Afferent lymph-derived T cells and dendritic cells use different CCR7-dependent routes for lymph node entry and intranodal migration

Transcription

1 Braun et al. Supplementary Information 1 Supplementary Information Afferent lymph-derived T cells and dendritic cells use different CCR7-dependent routes for lymph node entry and intranodal migration Asolina Braun, Tim Worbs, Leandros Moschovakis, Stephan Halle, Katharina Hoffmann, Jasmin Bölter, Anika Münk and Reinhold Förster

2 Braun et al. Supplementary Information 2 SUPPLEMENTARY MATERIALS Supplementary Figures Supplementary Figure 1. Intralymphatic injection. (a) Canulation of an afferent lymph vessel of the popln and i.l. injection of blue ink. (b) After i.l. injection, ink is drained strictly through the popln into its efferent lymphatics. (c) View of the lower abdominal cavity. The efferent lymphatics of the popln drain towards the medial iliac (2 nd level) and renal (3 rd level) LN. (d) Schematic overview of the lymphatic drainage of the murine hind leg via the popln. Lymph vessels connect the primary draining popln with downstream secondary (medial iliac) and tertiary (renal) LN.

3 Braun et al. Supplementary Information 3 Supplementary Figure 2. Positioning of DCs in primary popln 48h after i.l. injection. 1x10 5 cells of a 1:1 mixture of EGFP-expressing wt (green) and TAMRAlabeled (red) Ccr7 -/- BMDCs were injected i.l., 48h later the primary draining popln was isolated. Frozen sections were stained with anti-lyve1-mab (blue). Arrows indicate wt DCs or parts of DCs that have entered medullary sinuses. Representative image from n=12 LN from 5 independent experiments.

4 Braun et al. Supplementary Information 4 Supplementary Figure 3. Skin DCs express higher surface levels of CCR7 compared to BM-derived DCs. (a, b) FACS analysis of CCR7 expression levels on CD11c-YFP + /CD86 + double-positive mature DCs derived from in vitro BM cultures or split ear skin DC preparations from CD11c-YFP donor mice, respectively. (a) Isotype controls are shown in gray. (b) CCR7 expression levels (relative MFI values shown as fold isotype) of BMDCs as well as skin DCs of wild-type (Bl6), β-actin-egfp (ßact-EGFP) and CD11c-YFP donor mice. Data shown is representative for two independent experiments.

5 Braun et al. Supplementary Information 5 Supplementary Figure 4. Compartmental topology of the murine popln. Schematic overview summarizing topological and morphological features of the LN sinus compartment (grey) with relevance to the entry routes of afferent lymph-derived wt DCs (orange arrow) and wt T cells (yelllow arrow). While DCs reach the paracortical TCZ via interfollicular areas of the afferent side cortex, T cells migrate through the labyrinthine environment of the peripheral medulla (enlarged detail view) consisting of the peripheral medullary sinus system and peripheral medullary cords. Note the characteristic change in cellular composition and Lyve-1 staining of the sinus floor within the transition zone between afferent side SCS and capsule-lining medullary sinus. CD169 + macrophages are interspersed within the lumen of medullary sinuses (concentrating in particular within central medullary areas near the LN hilus), while cortical sinuses do not harbor any CD169 + macrophages. This illustration represents an idealized synopsis of extensive immunohistological analyses of the popln.

6 Braun et al. Supplementary Information 6 Supplementary Figure 5. Distribution of T cells to secondary draining illn after i.l. injection. Immunohistological analysis of illn at 2h and 4h after coinjection of 2x10 4 EGFP-expressing wt (green) and 2x10 4 TAMRA-labeled Ccr7 -/- (red) polyclonal CD4 + T cells (and vice versa, not shown). n=15 LN per time point from 5 independent experiments. Scale bars, 100 µm.

7 Braun et al. Supplementary Information 7 Supplementary Videos Supplementary Video 1. Ccr7 -/- BMDCs do not reach the paracortical TCZ after intralymphatic application. To highlight the divergent positioning of i.l. injected wt and Ccr7 -/- BMDCs (1x10 5 cells of a 1:1 mixture), a popln explanted 12h after cell transfer was sectioned completely and composite images of all 126 sections were acquired by fluorescence microscopy. A sequence of the successive slides is shown in the left panel. The images were further assembled in Fiji software to generate a 3D-reconstruction of the LN (right panel). EGFP-expressing wt are shown in green, TAMRA-labeled Ccr7 -/- BMDCs in red. Additionally, B cells were stained with anti- IgD antibody (blue). Note, that Ccr7 -/- DCs concentrate in the SCS on the afferent draining side of the LN, whereas wt DCs are widely distributed inside the TCZ. Supplementary Video 2. Long-term two-photon microscopical imaging of intranodal DC migration behavior. To exemplify the progression of i.l. injected BMDCs over time, a popln was explanted 40 min after i.l. injection of 3x10 3 TAMRAlabeled wt (red) and 3x10 3 EGFP-expressing Ccr7 -/- (green) BMDCs and the same LN area was imaged ex vivo at 1-2h, ~4h and ~8h p.i. to demonstrate long-term development of intranodal DC positioning (exact time p.i. is shown in the individual panels). SHG signal of the LN capsule is shown in blue. Acceleration of time lapse replay is 225-fold. Wt but not Ccr7 -/- DCs exhibit a pronounced inward directional migration; at ~8h most wt DCs have already left the imaging volume. Supplementary Video 3. Directional displacement and uropod formation of wt BMDCs is not reflected in Ccr7 -/- BMDCs. The left panel shows immigration of wt DC imaged ex vivo starting at 3h after i.l. injection of 2x10 3 EGFP-expressing wt BMDCs (green). The right panel shows EGFP + Ccr7 -/- BMDCs (green) which largely remain within or near the SCS and do not form uropods. Imaging was carried out at 4h20min to 5h after i.l. injection of 2.5x10 3 EGFP + Ccr7 -/- BMDCs, since at earlier time points nearly no DC were found beyond the SCS. Note that several Ccr7 -/- BMDCs migrated towards the SCS in the course of imaging. The tracks of both time lapse recordings are time-color coded, the acceleration is 225-fold. Ongoing imaging duration of the videos is depicted below the scale bar. SHG signal is shown in blue.

