Supplementary Figure 1 Lymphocytes can be tracked for at least 4 weeks after

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1 Supplementary Figure 1 Lymphocytes can be tracked for at least 4 weeks after photoconversion by using H2B-Dendra PPs of H2B-Dendra2 BM chimeras were photoconverted and analyzed 7 days (upper panel) or 28 days (lower panel) after photoconversion. Plots show all H2B-Dendra2-expressing lymphocytes in pln or PP conv (Representative of 3 independent experiments). Although the separation between D-Red + and D-Red - cells was still clear 28 days after photoconversion, D-Red signal decreased over time, even in naïve lymphocytes in pln. 1

2 Supplementary Figure 2 Viability and stress responses of lymphocytes after PP illumination. (a) Analysis of PP conv and PP ctrl by flow cytometry 1 day after the illumination of PPs in WT mice. Plots show expression of AnnexinV and DAPI signal on all lymphocytes. Numbers show frequency of each gate (representative plots from n = 4 mice, 8 PP conv and 9 PP ctrl samples, 2 independent experiments). (b) Frequency of AnnexinV + DAPI - (left) and AnnexinV + DAPI + (right) cells among all lymphocytes in PP conv and PP ctrl from experiments described in (a) (Mean±SD, unpaired Student s t-test, not significant (ns)). (c) Western blot analysis of stress response-associated proteins in WT PPs illuminated for 0, 5, 10 or 30 seconds. PPs were analyzed ~5 minutes after the illuminations (representative of 2 independent experiments). 2

3 Supplementary Figure 3 Proliferating cells loose D-Red and FITC signals as they divide. (a) Photoconverted (left) or non-photoconverted (right) H2B-Dendra2-expressing CD4 + T cells were labeled with efluor 450 Cell proliferation dye (Invitrogen), stimulated with anti-cd3/anti-cd28 antibodies for 3 days and analyzed by flow cytometry. Red color represents stimulated photoconverted cells, green color represents stimulated non-photoconverted cells and blue color represents cells that are not stimulated. Proliferation of these cells was analyzed by the dilution of the proliferation dye (representative of 3 independent experiments). (b) CD19 + B cells in PP inj 3 days after FITC injection in WT mice. CD62L lo activated B cells lost the FITC signals as they proliferate while CD62L hi naïve B cells stayed as FITC + cells (representative of 2 independent experiments). 3

4 Supplementary Figure 4 Different helper T cell subsets are enriched among resident cells in mln and PPs. Cumulative data and statistical analysis for Figure 3a-d. (a) Frequency of PD- 1 hi CXCR5 hi T FH cells among CD4 + CD62L lo T cells (left) and CD4 + CD62L lo D-Red + cells (right) in mln conv 7 days after (n = 4 mice in 2 independent experiments). (b) Frequency of PD-1 hi CXCR5 hi T FH cells among CD4 + CD62L lo T cells (left) and among CD4 + CD62L lo FITC + cells (right) in PP inj 7 days after (pooled samples from n = 18 mice in 3 independent experiments). (c) Frequency of Foxp3 + regulatory T cells for the populations as in (a). (d) Frequency of Foxp3 + regulatory T cells for the populations as in (b). Paired Student's t-test, *, P<0.05; **, P<0.01; not significant (ns). 4

5 Supplementary Figure 5 Most T FH cells stay in PPs as resident cells. Frequency of FITC + and FITC - cells among CD4 + PD-1 hi CXCR5 hi T FH cells in PP inj 7 days after as described in Figure 3b (pooled samples from n = 18 mice in 3 independent experiments, mean ± SD). Supplementary Figure 6 Expression levels of different surface markers for resident cells in PPs. Normalized geometric mean fluorescence intensity (gmfi) values for Figure 3e and f. gmfi values were normalized according to CD62L hi naïve CD4 + T cells so that they would have a value of 100. Normalized gmfi values for CCR7, IL-7Rα, ICOS and CD69 for CD4 + CD62L lo FITC - (left) and CD4 + CD62L lo FITC + cells (right) in PP inj 7 days after FITC injection (pooled samples from n = 18 mice in 3 independent experiments). Paired Student's t-test, *, P<0.05; not significant (ns). 5

