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1 doi:1.138/nature154 a -DTR -DTR WT WT 1,75,16 1,58 1,86 GFP b 87,5% Donor DC -DTR 12,5% Recipient DC WT 1,98%,54% CD45.1 Supplementary Fig. 1. In vivo depletion of + DCs in -DTR transgenic and chimeric mice. a, Depletionof + DCs in spleens of wild type and -DTR transgenic mice was analyzed by flow cytometry after 8 days of treatment with or. DCs were identified by flow cytometry as + GFP + cells. More than 9% DCs were depleted after treatment. b, Depletion of + DCs in spleens of -DTR bone marrow chimeras was analyzed by flow cytometry after 1 days of treatment with or.donordcswereidentifiedbyflowcytometryas + CD cells and recipient DCs as + CD cells. DCs were depleted by ~ 75% after treatment. Depletion of DCs was less efficient in -DTR bone marrow chimeras than in - DTR transgenic mice because wild-type donor DCs were not depleted after treatment. 1

2 doi:1.138/nature154 x DTR -DTR -DTR -DTR WT WT WT WT Cells/µl blood Leucocytes Lymphocytes x Cells/µl blood Erythrocytes Thrombocytes Supplementary Fig. 2. Haematological analyses of wild type and -DTR transgenic mice treated with or for 8 days. The number of leukocytes, lymphocytes, erythrocytes and thrombocytes in the peripheral blood was determined using an automated haematological analyzer. Data are mean values + SEM determined on 7 mice for each genotype and condition. In vivo depletion of + DCs in -DTR transgenic mice does not induce significant leukocytosis and lymphocytosis. 2

3 doi:1.138/nature154 a % 1 8 Body weight 6 4 WT -DTR Bone marrow -DTR ->WTWT Bone marrowwt->-dtr 2 b Cells / lymph node x Days post injection Cervical Brachial Inguinal MLN c Cells recruited / lymph node x Cervical Brachial Inguinal MLN Supplementary Fig. 3. Repeated injections of into lethally irradiated -DTR transgenic mice reconstituted with C57Bl/6:CD45.1 bone marrow, does not affect lymph node cellularity and lymphocyte homing. a, Weight loss after repeated treatment in -DTR transgenic and chimeric mice. b, c, -DTR mice reconstituted with C57Bl/6:CD45.1 wild-type bone marrow were treated with or for 6 days. Total cells in the different lymph nodes were determined by flow cytometry (b). CFSE-labeled lymphocytes from wild type mice were injected intravenously and homing was assessed 4h after injection (c), by quantifying the number of fluorescent lymphocytes in each lymph node (n= 4 mice/group). Treatment with during 6 days had no significant effect on cellularity and lymphocyte homing into mucosal and peripheral lymph nodes. Treatment for longer periods was not possible because chimeric mice succumbed to weight loss (a). 3

4 doi:1.138/nature154 X1 6 2,5 Cellularity 2 Cells/lymph node 1,5 1,5 Cervical Brachial Inguinal MLN Recruited cells/lymph node X Homing assay 2 Cervical Brachial Inguinal MLN Supplementary Fig. 4. Effects of DC depletion on lymph node cellularity and lymphocyte homing into lymph nodes were fully reversible. Homing assays were performed in - DTR chimeric mice, 3 weeks after the last injection (n=4/group). Fluorescently (CFSE)- labelled wild-type lymphocytes were injected into the indicated recipient mice and homing was assessed 4h after injection by flow cytometry. 4

