APCconjugated IL-17 and PE-conjugated Foxp3 antibodies were purchased from ebioscience. Isolation and culture of dendritic cells and macrophages. Briefly, spleens were injected with 1 mg of collagenase D (Gibco, Invitrogen), cut into small pieces and incubated at 37 C for 3 minutes. Single cell suspension was then passed trough a 7 µm cell strainer, washed and incubated with purified CD16/CD32-antibody to block non-specific FcR binding. CD11c + dendritic cells were then purified using CD11c-antibody coated microbeads (Miltenyi Biotec) and MAC3 + or F4/8 + cells were isolated using PEconjugated MAC3- or F4/8- antibodies followed by incubation with anti-pe microbeads (Miltenyi Biotec). For culture and phagocytosis assay, DCs or macrophages were plated into 24 well plates (.1 to 1 x 1 6 cells/well) and incubated in the absence or presence of apoptotic cells (5:1 ratio) overnight in a serum-free milieu x-vivo 2. Supernatants were then collected for cytokine quantification. Apoptotic cells were obtain from thymocytes after γ-irradiation (15 Rad) followed by a 3-8 h incubation in complete DMEM at 37 C. In some experiments, apoptotic cells were first labeled with carboxyfluoroscein succinimidyl ester (CFSE, 2.5 µm) and then incubated with splenic DC or macrophages overnight. Cells were then stained with PE-conjugated CD11c- or F4/8- antibodies and then analyzed under immunofluorescence microscopy. For phagocytosis assay in vivo,

11 syngenic CD4 + T cells were labeled with CFSE and injected into Rag1 -/- mice followed by CD3-antibody treatment. 1-2 h later, spleens, lungs and liver were harvested and DCs or macrophages were stained with purified CD11c- or F4/8- antibodies followed by Cy5-goat anti-hamster IgG or Rhod-Red goat anti-rat IgG (Jackson Lab) secondary staining respectively and examined under the confocal microscopy. For coculture experiments DC (.2 x 1 6 cells) plus apoptotic cells (1 6 cells) were placed in the upper level of a transwell culture insert (.4 µm pore size; BD Biosciences) and CD4 + CD25 - naïve purified T cells (.5 x 1 6, >98-99% purity, Miltenyi Biotec) were plated below the transwell in CD3 antibody-coated wells (5 µg/ml) with soluble CD28-antibody (2 µg/ml) for 3-4 d. In some wells, TGF-β 1,2,3 antibody (5 µg/ml; clone: 1D11; R&D Systems) was added. T cells were counted and prepared for Foxp3 mrna quantification by realtime PCR or staining with Foxp3-antibody 45. Phagocyte depletion. In some experiments, mice were scarified at 24 hours and DCs were stained with FITC-CD11c- together with PE-CD8, -CD86,- or -CD83 antibodies to examine the phenotype of the DC subsets. In other experiments, DO11.1 OVA peptide TCR transgenic mice were injected i.p. with or OVA protein (2 mg /mouse). On days 4-5, blood samples were collected and leukocytes were stained immediately with CD4- and CD8- antibodies together with Annexin V and 7-AAD ( BD Biosciences) to detect apoptosis. Some OVA-treated mice were then injected with clodronate on day 5 to deplete phagocytes. On day 1, all mice were scarified and spleen cells were isolated to determine the OVA-specific T cell proliferation and Foxp3 + Tregs. In other experiments, C57BL/6 mice were injected with - or clodronate-loaded liposomes followed by CD3-antibody treatment one day later. T cells were isolated (day

12 4) and stained with CD4-, CD8-,CD25- and Foxp3- antibodies and analyzed by FACS calibur. T cell culture and proliferation assay. 1 6 T cells/ml of complete DMEM were stimulated either by soluble CD3-specific antibody (5 µg/ml) or MOG peptide (.1 to 1 µg/ml) or PLP peptide (.1 to 1 µg/ml). Cells were cultured at 37 C in 5% CO 2 for 3 to 5 d and pulsed with 1 µci [ 3 H] thymidine for the last 6 16 h for proliferation assay. Radioactivity incorporation was counted using a flatbed β-counter (Wallac). For cytokine assay, cells were culture in complete DMEM in the same peptide- or TCRstimulations as proliferation assay for 3 or 5 d and supernatants were collected for TNF and IFN-γ quantification. For IL-17 expressing cell detection, phorbol 12-myristate 13- acetate (PMA, 5 ng/ml, Sigma), ionomycine (25 ng/ml, Sigma) and GolgiPlug (1 µl/ml, BD Biosciences) were added for the last 5 h of culture. Then cells were prepared for intracellular staining. Flow cytometry analysis of LAP-TGF-β. For analysis of the latent form of TGF-β (LAP-TGF-β) on macrophages and DCs, C57BL/6 mice were injected with or CD3-antibody (5 µg/per mouse). Spleen cells were isolated 24 h later and incubated with CD16/32 FcR blocking antibody (BD Biosciences). Cells were then stained with antibodies against F4/8 or CD11c together with biotinylated-lap-tgf-β antibody (.5 µg/ per 1 6 cells, R&D systems) followed by Streptavidin-APC staining (.2 µg/ 1 6 cells).

13 EAE induction, scoring and analysis. For induction of EAE, C57BL/6 mice were immunized subcutaneously on day with 2 µg/mouse of MOG peptide (Research Genetics), emulsified in CFA (IFA supplemented with 4 µg/ml heat-killed M. tuberculosis) at two sites in the lower back as well as in each rear footpads (5 µg per site). Mice received also 2 ng of Bordetella pertussis (List Biological Lab) i.p. on the day of immunization and 2 d later. For induction of relapsing-remitting EAE, SJL mice ( Jackson Laboratory) were immunized with with PLP peptide ( 75 µg/mouse) in CFA ( 2 µg/mouse) at three sites on the back followed by i.p. injection of Bordetella pertussis toxin ( 1 ng per mouse) 2 d later. All the immunized mice developed the acute disease on d Since some mice died from the acute EAE, the surviving mice were then randomly divided into the indicated groups to ensure that each group had similar average score before the treatments. As indicated, - or clodronate- loaded liposomes were injected on day 13, intact CD3-antibody (5 µg) was injected once on day 14 and CD3-IgG3 antibody ( 5 µg per mouse per day) was injected for 5 d ( d 14-18). The mice were monitored and scored daily for 33 or 62 d. Whenever a mouse died of score 5, the mean EAE score of the involved group did not include the dead mouse during the following days. The basic scoring system used was : no disease; 1: limp tail; 2: weak/partially paralyzed hind legs; 3: completely paralyzed hind legs; 4: complete hind and partial front leg paralysis and 5: complete paralysis/death. Animals were scored twice a week or daily when the disease started to occur. On day 21 after MOG immunization or days 33 or 62 after PLP peptide immunization, spinal cords were harvested and a part was fixed with neutral 1% formalin, extracted, embedded in paraffin and cut in 5 µm sections for H&E staining whereas the other part was submitted

14 to collagenase D digestion (1 mg/ml) for 3 min at 37 C. Then the solution was passed through a 7 µm cell strainer, washed once with and resuspended in 38% Percoll solution for 2 min at 2 rpm. Then pellets of cells were resuspended in the culture medium for further staining or culture.