SUPPLEMENTARY INFORMATION

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Nicholas Lane
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1 SUPPLEMENTARY INFORMATION CD169 + MACROPHAGES PRESENT LIPID ANTIGENS TO MEDIATE EARLY ACTIVATION OF INVARIANT NKT CELLS IN LYMPH NODES Ptrii Brrl, Polo Polzell, Andres Brukuer, Nio vn Rooijen, Gurdyl S. Besr, Vinenzo Cerundolo nd Fundo D. Btist Nture Immunology: doi:1.138/ni.1853

Supplementry Figure 1 Before sort After sort 79% 21% TCR- TCR- 97% NK1.1 SNARF-1 SNARF-1 d 78.6% 21.3% CD45.2 NK1.

Supplementry Figure 1. Adoptive trnsfer of inkt-s1 ells. (-) Single ell suspensions were prepred from LNs of Vα14 trnsgeni mie nd depleted of B22 + ells.

() 3 x 1 6 purified inkt ells were lelled with SNARF-1 nd doptively trnsferred into CD45.1 ongeni reipient mie.

(d-e) inkt-s1 ells purified from LNs (green) were lelled nd doptively trnsferred into WT reipients longside CD4 + T ells (red) nd B ells (lue).

2 Supplementry Figure 1 Before sort After sort 79% 21% TCR- TCR- 97% NK1.1 SNARF-1 SNARF-1 d 78.6% 21.3% CD45.2 NK1.1 inkt-s1 ells B ells CD4 + T ells SCS B ell follile B ell folliles e 1 Prortex inkt ells (%) 5 Prortex Prortex B ell follile Interfolliulr Supsulr Undefined Supplementry Figure 1. Adoptive trnsfer of inkt-s1 ells. (-) Single ell suspensions were prepred from LNs of Vα14 trnsgeni mie nd depleted of B22 + ells. inkt ells were identified s TCR-β + CD1d- Tet + ells () nd sorted for purifition. () Purified ells (left) were stined with NK1.1 (right). () 3 x 1 6 purified inkt ells were lelled with SNARF-1 nd doptively trnsferred into CD45.1 ongeni reipient mie h lter trnsferred inkt ells were deteted s SNARF-1 + -CD ells in the LNs (left pnel) of reipient nimls nd stined with NK1.1 (right pnel). (d-e) inkt-s1 ells purified from LNs (green) were lelled nd doptively trnsferred into WT reipients longside CD4 + T ells (red) nd B ells (lue). LNs were removed, fixed nd imged y multi-photon mirosopy. (d) Three-dimensionl reonstrution from 2 μm setion of poplitel LN showing the distriution of inkt ells (white rrowheds) in the resting LN. (e) Quntifition of the perentge of inkt ells in vrious lotions of the LN. Nture Immunology: doi:1.138/ni.1853

Supplementry Figure 2 Before sort After sort After sort TCR- TCR- %Mx NK1.1 NK1.1 Lymph nodes Spleen Liver CFSE CFSE CFSE CD45.2 CD45.2 CD45.2 Supplementry Figure 2.

1 + ells (left pnel) nd sorted for purifition. Purified ells (middle pnel) were stined with (Right pnel).

3 Supplementry Figure 2 Before sort After sort After sort TCR- TCR- %Mx NK1.1 NK1.1 Lymph nodes Spleen Liver CFSE CFSE CFSE CD45.2 CD45.2 CD45.2 Supplementry Figure 2. Adoptive trnsfer of inkt-s2 ells. () Single ell suspensions were prepred from spleen nd liver of Vα14 trnsgeni mie nd depleted of B22 + ells. inkt ells were identified s TCR-β + NK1.1 + ells (left pnel) nd sorted for purifition. Purified ells (middle pnel) were stined with (Right pnel). () 3-5 x 1 6 purified inkt ells were lelled with CFSE nd doptively trnsferred into CD45.1 ongeni reipient mie h lter trnsferred inkt ells were deteted s CFSE + -CD ells in LNs (left pnel), spleen (middle pnel) nd liver (right pnel) of reipient nimls. Nture Immunology: doi:1.138/ni.1853

Supplementry Figure 3 Speed verge (mm/min) 18 9 *** Frequeny (%) 8 4 inkt-s1 CD4 + T inkt-s1 CD4 + T inkt-s1 CD4 + T -5 5-1

inkt-s1 mm 15-15 CD4 + T mm 15 15 mm -15 Men displement (mm) 1 5 inkt-s1 CD4 + T 2. 4.

