E-selectin ligands in the human mononuclear phagocyte system: Implications for infection, inflammation, and immunotherapy

Janua y 2018 | Volume 8 | A icle 18781 Re iew published: 19 Janua y 2018 doi: 10.3389/ immu.2017.01878 F on ie s in Immunology | www. on ie sin.o g Edi ed by: Yoann Rombou s, UMR5089 Ins i u de Pha macologie e de Biologie S uc u ale (IPBS), F ance Re iewed by: Michael Hickey, Monash Uni e si y, Aus alia Luciana Ba os A uda, Uni e sidade Fede al do Rio de Janei o, B azil *Co espondence: Paula A. Videi a [email p o ec ed]; Robe Sacks ein [email p o ec ed] †Co- i s au ho ship. Special y sec ion: This a icle was submi ed o Mic obial Immunology, a sec ion o he jou nal F on ie s in Immunology Recei ed: 30Sep embe 2017 Accep ed: 08Decembe 2017 Published: 19Janua y2018 Ci a ion: Sil aM, Videi aPA and Sacks einR (2018) E-Selec in Ligands in he Human Mononuclea Phagocy e Sys em: Implica ions o In ec ion, In lamma ion, and Immuno he apy. F on . Immunol. 8:1878. doi: 10.3389/ immu.2017.01878 e-Selec in Ligands in he Human Mononuclea Phagocy e Sys em: implica ions o in ec ion, in lamma ion, and immuno he apy Ma iana Sil a1,2†, Paula A. Videi a3,4*† and Robe Sacks ein1,2,5* 1 Depa men o De ma ology, Ha a d Skin Disease Resea ch Cen e , B igham and Women’s Hospi al, Ha a d Medical School, Bos on, MA, Uni ed S a es, 2 P og am o Excellence in Glycosciences, Ha a d Medical School, Bos on, MA, Uni ed S a es, 3 UCIBIO, Depa amen o Ciências da Vida, Faculdade de Ciências e Tecnologia, Uni e sidade NOVA de Lisboa, Lisboa, Po ugal, 4 P o essionals and Pa ien Associa ions In e na ional Ne wo k (CDG & Allies – PPAIN), Faculdade de Ciências e Tecnologia, Uni e sidade NOVA de Lisboa, Lisboa, Po ugal, 5 Depa men o Medicine, B igham and Women’s Hospi al, Ha a d Medical School, Bos on, MA, Uni ed S a es The mononuclea phagocy e sys em comp ises a ne wo k o ci cula ing monocy es and dend i ic cells (DCs), and “his iocy es” ( issue- esiden mac ophages and DCs) ha a e de i ed in pa om blood-bo ne monocy es and DCs. The capaci y o ci cula - ing monocy es and DCs o unc ion as he body’s i s -line de ense agains o ending pa hogens g ea ly depends on hei abili y o eg ess he bloods eam and in il a e in lamma o y si es. Ex a asa ion in ol es a sequence o coo dina ed molecula e en s and is ini ia ed by E-selec in-media ed decele a ion o he ci cula ing leukocy es on o mic o ascula endo helial cells o he a ge issue. E-selec in is inducibly exp essed by cy okines ( umo nec osis ac o -α and IL-1β) on in lamed endo helium, and binds o sialo ucosyla ed glycan de e minan s displayed on p o ein and lipid sca olds o blood cells. E icien ex a asa ion o ci cula ing monocy es and DCs o in lamed issues is c ucial in acili a ing an e ec i e immune esponse, bu also uels he immunopa hology o se e al in lamma o y diso de s. Thus, insigh s in o he s uc u al and unc ional p op- e ies o he E-selec in ligands exp essed by di e en monocy e and DC popula ions is key o unde s anding he biology o p o ec i e immuni y and he pa hobiology o se e al acu e and ch onic in lamma o y diseases. This e iew will add ess he ole o E-selec in in ec ui men o human ci cula ing monocy es and DCs o si es o issue inju y/in lamma ion, he s uc u al biology o he E-selec in ligands exp essed by hese cells, and he molecula e ec o s ha shape E-selec in ligand cell-speci ic display. In addi ion, he apeu ic app oaches a ge ing E-selec in ecep o /ligand in e ac ions, which can be used o boos hos de ense o , con e sely, o dampen pa hological in lamma o y condi ions, will also be discussed. Keywo ds: mononuclea phagocy e, HCeLL, e-selec in ligand, cell mig a ion, e-selec in, sialyl Lewis X iNTRODUCTiON The mononuclea phagocy e sys em (MPS) comp ises monocy es, dend i ic cells (DC), and