Instant effect of soluble antigen on effector T cells in peripheral immune organs during immunotherapy of autoimmune encephalomyelitis

Odoardi et al. 10.1073/pnas.0608383104.

Supporting Information

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SI Table 1
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SI Figure 6

Fig. 6. i.v. MBP prevents CNS inflammation. (A) T_MBP-GFP cells (green), W3/13⁺ T cells (red, left panels), and ED1⁺ monocytes/macrophages (red, right panels) in EAE lesions after 48 h i.v. OVA (OVA 48 h) or i.v. MBP (MBP 48 h) treatment. Arrows indicate GFP⁺ W3/13⁺ T cells (closed arrows), recruited W3/13+ cells, and ED1⁺ monocytes/macrophages. Immunohistochemistry, spinal cord 96 h p.t.. Confocal microscopy. Representative data of three independent experiments. Magnification bars: 10 mm. (B) Numbers of T_MBP-GFP cells, CD4⁺, CD8⁺, B cells⁺ (OX 33), CD11a⁺, CD11b⁺, CD11c⁺, and MHC class II⁺ (OX 6) cells in the spinal cord 100 h p.t.. Animals received either i.v. MBP (black) or OVA (white) 48 h p.t.. Means +/-SD of two independent experiments including three animals/ treatment. (C) Quantitative PCR of spinal cords snap-frozen 100 h p.t.. Animals received either i.v. MBP (black bar) or OVA (white bar) at 48 h p.t.. Representative data of three independent experiments. Samples were measured in two independent quantitative PCR reactions.

SI Figure 7

Fig. 7. Antigen responsiveness after i.v. MBP infusion. T_MBP-GFP cells in the spleen after i.v. MBP are non-responsive. T_MBP-GFP cells sorted from the spleen 72 h p.t., i.e., 24 h after i.v. treatment with MBP (diamonds) or OVA (circles) and resting in vitro T_MBP-GFP (squares) were exposed to MBP (black) or OVA (white) in the presence of irradiated professional thymic APCs. Cytofluorometric counts of T_MBP-GFP cells at time points 2, 3, 4, and 5 days after co-culture with APCs. Amplification rate: N_GFP cells days 2-5/N_GFP cells day 0. Triplicate measurements +/- SD. Representative data from two independent experiments including two animals/group.

SI Figure 8

Fig. 8. Effector T cell distribution within the spleen. (A) Distribution of T_MBP-GFP cells in the spleen 60 h p.t.. T_MBP-GFP cells (anti-GFP immunohistochemistry, brown diaminobenzidine staining) are mainly distributed in the red pulp (RP; A,C) and the T cell areas (periarteriolar lymphocyte sheaths, PALS; B,D) of the spleen. Less cells are located within the B cell follicles (Foll). (A,C) No i.v. MBP treatment, (B,D) i.v. MBP at 48 h p.t. Hematoxylin/Eosin staining. (C) and (D) represent magnifications of (A) and (B), respectively. Magnification bars (A and B): 50 mm; (C and D): 10 mm. Arrows in C and D point towards representative T_MBP-GFP cells.

SI Figure 9

Fig. 9. Random walk of T_GFP cells within the spleen. (A) Superimposed trajectories (each line represents one cell) of 164 T_MBP-GFP cells (left), 146 T_OVA-GFP cells (middle), 162 T_MOG-GFP cells (right), before (upper plots) and 60 min after (lower plots ) i.v. specific antigen injection. Cells were tracked over a 10-min period. S: trajectory vector calculated from the sum of all cell trajectories divided by the number of cells. (B) Plots show the square roots of mean absolute displacements of individual T_MBP-GFP cells (left), T_OVA-GFP cells (middle), and T_MOG-GFP cells (right), away from their starting point as a function of time, before (blue diamonds) and 60 min after (red diamonds) i.v specific antigen injection. The linear regression on this transformed scale displays straight lines, as expected for a random walk process. Representative data of three independent experiments/cell line, in each of which at least 55 cells were evaluated.