6 Supplementary Figure 7 Resident CD4 + T cells are as motile as other T cells. BM chimeras were prepared by transferring a 1:1 mixture of MigH2BD2-transduced μmt -/- BM and nontransduced WT BM into irradiated WT mice. In these chimeras, B cells did not express H2B- Dendra2 protein. PPs of these BM chimeras were photoconverted and analyzed 7 days later by twophoton microscopy as described in Supplementary Methods. (a) Representative images from the analysis of PP conv with live cell imaging (representative of 2 independent experiments, n = 2 mice, 2 PP conv s, scale bar: 10 μm). (b) On the left, mean track speeds of D-Red - and D-Red + (resident cells) and on the right, track straightness of D-Red - and D-Red + (resident cells). Each symbol represents the average value from the analysis of one movie (n = 2 mice in 2 independent experiments, 3 movies from 2 PP conv s, paired Student's t-test, not significant (ns)). 6

7 Supplementary Figure 8 Analysis of the homing of resident cells to PPs and lamina propria. (a) WT mice were FITC injected into PPs. 7 days later, PP inj from these mice were pooled and 12x10 6 cells out of this pool were adoptively transferred into unmanipulated mice. 2 days after the transfer, PPs and small intestinal lamina propria of the transferred mice were analyzed by flow cytometry. (b and c) Analysis of the transferred PP inj pool cells, showing the frequency of CD4 + and CD19 + cells among the FITC + cells (b), as well as CD62L expression on these cells (c). (d and e) Frequency of FITC + cells among CD19 + (left) and CD4 + (right) in PPs (d) and lamina propria (e) of the transferred mice (n =3 transferred and one non-transferred mice in one experiment). 7

8 Supplementary Figure 9 Frequency of FITC + cells in PP inj samples after in vitro stimulation. Seven days after FITC injection into PPs, PP inj cells were cultured in the presence of Brefeldin A, with PMA/Ionomycin (stimulated) or without (not stimulated). After 4 hours of culture, cells were analyzed by flow cytometry. CD4 + (left) and CD19 + (right) cells among DAPI - live lymphocytes are shown. Gates show the frequencies of FITC + cells. Representative of 2 independent experiments with n= 7 mice. 8

9 Supplementary Figure 10 Generation of resident CD4 + T cells is independent of CD69. Data from two of the experiments reported in Figure 5a with 3 mice (all analysis were done among D- Green + cells, WT: CD45.1 +, CD69 -/- : CD ). (a) Frequency of CD62L lo cells among WT and CD69 -/- CD4 + T cells in PP conv (paired Student's t-test, not significant (ns)). (b) Ratio of the frequencies of CD62L lo /CD62L lo D-Red + cells among WT and CD69 -/- CD4 + T cells in PP conv 7 days after photoconversion (paired Student's t-test, not significant (ns)). (c) Ratio of the frequencies of CD62L lo CD /CD62L lo CD D-Red + cells for WT and CD62L lo CD /CD62L lo CD D- Red + for CD69 -/- CD4 + T cells in PP conv 7 days after photoconversion (paired Student's t-test, not significant (ns)). (d) Frequency of CD62L lo cells among WT and CD69 -/- CD4 + T cells in mln (paired Student's t-test, not significant (ns)). 9

10 Supplementary Figure 11 TCR Vβ5 - cells accumulate in PPs of naive RAG-sufficient OT-II mice as CD62L lo effector/memory CD4 + T cells. Statistical analysis for Figure 7a and b. (a) Frequency of Vβ5 - cells among all CD4 + T cells in pln, mln and PPs (n = 5 mice in 2 independent experiments, mean ± SD). One-way ANOVA followed by Tukey s post-test, ***, P<0.001; not significant (ns). (a) Frequency of CD62L lo cells among Vβ5 + and Vβ5 - CD4 + T cells in pln, mln and PPs (n = 5 mice in 2 independent experiments, mean ± SD). Paired Student's t-test, ***, P<

