su pplementar y information - buffalo, ny · cd11c-dtr 12,5% recipient dc wt gfp cd11c cd11c-dtr...

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Supplementary Fig. 1. In vivo depletion of CD11c+ DCs in CD11c-DTR transgenic andchimeric mice. a, Depletion of CD11c+ DCs in spleens of wild type and CD11c-DTRtransgenic mice was analyzed by flow cytometry after 8 days of treatment with DTX orPBS. DCs were identified by flow cytometry as CD11c+GFP+ cells. More than 90% DCswere depleted after DTX treatment. b, Depletion of CD11c+ DCs in spleens of CD11c-DTRbone marrow chimeras was analyzed by flow cytometry after 10 days of treatment withDTX or PBS. Donor DCs were identified by flow cytometry as CD11c+CD45.1- cells andrecipient DCs as CD11c+CD45.1+ cells. DCs were depleted by ~ 75% after DTX treatment.Depletion of DCs was less efficient in CD11c-DTR bone marrow chimeras than in CD11c-DTR transgenic mice because wild-type donor DCs were not depleted after DTX treatment.

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SUPPLEMENTARY INFORMATIONdoi:10.1038/nature10540

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Supplementary Fig. 2. Haematological analyses of wild type and CD11c-DTRtransgenic mice treated with DTX or PBS for 8 days. The number of leukocytes,lymphocytes, erythrocytes and thrombocytes in the peripheral blood was determined usingan automated haematological analyzer. Data are mean values + SEM determined on 7mice for each genotype and condition. In vivo depletion of CD11c+ DCs in CD11c-DTRtransgenic mice does not induce significant leukocytosis and lymphocytosis.


SUPPLEMENTARY INFORMATIONRESEARCHdoi:10.1038/nature10540

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Supplementary Fig. 3. Repeated injections of DTX into lethally irradiated CD11c-DTRtransgenic mice reconstituted with C57Bl/6:CD45.1 bone marrow, does not affect lymphnode cellularity and lymphocyte homing. a, Weight loss after repeated DTX treatment inCD11c-DTR transgenic and chimeric mice. b, c, CD11c-DTR mice reconstituted withC57Bl/6:CD45.1 wild-type bone marrow were treated with DTX or PBS for 6 days. Total cells inthe different lymph nodes were determined by flow cytometry (b). CFSE-labeled lymphocytesfrom 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 DTX during 6 days had no significant effect on cellularity and lymphocytehoming into mucosal and peripheral lymph nodes. Treatment for longer periods was not possiblebecause chimeric mice succumbed to weight loss (a).



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Supplementary Fig. 4. Effects of DC depletion on lymph node cellularity and lymphocytehoming into lymph nodes were fully reversible. Homing assays were performed in CD11c-DTR chimeric mice, 3 weeks after the last DTX injection (n=4/group). Fluorescently (CFSE)-labelled wild-type lymphocytes were injected into the indicated recipient mice and homingwas assessed 4h after injection by flow cytometry.



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Supplementary Fig. 5. Adoptive transfer of BMDCs into DTX-treated CD11c-DTR transgenicmice. a, b, CMTMR-labelled BMDCs (a) were injected into the footpad and the numbers ofBMDCs that reached the draining (homolateral) and non-draining (contralateral) inguinal lymphnodes at day 8 were quantified by flow cytometry (b). c, HEV phenotype was analyzed byimmunofluorescence staining of inguinal lymph node sections with MECA-79 mAb. d, Cellularityand lymphocyte homing into draining (inguinal) lymph nodes after adoptive transfer of wild-typeBMDCs, prepared in the absence of LPS, were compared to the levels observed after adoptivetransfer of wild-type BMDCs prepared in the presence of LPS (n=3/group). Non-draining lymphnodes (brachial) are shown as control. Bars represent means +/- SEM. *, P<0.05; **, P<0.01. e,Wild-type and CCR7-/- BMDCs (-LPS) were injected into the right and left footpads, respectively,of DTX-treated CD11c-DTR transgenic mice. Cellularity and lymphocyte homing into draining(popliteal) lymph nodes after adoptive transfer of CCR7-/- BMDCs were compared to the levelsobserved after adoptive transfer of wild-type BMDCs (n=3/group). CCR7-/- BMDCs, which do notmigrate efficiently to draining lymph nodes45, had a reduced capacity to recover cellularity andlymphocyte homing in DC-depleted mice. Bars represent means +/- SEM.



a

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Mesenteric lymph nodeCD11c-DTR transgenic mice