SI Figure 10

Fig. 10. Effector T cell arrest after i.v. antigen. Distribution of instantaneous velocities of T_MBP-GFP cells (upper plots), T_OVA-GFP cells (middle plots), and T_MOG-GFP cells (lower plots) measured before (white bars), 60 min (gray bars), and 90 min (black bars) after i.v injection of specific antigen (left plots) or control antigen (right plots). T_MBP-GFP cells: means +/- SD from three independent experiments/antigen including 312 cells and 37440 time points from six videos. T_OVA-GFP cells: means +/- SD from three independent experiments/antigen including 326 cells and 38400 time points from six videos. T_MOG-GFP cells: means +/- SD from three independent experiments/time point including 308 cells and 36960 time points from six videos.

SI Figure 11

Fig. 11. Stop-and-go mode of T_GFP cell locomotion. Velocity fluctuations of individual T_MBP-GFP cells within the spleen. Fluorescence video recording. Four representative cells out of 312 are shown. Velocities were analyzed point by point from positions during consecutive 30-sec intervals for a period of 2 h.

SI Figure 12

Fig. 12. Antigen presentation capacity of ex vivo isolated splenocytes after i.v. antigen. (A) Numbers of DQ OVA^TM processing splenocytes 0 (top plots) and 30 min (bottom dot plots) after application of the quenched dye. 9% of splenocytes became fluorescent (lower left panel); 84% of these are MHC II⁺ (lower right). Cytofluorometry. IgG: isotype control. Representative data of three independent experiments. (B) Quantitative PCR of IL-2 receptor (IL2R) and IFN-g mRNA isolated from T_MBP-GFP cells after 48-h coculture with splenocytes. The splenocytes were harvested ex vivo 120 min after i.v. OVA (white bars, Ctr) or 30 (gray bars) or 120 (black bars) min after i.v. MBP infusion, and were immediately fixed with 0.025% glutaraldehyde (Sigma Aldrich) in PBS for 3 min. The reaction was stopped with 0.2 M glycine (Sigma-Aldrich) in PBS v/v for 10 min at room temperature. Cells were washed three times before incubation with T_MBP-GFP cells. All steps were performed at 4°C. (C) Corresponding quantification of intracellular IFNg production. FACS measurement of intracellular IFNg of T_MBP-GFP cells after 48-h coculture with glutaraldehyde-fixed splenocytes isolated 120 min after i.v. OVA (filled histograms) or 30 (left) or 120 (right) min after i.v. MBP infusion (overlays).

SI Figure 13

Fig. 13. Kinetics of T cell activation after i.v. antigen infusion. (A) Surface protein expression of T_MBP-GFP in spleen after i.v. MBP. CD4, IL-2 receptor (IL2R), abT cell receptor (TCR), and OX40-antigen (OX40) were determined at the indicated time points after i.v. antigen (red overlay histograms) infusion. Filled histograms: controls (before i.v. antigen). Representative data of four independent experiments. (B) IFNg (left), IL-2 (middle), and IL-2 receptor (right) mRNAs in spleens snap-frozen 3 days after T_OVA-GFP transfer were quantified in relation to the housekeeping b-actin mRNA. Numbers indicate time points of sampling after i.v. OVA. Representative data of two independent experiments. (C) Intracellular IFNg staining in T_OVA-GFP from spleens 0.5, 1, and 2 h after i.v. OVA. Filled histogram: IFNg control (no antigen), blue overlay histogram: IFNg after specific i.v. antigen. Representative data of three independent experiments are shown. (D) Surface protein expression of T_OVA-GFP in spleen after i.v. OVA. CD4, IL-2 receptor (IL2R), abT cell receptor (TCR), and OX40-antigen (OX40) were determined at the indicated time points after i.v. specific antigen (red overlay histograms) infusion. Filled histograms: controls (before i.v. OVA). Representative data of four independent experiments.