11 Supplementary Figure 12 Resident OT-II cells induced by continuous OVA treatment contain different functional sub-populations. Experiments were done as described in Figure8d. Briefly, WT mice transferred with congenically marked (CD ) OT-II cells were immunized with OVA+CT and given OVA in drinking water for either 1-week (DW 1W) or 3-weeks (DW 3W). 14 days after the immunizations, FITC injected into PPs of these mice and analyzed 7 days later (21 days after immunizations) and FITC+ resident OT-II cells were analyzed by flow cytometry (n = 3-6 mice per group in 2 independent experiments, mean ± SD). (a) Frequency of Vβ5 + cells among CD4 + CD CD62L lo FITC + T cells in pooled PP inj s showing that virtually all of them were Vβ5 +. (b) Frequency of PD-1 hi CXCR5 hi T FH cells among CD4 + CD CD62L lo FITC + T cells in pooled PP inj s. Representative plot from a mouse in OVA+CT DW 3W group (left). Unpaired Student's t- test, not significant (ns). (c) Frequency of Foxp3 + cells among CD4 + CD CD62L lo FITC + T cells in pooled PP inj s. Representative plot from a mouse in OVA+CT DW 3W group (left). Unpaired Student's t-test, not significant (ns). 11

12 Supplementary Methods Annexin V staining Annexin V staining was performed with Annexin V-biotin Apoptosis Detection kit (ebioscience). Single cell suspensions of illuminated or non-illuminated PPs were incubated with Annexin V- biotin in 1X Annexin V binding buffer for 15 minutes at room temperature. Then the cells were washed and stained with fluorescent dye-conjugated streptavidin (BioLegend) and antibodies. After the antibody stainings, cells were also stained with DAPI (4',6-diamidino-2-phenylindole) for live/dead staining. Western Immunoblotting Individual Peyer s patches were isolated, washed with PBS and boiled in 2x SDS loading buffer. Soluble protein extracts were run on gradient SDS-PAGE gels and transferred to nitro-cellulose membranes. Primary antibody incubations were performed overnight at 4 C followed by 1 hour incubation with horseradish peroxidase-conjugated secondary antibodies at room temperature (GAPDH (Millipore #MAB374), p38 (Cell Signaling Technology #9212), phospho-p38 (Cell Signaling Technology #9211), Hsc70 (Stressgen SPA815) and HRP labeled secondary antibodies from Santa Cruz Biotechnology.). Blots were developed with an ECL detection kit (Santa Cruz Biotechnology), and the digital chemiluminescence images were taken by a Luminescent Image Analyzer LAS-3000 (Fujifilm). Two-photon imaging of Peyer s patch sections. Intestinal pieces containing Peyer s patches were placed in 4% UltraPure Low Melting Point Agarose (Invitrogen; w/v, in PBS). Small PP-containing agarose cubes were cut into 400 µm thick sections using a vibrating blade microtome (Leica). These sections were glued into a custom-made 12

13 imaging chamber using tissue adhesive (Nexaband, Abbott Animal Health). Until this point, PPcontaining agarose pieces were kept on ice or in ice-cold PBS. For two-photon microscopy, the tissue pieces in the imaging chamber were superfused with RPMI1640 medium supplemented with 5 g/l glucose, warmed to 37 C and supplied with Carbogen (95%O 2 +5%CO 2, Linde Gas, flow rate <0.25 L/min.). The imaging setup is based on a TriMScope (LaVision Biotec) and an upright microscope (BX51, Olympus) with a 20x/0.95 water immersion objective. D-Green was excited at 865 nm using a pulsed infrared laser (MaiTai Ti:Sa, Spectra Physics) and D-Red was excited at 1100 nm using an identical laser coupled to an optical parametric oscillator (OPO). D-Green signal was detected using a 525/50 band-pass filter and D-Red signal above 550nm was separated using a 550+ beam splitter. Imaging analysis was performed with Bitplane Imaris. To determine mean track speed and track straightness of D-Red - (D-Green + D-Red - ) and D-Red + cells, within a defined 3D volume and time frame, the movement of all cells that could be unequivocally identified as D-Red - or D-Red + was analyzed using the Manual tracking option in Imaris. Cells that could be followed for less than 3 timepoints were excluded from the analysis. Mean track speeds and track straightness values of all D-Red - and D-Red + cells were averaged per movie. Track straightness values can range between 0 and 1 corresponding to no displacement and maximal displacement, respectively. 13