5 doi:1.138/nature154 a Flow cytometry analysis of bone marrow-derived DCs 94,34 79,62 b DC CD45.1+ / Lymph Node MHC-II c Ing H Ing C d Gr-1 Cellularity % 12 BMDCs (+LPS) draining LN BMDCs (-LPS) draining LN Non-draining LN 1 ** % Homing * e % MECA-79 Inguinal Homolateral (draining) lymph node Cellularity WT BMDCs (-LPS) CCR7-/- BMDCs (-LPS) MECA-79 Inguinal Contralateral (non-draining) lymph node % Homing Draining LN Draining LN Supplementary Fig. 5. Adoptive transfer of BMDCs into -treated -DTR transgenic mice. a, b, CMTMR-labelled BMDCs (a) were injected into the footpad and the numbers of BMDCs that reached the draining (homolateral) and non-draining (contralateral) inguinal lymph nodes at day 8 were quantified by flow cytometry (b). c, HEV phenotype was analyzed by immunofluorescence staining of inguinal lymph node sections with MECA-79 mab. d, Cellularity and lymphocyte homing into draining (inguinal) lymph nodes after adoptive transfer of wild-type BMDCs, prepared in the absence of LPS, were compared to the levels observed after adoptive transfer of wild-type BMDCs prepared in the presence of LPS (n=3/group). Non-draining lymph nodes (brachial) are shown as control. Bars represent means +/- SEM. *, P<.5; **, P<.1. e, Wild-type and CCR7 -/- BMDCs (-LPS) were injected into the right and left footpads, respectively, of -treated -DTR transgenic mice. Cellularity and lymphocyte homing into draining (popliteal) lymph nodes after adoptive transfer of CCR7 -/- BMDCs were compared to the levels observed after adoptive transfer of wild-type BMDCs (n=3/group). CCR7 -/- BMDCs, which do not migrate efficiently to draining lymph nodes 45, had a reduced capacity to recover cellularity and lymphocyte homing in DC-depleted mice. Bars represent means +/- SEM. 5

6 doi:1.138/nature154 a MAdCAM-1 MAdCAM-1 b FucT-VII Mesenteric lymph node -DTR transgenic mice FucT-VII GlyCAM-1 GlyCAM-1 GlcNAc6ST-2 Peripheral lymph node -DTR chimeric mice GlcNAc6ST-2 Supplementary Fig DCs play a critical role in the homeostatic maintenance of HEV phenotype in both mucosal and peripheral lymph nodes. a, In vivo depletion of + DCs in -DTR transgenic mice results in down-regulation of HEV-specific marker FucT-VII in mucosal lymph node HEV. Mesenteric lymph node sections from -DTR transgenic mice treated with or for 8 days were stained with the indicated antibodies. Contrary to FucT- VII, MAdCAM-1 expression in mesenteric lymph node HEV was maintained in the absence of + DCs. b, Ablation of + DCs in -DTR chimeric mice results in down-regulation of HEV-specific markers GlyCAM-1 and GlcNAc6ST-2 in peripheral lymph node HEV. Brachial lymph node sections from -DTR bone marrow chimeras treated with or for 1 days were stained by immunofluorescence with anti-glycam-1 or anti- GlcNAc6ST-2 antibodies. 6

7 doi:1.138/nature154 CD31 CD45-CD31+ endothelial cells / mouse PLNs (pool) X1 3 CD45 Gated on endothelial cells (CD45 - CD31 + ) CD MECA-79 Mean fluorescence intensity for MECA-79 DTR : 1784 / DTR : 4544 Supplementary Fig. 7. Isolation of MECA-79 + CD31 + HEV endothelial cells from peripheral lymph nodes of -DTR transgenic mice treated with or for 8 days. HEV endothelial cells (CD45 -,CD31 +,MECA-79 + ) and non-hev endothelial cells (CD45 -,CD31 +, MECA-79 - ) were isolated by cell sorting from pooled peripheral lymph nodes of 5 mice for each condition. Stromal cell suspensions were prepared and HEV endothelial cells (1 cells) were isolated from the CD45 - fraction by gating on CD31 + endothelial cells. Although the percentage of MECA-79 + CD31 + HEV endothelial cells was reduced after treatment (from % in -treated mice to 9.9 % in -treated mice) and the intensity of MECA-79 staining was also reduced on the remaining MECA-79 + cells (from 1784 in DTR - to 4544 in DTR treated mice), it was nevertheless possible to isolate these cells by cell sorting. In our experiments, MECA-79 expression was generally less downregulated after -treatment than other HEVmarkers such as GlyCAM-1. This is likely to be due to the fact that the sulphated sialomucins (CD34, podocalyxin, ) recognized by MECA-79 at the surface of HEV endothelial cells have a very long half-life, whereas GlyCAM-1 is a secreted molecule with a more rapid turnover. 7