issue- esiden mac ophages. MPS cells ha e specialized phagocy ic capabili ies, and an igen p o- cessing and p esen ing unc ions, he eby ini ia ing he immune esponse and linking inna e and adap i e immune sys ems (1). In addi ion o hei ole as key sen inels and egula o s o immuni y, FigURe 1 | P oposed model o mig a ion o human monocy es and dend i ic cell (DC) p ogeni o s in o issues in s eady-s a e and in lamma o y condi ions. A e di e en ia ion in he bone ba ow, p ecu so s o DCs and monocy es en e he blood s eam and a e dis ibu ed o lymphoid o gans [ h ough high endo helial enules (HEV)] and o a ious pe iphe al issues. In s eady s a e, non-classical monocy es a e p e e en ially ec ui ed in o he es ing ascula u e, whe e hey pa ol he endo helium and may con ibu e o he main enance o issue- esiden mac ophage and DC popula ions. Con en ional DCs (cDCs) eci cula e be ween pe iphe al issues and lymphoid o gans (mig a o y cDCs), pa icipa ing in he induc ion o pe iphe al ole ance, o eside in he lymphoid o gans (lymphoid- esiden cDCs). By con as , plasmacy oid DCs (pDCs) mos ly popula e lymphoid issues (lymphoid- esiden pDCs) and lack mig a o y abili y unde s eady-s a e condi ions. Upon in lamma ion, classical monocy es, cDCs, and pDCs a e ec ui ed o a ec ed issues. A e an igen up ake and di e en ia ion in o ully unc ional ma u e DCs, monocy e- de i ed DCs (moDCs), and cDCs en e d aining lymph nodes ia a e en lympha ics. pDCs can only access eac i e lymph nodes om he blood s eam ia HEVs. 2 Sil a e al. E-Selec in Ligands in he Human MPS F on ie s in Immunology | www. on ie sin.o g Janua y 2018 | Volume 8 | A icle 1878 mononuclea phagocy es a e also in ol ed in se e al pa hologi- cal in lamma o y condi ions, including au oimmune diseases, in ec ion, cance , and abno mal wound healing p ocesses (2). To access in lamma o y si es, ci cula ing monocy es and DCs mus i s engage he ascula endo helial ba ie agains he p e ailing o ces o hemodynamic shea , a p ocess ha occu s ia adhesi e in e ac ions be ween ascula E-selec in and i s glycan coun e - ecep o s (E-selec in ligands) on he ci cula - ing cells (3). This ini ial con ac esul s in e he ing and slow olling o he cells along he endo helial su ace a eloci ies well below ha o blood low (4). E-selec in-media ed slow oll- ing is a i al s ep in his cascade o e en s as i allows in ima e con ac be ween MPS cells and he in lamed endo helium, and he ecogni ion o in lamma o y molecules wi hin he milieu (3). Consequen ly, a g ea e knowledge o how E-selec in ligand display is elabo a ed by di e en ypes o ci cula ing mono- cy es and DCs is key o unde s anding he physiological and pa hological e en s associa ed wi h he MPS. In his e iew, we will p o ide in o ma ion on he s uc u al biology and ope a- ion o he wide a ie y o E-selec in-binding glycoconjuga es exp essed by ci cula ing MPS cells (i.e., blood monocy e and non- issue- esiden DC popula ions) in ligh o hei impac on pa hology and po en ial he apies. Fu he mo e, we will discuss he molecula basis o he biosyn hesis o hese glycoconjuga es, and how such knowledge can ame no el s a egies o inhibi o en o ce a icking o MPS cells. MONONUCLeAR PHAgOCYTe FAMiLY: HeTeROgeNeiTY AND MigRATORY CAPABiLiTieS Monocy es Monocy es cons i u e a he e ogeneous cell popula ion, comp is- ing app oxima ely 5–10% o o al pe iphe al blood leukocy es. These cells a ise om g anulocy e–mac ophage p ogeni o s in he bone ma ow and a e subsequen ly eleased in o pe iphe al blood, whe e hey ci cula e o se e al days (5). A s eady s a e (i.e., wi hou any in lamma o y cue), monocy es can en e non-lymphoid issues, and he e hey ei he e ain hei blood monocy ic beha io (6), o gene a e he immedia e p ecu so s o “monocy e-de i ed mac ophages and DCs,” which cons i u e a small po ion o