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GlyCAM-1 GlyCAM-1

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GlcNAc6ST-2 GlcNAc6ST-2

Supplementary Fig. 6. CD11c+ DCs play a critical role in the homeostatic maintenance ofHEV phenotype in both mucosal and peripheral lymph nodes. a, In vivo depletion of CD11c+

DCs in CD11c-DTR transgenic mice results in down-regulation of HEV-specific marker FucT-VIIin mucosal lymph node HEV. Mesenteric lymph node sections from CD11c-DTR transgenic micetreated with DTX or PBS 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 ofCD11c+DCs. b, Ablation of CD11c+ DCs in CD11c-DTR chimeric mice results in down-regulationof HEV-specific markers GlyCAM-1 and GlcNAc6ST-2 in peripheral lymph node HEV. Brachiallymph node sections from CD11c-DTR bone marrow chimeras treated with DTX or PBS for 10days were stained by immunofluorescence with anti-GlyCAM-1 or anti- GlcNAc6ST-2 antibodies.



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Supplementary Fig. 7. Isolation of MECA-79+CD31+ HEV endothelial cells from peripherallymph nodes of CD11c-DTR transgenic mice treated with DTX or PBS for 8 days. HEVendothelial 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 eachcondition. Stromal cell suspensions were prepared and HEV endothelial cells (10 000 cells) wereisolated from the CD45- fraction by gating on CD31+ endothelial cells. Although the percentage ofMECA-79+CD31+ HEV endothelial cells was reduced after DTX treatment (from 17.58 % inPBS-treated mice to 9.09 % in DTX-treated mice) and the intensity of MECA-79 staining wasalso reduced on the remaining MECA-79+ cells (from 17840 in DTR PBS- to 4544 in DTR DTX-treated mice), it was nevertheless possible to isolate these cells by cell sorting. In our experiments,MECA-79 expression was generally less downregulated after DTX-treatment than other HEV-markers 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 avery long half-life, whereas GlyCAM-1 is a secreted molecule with a more rapid turnover.



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Supplementary Fig. 8. In vivo depletion of CD11c+ DCs does not affect the expression ofseveral genes involved in the multi-step adhesion cascade. a, Ablation of CD11c+ DCs doesnot affect expression of CCL21 and ICAM-1 in HEV endothelial cells. qPCR analysis of CCL21and ICAM-1 gene expression was performed using total RNA from MECA-79+CD31+ HEVendothelial cells (HEV) and MECA-79-CD31+ non-HEV endothelial cells (Endo), isolated bycell sorting from DTX or PBS-treated CD11c-DTR transgenic mice. b, Ablation of CD11c+ DCsdoes 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 totalRNA from peripheral lymph node stromal cells (CD45- fraction), isolated from DTX or PBS-treated CD11c-DTR transgenic mice. The housekeeping genes YWHAZ (a) and GAPDH (b)were used as control genes for normalization. Mean and SD from triplicate qPCR runs areplotted. Data are representative of 2 independent experiments.



aFig. 4RAG2-/-WT MECA-79

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Supplementary Fig. 9. Analyses in RAG2-/- mice indicate that T and B lymphocytes arenot 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-typelymphocytes into peripheral lymph nodes from wild-type and RAG2-/- mice (n=4/group).Lymphocyte homing was increased in RAG2-/- mice. c, Ablation of CD11c+ DCs in RAG2-/-CD11c-DTR transgenic mice inhibits lymphocyte homing (n=3/group; 5 inguinal lymph nodesections/mouse). ***, P<0.001.



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Supplementary Fig. 10. Isolation of MECA-79+CD31+ HEV endothelial cells and CD11c+MHCclass II+ DCs from PLN of wild type mice. a, Stromal cell suspensions were prepared and HEVendothelial 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 (~ 30 000HEV endothelial cells were obtained from pooled PLN of 20 mice). qPCR analysis of GlyCAM-1,CD31 and VE-Cadherin expression in isolated cells was performed to verify the quality of theisolation procedure. Mean and SD from triplicate qPCR runs are plotted. b, CD11c+MHC class II+

DCs were isolated from single cell suspensions (pooled peripheral lymph nodes of 20 mice). CD11cand CD31 expression in isolated CD11c+MHC class II+ DCs and CD31+ endothelial cells wasanalyzed by qPCR. Mean and SD from triplicate qPCR runs are plotted. c, Flow cytometry profiles ofisolated CD11c+MHC class II+ DCs subsets from PLN of wild type mice. CD11c+MHC class II+ DCs,CD11chighMHC class IImed DCs (cDCs, classical DCs) and CD11cmedMHC class IIhigh DCs (mDCs,migratory DCs) were isolated from PLN single cell suspensions (pooled PLN of 5 mice digested withcollagenase D) by gating on CD11c+ and MHC class II+ cells.