8 doi:1.138/nature154 a mrna relative to YWHAZ b mrna relative to GAPDH 1,4 1,2 1,8,6,4,2 1,8 1,6 1,4 1,2 1,8,6,4,2 CCL21 Stromal fraction ICAM1 CD31 CXCL13 VCAM1 HEVEC DTR HEVEC DTR Endo DTR Endo DTR Supplementary Fig. 8. In vivo depletion of + DCs does not affect the expression of several genes involved in the multi-step adhesion cascade. a, Ablation of + DCs does not affect expression of CCL21 and ICAM-1 in HEV endothelial cells. qpcr analysis of CCL21 and ICAM-1 gene expression was performed using total RNA from MECA-79 + CD31 + HEV endothelial cells (HEV) and MECA-79 - CD31 + non-hev endothelial cells (Endo), isolated by cell sorting from or -treated -DTR transgenic mice. b, Ablation of + DCs does not affect expression of CXCL13 and VCAM-1 in peripheral lymph node stromal cells. qpcr analysis of CD31, CXCL13 and VCAM-1 gene expression was performed using total RNA from peripheral lymph node stromal cells (CD45 - fraction), isolated from or treated -DTR transgenic mice. The housekeeping genes YWHAZ (a) andgapdh (b) were used as control genes for normalization. Mean and SD from triplicate qpcr runs are plotted. Data are representative of 2 independent experiments. 8

9 doi:1.138/nature154 a WT RAG2-/- Fig. 4 MECA-79 5µm b c RAG2-/- -DTR mice Recruited cells/ lymph node section *** Supplementary Fig. 9. Analyses in RAG2-/- mice indicate that T and B lymphocytes are not essential for homeostatic maintenance of HEV phenotype and function. a, Immunofluorescence staining of peripheral lymph node sections from wild-type and RAG2-/- mice with the HEV-specific mab MECA-79. b, Homing of CFSE-labelled wild-type lymphocytes into peripheral lymph nodes from wild-type and RAG2-/- mice (n=4/group). Lymphocyte homing was increased in RAG2-/- mice. c, Ablationof + DCs in RAG2-/- -DTR transgenic mice inhibits lymphocyte homing (n=3/group; 5 inguinal lymph node sections/mouse). ***, P<

10 doi:1.138/nature154 a Stromal cell suspensions Gated on CD45- cells b Lymph nodes cell suspensions Sorted cells CD31 CD31 mrna relative to YWHAZ 1,4 1,2 1,8,6,4,2 CD45 Endothelial cells MECA-79 GlyCAM-1 Sorted cells HEV Endo MECA-79 HEV CD31 VE-Cadherin c mrna relative to YWHAZ MHC II+ MHCII MHCII CD31 Lymph Node DC Endo high MHC II med med MHC II high Supplementary Fig. 1. Isolation of MECA-79 + CD31 + HEV endothelial cells and + MHC class II + DCs from PLN of wild type mice. a, Stromal cell suspensions were prepared and HEV endothelial cells (CD45 -, CD31 +, MECA-79 + ) and non-hev endothelial cells (CD45 -, CD31 +, MECA-79 - ) were isolated from the CD45 - fraction by gating on CD31 + endothelial cells (~ 3 HEV endothelial cells were obtained from pooled PLN of 2 mice). qpcr analysis of GlyCAM-1, CD31 and VE-Cadherin expression in isolated cells was performed to verify the quality of the isolation procedure. Mean and SD from triplicate qpcr runs are plotted. b, + MHC class II + DCs were isolated from single cell suspensions (pooled peripheral lymph nodes of 2 mice). and CD31 expression in isolated + MHC class II + DCs and CD31 + endothelial cells was analyzed by qpcr. Mean and SD from triplicate qpcr runs are plotted. c, Flow cytometry profiles of isolated + MHC class II + DCs subsets from PLN of wild type mice. + MHC class II + DCs, high MHC class II med DCs (cdcs, classical DCs) and med MHC class II high DCs (mdcs, migratory DCs) were isolated from PLN single cell suspensions (pooled PLN of 5 mice digested with collagenase D) by gating on + and MHC class II + cells. 1

11 doi:1.138/nature154 mrna relative to CD31 1,2 1,8,6,4,2 GlyCAM-1 HEV D HEV Fc-Ig D11 HEV+3DC Fc-Ig D11 HEV+3DC LTBR-Ig D11 Endo+3DC Fc-Ig D11 Endo+3DC LTBR-Ig D11 Supplementary Fig DCs control HEV-specific gene expression through a LTβR-dependent pathway. MECA-79 + CD31 + HEV endothelial cells (HEV) and + MHC class II + DCs (DC) were isolated by cell sorting from pooled peripheral lymph nodes of wildtypemiceandco-culturedduring11days.co-culturesofhevanddc(1hev+3dc)were treated with LTβR-Ig or control Fc-Ig and HEV-specific gene expression was analyzed by qpcr using GlyCAM-1 as a reporter gene. Co-cultures of MECA-79 - CD31 + endothelial cells (Endo) and DC were used as controls. CD31 was used as a control gene for normalization. Mean and SD from triplicate qpcr runs are plotted. 11