8 Braun et al. Supplementary Information 8 Supplementary Video 4. Close-up view of cellular morphology during intranodal migration. During early directional migration, EGFP + wt BMDCs shown in the left panel are highly polarized, displaying a pronounced leading edge and uropod. EGFP + Ccr7 -/- BMDCs (right panel) do not show any pronounced cell polarization and reach the SCS-adjacent LN parenchyma only at later time points. Start of ex vivo videos p.i. is shown below the scale bar. Injected cell numbers were 2-2.5x10 3. SHG signal is shown in blue. Acceleration of time lapse replay is 225-fold. Supplementary Video 5. Wt and Ccr7 -/- BMDCs exhibit comparable dendrite movement and sweeping behavior. Dendrite morphology was analyzed after the directional migration of wt BMDCs had ceased. Start of ex vivo time lapse imaging is depicted in the lower left of the panels x10 3 EGFP + (left panel) or EGFP + Ccr7 -/- (right panel) BMDCs were injected. SHG signal is depicted in blue. Acceleration of time lapse replay is 225-fold. Supplementary Video 6. After i.l. application, primary skin-derived wt DC display an intranodal migration comparable to wt BMDCs. Ex vivo recording starts 2h after i.l. injection of 3.5x10 3 skin-emigrated CD11c-YFP DC (green), time scale of DC tracks is color-coded. SHG signal is shown in blue. Acceleration of time lapse replay is 225-fold. Supplementary Video 7. Transmigration of wt CD4 through lymphatic endothelium of the capsule-lining sinus floor. Examples of wt CD4 T cells (green) entering peripheral medullary cords by transmigration through Lyve-1 + endothelium (red, cyan outline). Lymphatic endothelium was visualized by i.l. injecting anti-lyve-1 antibody in situ prior to cell application. SHG signal of the LN capsule is shown in blue. Start of ex vivo time lapse is 1h20min (first part of the movie) or 35min (second part of the movie) after i.l. injection of 1.6-2x10 4 polyclonal EGFP-expressing wt CD4 T cells. Acceleration of time lapse replay is 113-fold. Supplementary Video 8. Transmigration of wt CD4 through medullar sinus endothelium. Example of a wt CD4 T cell (green) which was recorded at 2h p.i. entering peripheral medullary cords by transmigration through Lyve-1 + endothelium

9 Braun et al. Supplementary Information 9 (red) after extended migration within the lumen of a peripheral medullary sinus. Acceleration of time lapse replay is 45-fold. Supplementary Video 9. Upon exiting the capsule-lining sinus, wt CD4 preferably migrate within peripheral medullary cords. Intranodal migration behavior of EGFP-expressing polyclonal wt CD4 T cells (green with red tracks) within peripheral medullary areas is shown. Surface rendering (gray) of Lyve-1 + endothelium (blue) illustrates the 3D structure of peripheral medullary sinus lumen and the parenchyma of peripheral medullary cords. Note that the major dwelling of CD4 is found in peripheral medullary cords and is markedly confined by Lyve-1 + endothelium. Start of the ex vivo time lapse is 1h after i.l. injection of 1.6x10 4 CD4 T cells. Recordings shown are accelerated 225 fold. Supplementary Video 10. Intralymphatically administered CD4 T cells enter LN parenchyma in a CCR7-dependent manner primarily within peripheral medullary areas. While TAMRA-labeled wt CD4 T cells (red, cell tracks in red) display an inward migration towards the deep TCZ, the random walk-like migration of EGFP + Ccr7 -/- CD4 T cells (green, cell tracks in green) remains confined to peripheral medullary areas. Start of ex vivo time lapse recording is 45 min after i.l. injection of 2.5x10 4 total CD4 (1:1). SHG signal of the LN capsule is shown in blue. Acceleration of time lapse replay is 225-fold. Supplementary Video 11. Immigration of DC allows CD4 T cells to enter the LN at SCS. Left panel: Afferent lymph-derived CD4 T cells alone (green) do not transmigrate through the floor of the afferent side SCS. Start of ex vivo time lapse is 30min after i.l. injection of 2x10 4 wt CD4 + cells. Right panel: 3x10 3 TAMRA-labeled wt BMDCs (red) were i.l. injected 40 min prior to application of wt CD4 T cells (green). Start of ex vivo time lapse is 80 min after i.l. injection of 1.6x10 4 wt CD4 + cells. SHG signal of the LN capsule is shown in blue. Acceleration of time lapse replay is 225- fold. Supplementary Video 12. LPS-treated CD4 cells fail to enter popln via SCS. EGFP-expressing CD4 T cells (green) were incubated with 1 µg/ml LPS for 30 min. After washing, 2x10 4 CD4 + cells were i.l. injected. Time after cell injection is shown beneath the scale bar of the ex vivo time lapse movie. SHG signal of the LN capsule

10 Braun et al. Supplementary Information 10 is shown in blue. Acceleration of time lapse replay is 225-fold. Representative movie of 10 LN analyzed in 2 independent experiments