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Supplementary Fig. 11. CD11c+ DCs control HEV-specific gene expression through aLTβR-dependent pathway. MECA-79+CD31+ HEV endothelial cells (HEV) and CD11c+MHCclass II+ DCs (DC) were isolated by cell sorting from pooled peripheral lymph nodes of wild-type mice and co-cultured during 11 days. Co-cultures of HEV and DC (1 HEV + 3 DC) weretreated with LTβR-Ig or control Fc-Ig and HEV-specific gene expression was analyzed by qPCRusing 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 andSD from triplicate qPCR runs are plotted.



GFPC

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Supplementary Fig. 12. Injection of DTX into CD11c-DTR/LT-/- or CD11c-DTR/LT+/+ mixedbone marrow chimeras induces depletion of LT+/+ DCs (GFP+) harbouring the CD11c-DTR-GFP transgene, sparing non-transgenic LT-/- or LT+/+ DCs (GFP-). a, Depletion of CD11c+ DCsin spleens of CD11c-DTR/LT-/- and CD11c-DTR/LT+/+ mixed (50/50) bone marrow chimeras wasanalyzed by flow cytometry after 10 days of treatment with DTX or PBS. CD11c+ (LT+/+) DCsharbouring the CD11c-DTR-GFP transgene were identified by flow cytometry as CD11c+GFP+ cellsand non-transgenic LT-/- or LT+/+ DCs as CD11c+GFP - cells. Transgenic CD11c+GFP+ DCs weredepleted after DTX treatment, whereas non-transgenic CD11c+GFP- DCs were not affected. b,Cellularity and lymphocyte homing into the spleen in DTX-treated chimeric mice. Cellularity inspleen was determined by flow cytometry after 10 days of DTX treatment in CD11c-DTR/LT-/- andCD11c-DTR/LT+/+ chimeric mice. CFSE-labeled lymphocytes from wild type mice were injectedintravenously and homing was assessed 4h after injection, by quantifying the number of fluorescentlymphocytes in spleen (n= 4 mice/group). Data are representative of 2 independent experiments.



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-

TVII13 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 icroenvironment15. Together, these results indic ated that th e

lymphoid tissue m icroenvironment is critical fo r maintenance of HEV characteristics during

homeostasis4,15.

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 lymph11. 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

lymphatics12,38. 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



nodes12. 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 liposomes40.

Possible effects of DTX on non-DC populations in CD11c-DTR transgenic mice

CD11c+ DC are efficiently dep leted after DTX treatm ent in CD11c-DTR transg enic and

chimeric m ice. However, som e alternative cel l subsets expressing CD11c m ight also be

depleted upon injection of DTX (although this pr oblem is lim ited by the use of a prom oter

that is only expressed in CD11c high cells). Indeed, this has b een clearly dem onstrated for

lymph node MOMA-1 + (CD169, sialoadhesin, Siglec-1) subcapsular sinus m acrophages41,42.

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 observed40. 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 DTX i n CD11c-DTR transgenic mice41. In our study, the num bers of T and

B cells in lymph nodes were decreased after 8 da ys of DTX 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 CD11c-DTR trans gene after in vivo stim ulation and could be depleted by

DTX treatment16, 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 inflammation22, 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



or T cells does not affect the HEV differentiation program23. Finally, NK cells have also been

reported to express CD11c but they are unlikely to play a dominant role in the control of HEV

phenotype because they are no t dep leted in DTX-treated CD11c-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 CD11c-DTR m ice (as well as our HEV- DCs co-c ulture experim ents), support our

conclusion that CD11c+ 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 DTX injection on lymph node cellularity and

lymphocyte hom ing we re often m ore pronounced in CD11c-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 > 90% 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

CD11c+ 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.



Supplementary References

38 Drayson, M. T. & Ford, W. L. Afferent lymph and lymph borne cells: their influence

on lymph node function. Immunobiology 168, 362-379 (1984).

39 Liu, K. et al. In vivo analysis of dendritic cell development and homeostasis. Science

324, 392-397 (2009).

40 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, 277-291 (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, 398-404 (2005).

42 Iannacone, M. et al. Subcapsular sinus m acrophages prevent CNS invasion on

peripheral infection with a neurotropic virus. Nature 465, 1079-1083 (2010).

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, 503-517 (2007).

44 Walzer, T. et al. Identification, activation, and select ive in vivo ablation of mouse NK

cells via NKp46. Proc Natl Acad Sci U S A 104, 3384-3389 (2007).

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, 615-621 (2003).



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