12 doi:1.138/nature154 a -DTR/LT -/- mixed bone marrow chimeras -DTR/LT +/+ mixed bone marrow chimeras LT-/- DCs -DTR DCs LT+/+ DCs -DTR DCs GFP b Cells / spleen DTR/LT-/- -DTR/LT-/- -DTR/LT+/+ GFP Cellularity Homing assay X1 3 X1 6 Recruited cells / spleen 45 -DTR/LT+/+ -DTR/LT-/ Supplementary Fig. 12. Injection of into -DTR/LT -/- or -DTR/LT +/+ mixed bone marrow chimeras induces depletion of LT +/+ DCs (GFP + ) harbouring the -DTR- GFP transgene, sparing non-transgenic LT -/- or LT +/+ DCs (GFP - ). a, Depletion of + DCs in spleens of -DTR/LT -/- and -DTR/LT +/+ mixed (5/5) bone marrow chimeras was analyzed by flow cytometry after 1 days of treatment with or. + (LT +/+ )DCs harbouring the -DTR-GFP transgene were identified by flow cytometry as + GFP + cells and non-transgenic LT -/- or LT +/+ DCsas + GFP - cells. Transgenic + GFP + DCs were depleted after treatment, whereas non-transgenic + GFP - DCs were not affected. b, Cellularity and lymphocyte homing into the spleen in -treated chimeric mice. Cellularity in spleen was determined by flow cytometry after 1 days of treatment in -DTR/LT -/- and -DTR/LT +/+ chimeric mice. CFSE-labeled lymphocytes from wild type mice were injected intravenously and homing was assessed 4h after injection, by quantifying the number of fluorescent lymphocytes in spleen (n= 4 mice/group). Data are representative of 2 independent experiments. 12

13 doi:1.138/nature154 Supplementary Discussion Critical role of the lymphoid tissue microenvironment in the maintenance of HEV characteristics during homeostasis Studies perform ed in rodents 25 years ago, have revealed that, when lym ph nodes are deprived of afferent lymph, HEV convert to a flat-walled morphology and lose their ability to support lym phocyte traffic 11,38. Later studies showed this inhibition of lym phocyte hom ing correlates with downregulation of HEV-sp ecific differentiation m arkers, MECA-79 12, Fuc- TVII 13 and GlyCAM-1 14, a m ajor PNAd component 6. More recently, the use of HEV endothelial cells, freshly purif ied from hum an tissues, has c onfirmed these cells exhibit a remarkable plasticity in adult tis sues and rapi dly lose their specialized characteristics when isolated from their natural m icroenvironment 15. Together, these results indic ated that th e lymphoid tissue m icroenvironment is critical fo r maintenance of HEV characteristics during homeostasis 4,15. The rodent studies suggested that lymph contains cells or factors that regulate HEV phenotype and function. One of these factors co uld be antigen draining into the lym ph from peripheral tissues. Consistent with this hypothesis, direct injection of antigen into the node has been shown to restore the typical HEV m orphology in peripheral lym ph nodes deprived of afferent lymph 11. Dendritic cell precursors (pre-cdc) have been repor ted to enter in to lymph nodes through HEVs 39, but these precursors m ay need some signals from the afferent lym ph (antigen) for differentiation, activation or survival in lymph nodes. Regarding the effects of lymph flow on cellular composition of lymph nodes, previous studies have revealed th at the num bers of paracortical de ndritic cells and subcapsular sinus macrophages in lym ph nodes are greatly redu ced after the occlusion of afferent lymphatics 12,38. Interestingly, after reversal of occl usion, the restoration of HEV phenotype and function correlated with the reappearance of dendritic cells and macrophages in the lymph 13