Dynmi ehviour of inkt-s1 ells in response to lipid ntigen (-f) inkt- S1 ells (red) longside CD4 + T ells (lue) were doptively

The verge speed (), instntneous speed distriution (), rrest oeffiient (), onfinement index (d), representtive migrtory trks

4 Supplementry Figure 3 Speed verge (mm/min) 18 9 *** Frequeny (%) 8 4 inkt-s1 CD4 + T inkt-s1 CD4 + T inkt-s1 CD4 + T Speed (mm/min) inkt-s1 CD4 + T Arrest oeffiient (%) 1 5 *** d Confinement index (%) 1 5 ** e f mm inkt-s1 mm CD4 + T mm mm -15 Men displement (mm) 1 5 inkt-s1 CD4 + T Squre root of time (min 1/2 ) Supplementry Figure 3. Dynmi ehviour of inkt-s1 ells in response to lipid ntigen (-f) inkt- S1 ells (red) longside CD4 + T ells (lue) were doptively trnsferred into WT reipients tht were injeted i.p. with prtiles oted with α-glcer. The verge speed (), instntneous speed distriution (), rrest oeffiient (), onfinement index (d), representtive migrtory trks (e) nd men displement (f) for inkt ells (red) nd CD4 + T ells (lue) re shown t 16 h fter ntigen dministrtion. Dt were otined from two independent experiments per ondition nd were ompred with two-tiled unpired Mnn-Whitney test., ***p<.1;, ***p<.1; d, p**=.73 Nture Immunology: doi:1.138/ni.1853

Supplementry Figure 4 prtiulte lipids 3 min prtiulte lipids 2h prtiulte lipids 6h 3 mm 3 mm 3

Reltive MFI 5 1 1 min 3 min 2h 1 In Md Ms 1 1 min 3 min 2h 1 In Po Ms f CD169 prtiulte lipids

mm CD169 prtiulte lipids CD3 prtiulte lipids Poplitel LN B22 prtiulte lipids LyVE-1 prtiulte

Prtiulte lipid ntigen rrivl into drining LNs () Confol mirosopy imges of medistinl LNs t

(-e) Flow ytometry ws used to monitor the rrivl of fluoresent prtiulte lipids t vrious times

MFI were normlized to noninjeted mie. (,) After s.

p injetion drining (d) nd non-drining (e) LNs were exmined.

5 Supplementry Figure 4 prtiulte lipids 3 min prtiulte lipids 2h prtiulte lipids 6h 3 mm 3 mm 3 mm 1 Poplitel LN 1 2h Medistinl LN d 1 e 1 2h Reltive MFI 5 Reltive MFI 5 Reltive MFI 5 Reltive MFI min 3 min 2h 1 In Md Ms 1 1 min 3 min 2h 1 In Po Ms f CD169 prtiulte lipids Medistinl LN CD3 prtiulte lipids B22 prtiulte lipids LyVE-1 prtiulte lipids 3 mm 3 mm 3 mm 3 mm CD169 prtiulte lipids CD3 prtiulte lipids Poplitel LN B22 prtiulte lipids LyVE-1 prtiulte lipids 2 mm 2 mm 2 mm 2 mm Supplementry Figure 4. Prtiulte lipid ntigen rrivl into drining LNs () Confol mirosopy imges of medistinl LNs t vrious times fter i.p. injetion of mie with fluoresent prtiulte lipids (green). (-e) Flow ytometry ws used to monitor the rrivl of fluoresent prtiulte lipids t vrious times in different LNs (Po, poplitel; In, Inguinl; Md, medistinl; Ms, mesenteri). MFI were normlized to noninjeted mie. (,) After s.. injetion drining () nd non-drining () LNs were exmined. (d,e) After i.p injetion drining (d) nd non-drining (e) LNs were exmined. (f) Confol mirosopy imges of drining LNs from nimls injeted i.p. (top pnel) or s.. (ottom pnel) with fluoresent prtiulte lipids (green). Two hours fter immuniztion drining LNs were removed, proessed nd stined with CD169, CD3, B22 or LyVE-1 (ll lue). Nture Immunology: doi:1.138/ni.1853

Supplementry Figure 5 inkt-s2 ells B ells GlCer prtiles 2 h 2 h inkt-s2

(-) inkt-s2 (,) or inkt-s1 () ells longside purified B ells (lue,,) or CD4

nd llowed to home for 14-16 h. After i.p.