SI Figure 14

Fig. 14. Intravital spleen imaging. Settings used for A an inverted and B an upright stage. The spleen is placed in a temperature and oxygen controlled chamber.

SI Movie 1

Movie 1. Effector T cell motility in the spleen. Intravital video microscopy of T_MBP-GFP cells in the spleen 60 h p.t.. I.v. OVA injection was performed 12 min after recording start (red frame). Observation time: 80 min. 30-sec intervals. Green: T_MBP-GFP cells.

SI Movie 2

Movie 2. Effector T cell motility in the spleen. Two-photon microscopy of T_MBP-GFP cell motility in the spleen 60 h p.t.. I.v. OVA and Texas red dextran (60 mg) were injected 18 min after recording start (red frame). Yellow circles: motile T_MBP-GFP.cells. Orange circles: stationary T_MBP-GFP cells. Observation time: 100 min. 39-sec intervals. Green: T_MBP-GFP cells. Yellow: autofluorescence. Red: Texas red dextran⁺ splenocytes.

SI Movie 3

Movie 3. Effect of i.v. MBP on T_MBP-GFP cell locomotion in the spleen. Intravital video microscopy of T_MBP-GFP cells in the spleen 60 h p.t. I.v. MBP treatment was performed 16 min after recording start. Observation time: 110 min. 30-sec intervals. Arrows point toward clusters formation.

SI Movie 4

Movie 4. Effect of i.v. MBP on T_MBP-GFP cell locomotion in the spleen. Two-photon microscopy of T_MBP-GFP cell arrest after i.v. MBP infusion. Intravital imaging of the spleen 60 h p.t.. I.v. injection of MBP and Texas Red dextran (150 mg) was performed 15 min after recording start. Observation time: 1 h. 47-sec intervals. Green: T_MBP-GFP cells. Yellow: autofluorescence. Red: Texas red dextran⁺ splenocytes. Yellow circles: motile T_MBP-GFP cells Orange circles: stationary T_MBP-GFP cells.

SI Movie 5

Movie 5. Arrest of T_OVA-GFP cells after i.v. infusion of OVA. Intravital video microscopy of T_OVA-GFP cells in the spleen 60 h p.t.. I.v. OVA and Texas Red dextran (150 mg) were infused 12 min after recording start (red frame). Observation time: 110 minutes. 30-sec intervals. Green: T_OVA-GFP cells. Red: Texas red⁺ splenocytes. Arrows point toward cluster formation.

SI Movie 6

Movie 6. Cluster formation of T_MBP-GFP cells after i.v. MBP infusion. Intravital confocal microscopy of T_MBP-GFP cells in the spleen 60 h p.t., 40 minutes after i.v injection of MBP and Texas red dextran (150 mg). Observation time: 10 minutes. 30-sec intervals. Green: T_MBP-GFP cells. Red: Texas red⁺ splenocytes.

SI Movie 7

Movie 7. Short interactions of effector T cells with splenic phagocytes. Detail of video 2. Intravital 2-photon microscopy of a T_MBP-GFP cell in the spleen transiently contacting a Texas red dextran labeled splenic phagocytic cell. Observation time: 33 min.

SI Movie 8

Movie 8. Long-lasting contacts of effector T cells with splenic phagocytes after i.v. antigen. Detail of video 5. Intravital 2-photon microscopy of a T_MBP-GFP cell in the spleen establishing a stable contact with a Texas red dextran labeled splenic phagocytic cell. Observation time: 33 min.

SI Movie 9

Movie 9. Antigen processing by splenic phagocytic cells. Intravital video microscopy of the spleen 60 h p.t.. I.v. DQ OVA^TM treatment was performed 13 min after recording start (red frame). Circles indicate fluorescent splenocytes, i.e., phagocytic cells proteolytically digesting DQ OVA^TM. Green: DQ OVA^TM proteolysis. Red: T_OVA-GFP cells. Observation time: 1.5 h. 30-sec intervals.