14 doi:1.138/nature154 nodes 12. However, macrophages do not appear to be essential for maintenance of HEVs, since no effects on the capacity of HEVs to bind lymphocytes were observed after in vivo depletion of macrophages from lymph nodes using clodronate liposomes 4. Possible effects of on non-dc populations in -DTR transgenic mice + DC are efficiently dep leted after treatm ent in -DTR transg enic and chimeric m ice. However, som e alternative cel l subsets expressing m ight also be depleted upon injection of (although this pr oblem is lim ited by the use of a prom oter that is only expressed in high cells). Indeed, this has b een clearly dem onstrated for lymph node MOMA-1 + (CD169, sialoadhesin, Siglec-1) subcapsular sinus m acrophages 41,42. However, as indicated above, when this same macrophage popula tion was specifically depleted from lymph nodes using clodronate liposomes, no effects on the capacity of HEVs to bind lymphocytes were observed 4. Probst et al. reported that, in contrast to macrophages, the number and localization of CD4 +, CD8 + and CD19 + cells in lymph nodes was not affected by administration of i n -DTR transgenic mice 41. In our study, the num bers of T and B cells in lymph nodes were decreased after 8 da ys of treatm ent but we believe this a consequence of DC depletion and downregulation of the HEV phenotype since no decrease in lymph node cellularity was observe d at day 2. Jung et al have reported that activated CD8 + cells expres s the -DTR trans gene after in vivo stim ulation and could be depleted by treatment 16, but our studies were performed under homeostatic conditions in the absence of lymph node stim ulation. In addition, although B cells have been shown to be involved in HEV remodeling during inflammation 22, our analyses in RAG2-/- mice (Supplementary Fig. 9) indicate that a m ajor role of B cells or T cells in the homeosta tic maintenance of HEVs is unlikely. Similar to our observations, Browni ng and colleagues analyzed the HEV phenotype in mice deficient in only B cells or o nly T cells and reported that the complete loss of B cells 14

15 doi:1.138/nature154 or T cells does not affect the HEV differentiation program 23. Finally, NK cells have also been reported to express but they are unlikely to play a dominant role in the control of HEV phenotype because they are no t depleted in -treated -DTR transgenic mice 43. In addition, analyses in NK-DTR transgenic mice have shown that in vivo depletion of NK cells for at least 7 days does not affect T and B cell populations in lym ph nodes 44. Collectively these observations, together with our experim ents with adoptiv ely transferred wild-type DCs in -DTR m ice (as well as our HEV- DCs co-c ulture experim ents), support our conclusion that + DCs rather than other hem atopoietic cells (macrophages, NK, T or B cells) regulate HEV phenotype and function in lymph nodes during homeostasis. We observed that the effects of injection on lymph node cellularity and lymphocyte hom ing we re often m ore pronounced in -DTR trans genic m ice than in chimeric m ice. W e believe th is is due to the fact that DC deple tion was less ef ficient in chimeric m ice, due to the incom plete ch imerism (75% depletion co mpared to > 9% in transgenic m ice; see Supplem entary Fig. 1a and 1b). Although we strongly believe the differences observed between transgenic and chim eric mice are du e to the dif ferent levels of + DC depletion, we cannot exclude at this point the possibility that non-hem atopoietic stromal cells may also play a role in the regulation of HEV phenotype and function. 15

16 doi:1.138/nature154 Supplementary References 38 Drayson, M. T. & Ford, W. L. Afferent lymph and lymph borne cells: their influence on lymph node function. Immunobiology 168, (1984). 39 Liu, K. et al. In vivo analysis of dendritic cell development and homeostasis. Science 324, (29). 4 Mebius, R. E., Bauer, J., Twisk, A. J., Brev e, J. & Kraal, G. The functional activity of high endothelial venules: a role for the subc apsular sinus m acrophages in the lym ph node. Immunobiology 182, (1991). 41 Probst, H. C. et al. H istological analysis of CD 11c-DTR/GFP m ice af ter in v ivo depletion of dendritic cells. Clin Exp Immunol 141, (25). 42 Iannacone, M. et al. Subcapsular sinus m acrophages prevent CNS invasion on peripheral infection with a neurotropic virus. Nature 465, (21). 43 Lucas, M., Schachterle, W., Oberle, K., Aich ele, P. & Diefenbach, A. Dendritic cells prime natural killer cells by trans-presenting interleukin 15. Immunity 26, (27). 44 Walzer, T. et al. Identification, activation, and selective in vivo ablation of mouse NK cells via NKp46. Proc Natl Acad Sci U S A 14, (27). 45 MartIn-Fontecha, A. et al. Regulation of dendritic cell migration to the draining lymph node: impact on T lymphocyte traffic and priming. J Exp Med 198, (23). 16