The imges represent threedimensionl reonstrution of 9 μm () or 15 μm setion

6 Supplementry Figure 5 inkt-s2 ells B ells GlCer prtiles 2 h 2 h inkt-s2 ells B ells GlCer prtiles 16 h 16 h inkt-s1 ells CD4 + T ells GlCer prtiles 16 h 16 h Supplementry Figure 5. inkt ells re onfined in the SCS in response to prtiulte lipid ntigen. (-) inkt-s2 (,) or inkt-s1 () ells longside purified B ells (lue,,) or CD4 + T ells (lue, ), were lelled nd doptively trnsferred into WT reipient mie nd llowed to home for h. After i.p. injetion of prtiles oted with αglcer (green), medistinl LNs were removed nd imged y multi-photon mirosopy. Snpshots of movies re shown 2 h () nd 16 h (,) fter injetion. The imges represent threedimensionl reonstrution of 9 μm () or 15 μm setion (,). inkt ells re pointed with rrowheds. Representtive trks of ell movement orresponding to 3 min movie re tred nd oloured ording to ell type. Long tiks represent 2 () or 5 μm (,). Nture Immunology: doi:1.138/ni.1853

Supplementry Figure 6 Popultion % in PoLN 8. 4.

F4/8 B22 CD11 B22 CD3 B22 -CLL 3 mm 3 mm 3 mm 3mm

B22 GlCer prtiles SCS 3 mm 3 mm 1 mm SCS 1 mm 1 mm

dys prior to removl nd nlysis of drining LNs.

mononuler ells were deteted y flow ytometry.

expression (right) in LN inkt ells from CLL injeted

(lue), CD169, CD11, F4/8 nd/or CD3 (ll red).

7 Supplementry Figure 6 Popultion % in PoLN ns ns ns -CLL+CLL -CLL +CLL -CLL+CLL TCR- - CLL.3% + CLL.3% % Mx CD25 - CLL + CLL B ells T ells DCs CD169 B22 F4/8 B22 CD11 B22 CD3 B22 -CLL 3 mm 3 mm 3 mm 3mm +CLL 3 mm 3mm 3 mm 3 mm d CD169 B22 GlCer prtiles B22 GlCer prtiles SCS 3 mm 3 mm 1 mm SCS 1 mm 1 mm -CLL +CLL +CLL +CLL +CLL Supplementry Figure 6. LN morphology fter CLL tretment. WT mie were injeted with CLL (+CLL) or not (-CLL) 6 dys prior to removl nd nlysis of drining LNs. () Perentge of B ells, T ells nd DCs from totl mononuler ells were deteted y flow ytometry. () inkt ell popultion (left nd middle) nd CD25 expression (right) in LN inkt ells from CLL injeted (+CLL, red) nd ontrol (-CLL, lk) nimls 6 dys fter injetion. () Confol mirosopy imges from LNs stined with B22 (lue), CD169, CD11, F4/8 nd/or CD3 (ll red). (d) Confol mirosopy imges were olleted 4 h fter injetion with fluoresent prtiles oted with αglcer (green, rrowheds). LNs were removed, proessed nd stined for B22 (lue) nd CD169 (red). Nture Immunology: doi:1.138/ni.1853

Supplementry Figure 7 CD169 + CD11 int ells in LN SCS Mf Med Mf CD169 + CD11 int ells fter depletion of F4/8 ells CD169 CD11 SCS Mf fter purifition CD11 d F4/8 84.

Purifition of SCS mrophges () LN mrophges were identified s CD169 + CD11 + CD11 int ells (left pnel) nd lssified s F4/8 + (medullry, Med) nd F4/8 - (supsulr, SCS)

8 Supplementry Figure 7 CD169 + CD11 int ells in LN SCS Mf Med Mf CD169 + CD11 int ells fter depletion of F4/8 ells CD169 CD11 SCS Mf fter purifition CD11 d F4/8 84.5% 15.5% 99.3%.7% SCS Mf (F4/8 - ) Med Mf (F4/8 + ) CD11 F4/8 99%.8% CD11 F4/8 % Mx FSC Supplementry Figure 7. Purifition of SCS mrophges () LN mrophges were identified s CD169 + CD11 + CD11 int ells (left pnel) nd lssified s F4/8 + (medullry, Med) nd F4/8 - (supsulr, SCS) (right pnel). () CD169 + CD11 + CD11i int ells fter mgneti depletion of F4/8 + ells () SCS mrophges fter purifition (d) FSC profiles for SCS (lue) nd Med (red) mrophges s gted in (, right pnel). Nture Immunology: doi:1.138/ni.1853

Supplementary Figure S1

Supplementary Figure S1 Supplementry Figure S1 - UTR m - 3HA - 2-1 hgh - 1 Uiquitin *! *! lk distl promoter m K3R/ K121R-3HA UTR hgh founder lines - HA - - founder lines TG- E1 L A2 B1 F9 G6 H4 H6 B C D2 G1 H3 J2 L - 7 IP: lk