issue- esiden mac ophage and DC popula- ions (7–9). On he o he hand, unde in lamma o y condi ions, monocy es ansmig a e in o inju ed issues, whe e hey hen di ec ly media e an imic obial ac i i y o , depending on he local biochemical milieu, di e en ia e in o in lamma o y mac ophages o monocy e-de i ed DCs (moDCs) (10) (Figu e1). Ci cula ing 3 Sil a e al. E-Selec in Ligands in he Human MPS F on ie s in Immunology | www. on ie sin.o g Janua y 2018 | Volume 8 | A icle 1878 monocy es, hus, unc ion as a sys emic ese oi o issue- esiden myeloid cells (11, 12). The e a e h ee subse s o human monocy es, each o which dis- play di e en unc ional and mig a o y abili ies and can be dis in- guished based on hei exp ession o speci ic chemokine ecep o s, CD14 [ he lipopolysaccha ide (LPS) ecep o ], and CD16 (Fcγ RIII) (13). “Classical” monocy es (CD14++CD16−), which accoun o abou 90% o ci cula ing monocy es in heal hy indi iduals, exp ess high le els o he C-C chemokine ecep o ype 2 (CCR2), display high phagocy ic and myelope oxidase ac i i ies, gene a e eac i e oxygen species, and p oduce in lamma o y cy okines, such as in e leukin (IL)-1β, IL-6, and umo nec osis ac o (TNF)-α (14). On he o he hand, he “non-classical” monocy es (CD14+CD16++) comp ise a popula ion ha exhibi s low phago- cy ic and myelope oxidase ac i i ies (15, 16). Impo an ly, while classical monocy es a e ec ui ed p e e en ially o dis essed issues (17), non-classical monocy es a e ec ui ed o non-in lamed a eas, whe e hey pa ol he mic o ascula u e ia he CX3C chemokine ecep o 1 (CX3CR1) and leukocy e unc ion-associa ed an igen (LFA)-1, moni o ing he luminal su ace o es ing endo helium o signs o issue damage o in ec ion (18–20). In addi ion, non- classical monocy es a e mainly esponsi e o i us-associa ed signals, ia oll-like ecep o s (TLRs) 7 and 8, whe eas classical monocy es espond mos ly o bac e ia-associa ed signals (21). An in e media e subse o monocy es, cha ac e ized as CD14++CD16+, is iewed as being a ansi ional popula ion be ween classical and non-classical monocy e subse s, displaying signi ican p oduc ion o TNF-α and IL-1β, bu low pe oxidase ac i i y (16, 22). While he mig a o y abili y o he in e media e subse is con o e sial, hey exp ess he chemokine ecep o CCR2, a ea u e suppo ing hei abili y o in il a e si es o in lamma ion (23). S ill, o e all, he in e media e monocy e popula ion epo edly displays weake abili y o mig a e ac oss es ing endo helium compa ed o he o he wo monocy ic subse s (24). Dend i ic Cells Dend i ic cells a e he an igen-p esen ing cells pa excellence, showing a unique capaci y o ini ia e immune esponses. These specialized an igen-p esen ing cells cons i u e a unique leukocy e popula ion ha display high mo phological and unc ional he - e ogenei y (25). DCs can be o igina ed om common myeloid o lymphoid p ecu so s and a e di ided in o wo main g oups: con en ional DCs (cDCs) and plasmacy oid DCs (pDCs) (26). A e being eleased in o he bloods eam, hey a e dis ibu ed o lymphoid o gans (lymph nodes, spleen, and hymus) and a ious pe iphe al issues. DC unc ion is in insically ela ed o hei ana omical localiza ion, and, he e o e, a s ingen DC unc ional-ana omical classi ica ion needs o be de ined (Figu e1). A s eady s a e, DCs a e ound o be imma u e (as indica ed by high phagocy ic and endocy ic capaci y and low exp ession o MHC and cos imula o y molecules) and can be classi ied as ei he mig a o y o lymphoid- esiden DCs (27, 28). Mig a o y DCs se e as immune sen inels sc eening pe iphe al issues o signals o dange . They can also cap u e apop o ic cells o sel -an igens in non-in lamed issues and, a e en e ing lymph nodes ia a e en lympha ics, p esen hese o Tcells in he lymph nodes, hus playing a key ole in an igen-media ed pe iphe al ole ance (29–31). On he o he hand, lymphoid- esiden DCs di e en ia e wi hin lymphoid o gans di ec ly om blood DC p ecu so s, and hey unc ion o con inuously su ey blood o lymph (27, 32). Bo h cDC and pDC hema opoie ic p ogeni o s con ibu e o he lymphoid- esiden DC pool, whe eas mos mig a o y DCs a ise om blood cDCs (33). Unde in ec ion o s e ile in lamma o y ci cums ances, bo h ci cula ing cDCs and classical monocy es en e in lamed issues, whe e hey cap u e an igens and di e en ia e in o highly unc ional ma u e DCs. The ma u e DCs mig a e o he lymph nodes ia he a e en lymph, ini ia ing Tcell-media ed immune esponses. In addi ion o cDCs and pDCs, a dis inc subse o DCs a e de i ed om monocy es (known as “moDCs”) which a e consid- e ed o be “in lamma o y DCs”; hese cells p ominen ly p oduce TNF-α, ni ic oxide, and IL-23, and a e po en induce s o TH17 cells (34–37). In e es ingly, al hough pDCs a e belie ed o be absen om pe iphe al issues unde s eady-s a e condi ions, a numbe o ecen publica ions epo ed pDC ex a asa ion in o some in lamed issues, whe e hey sec e e la ge amoun s o ype I in e e on (38–42). In con as o cDCs, pDCs do no en e eac- i e seconda y lymphoid o gans a e a icking om pe iphe al issue ia a e en lympha ics; ins ead, hey appa en ly mig a e di ec ly om he bloods eam ia high endo helial enules (HEVs) by an E-selec in-dependen mechanism (43–47). Mac ophages Mac ophages a e a he e ogeneous and e sa ile popula ion o issue- esiden cells, mos ly o igina ing om sel - enewing emb yo-de i ed p ogeni o s and om blood monocy es ha ha e colonized issues (48, 49). They exis i ually in e e y issue h oughou he body, whe e hey su ey o po en ial signs o in ec ion/dange and pe o m phagocy ic clea ance o dying cells (50). In addi ion, mac ophages play a ole in adap- i e immuni y h ough an igen p esen a ion and p oduc ion o cy okines (51, 52). The e a e wo main mac ophage subse s, he M1 and he M2 mac ophages, wi h dis inc esponses o en i onmen al signals. The M1 subse p oduces high amoun s o p o-in lamma o y cy okines and eac i e oxygen and ni ogen species, hus play- ing a c ucial ole in Th1 pola iza ion and p omo ion o cellula immuni y. M2 mac ophages a e cha ac e ized by hei abili y o s imula e humo al immune esponses, igh ex acellula pa asi e in ec ions, and p omo e issue epai , angiogenesis, and umo p o- g ession (53, 54). Whe eas he majo unc ion o mac ophages is o igh in ec ions and kill a ge cells, hey do no ypically display hema ogenous mig a ion, no lea e si es o issue inju y (11, 55). MPS ex a asa ion Cascade: The Mul is ep Model Rec ui men o ci cula ing cells om blood o in lamed is- sue in ol es a sequen ial and coo dina ed se ies o molecula ac ions media ed by adhesi e in e ac ions be ween ci cula ing sen inels and endo helial cells in pos -capilla y enules (56). He e, we e iew he molecula e ec o s ha egula e he ini ial phagocy e–endo helial binding in e ac ions, which a e essen ial o ansendo helial mig a ion o blood monocy es and DCs o si es o inju y. FigURe 3 | The selec in amily. Selec ins a e a amily o h ee ca bohyd a e- binding p o eins: P-selec in, exp essed on ac i a ed pla ele s and endo helial cells, E-selec in exp essed on ac i a ed endo helial cells, and L-selec in exp essed on leukocy es. The igu e ep esen s he i e domains sha ed by selec ins: C- ype lec in domain, epide mal g ow h ac o -like domain (EGF), a a ying numbe o sho consensus epea s ha ing homology o complemen egula o y p o eins, a ansmemb ane egion, and a cy oplasma ic domain. FigURe 2 | Mul is ep model o ci cula ing blood cell adhesion and mig a ion along he ascula endo helium. Cells make adhesi e con ac s on o he in lamed endo helial su ace h ough engagemen o hei sialo ucosyla ed glycan de e minan s o ascula E-selec in (S ep 1— e he ing and olling). Subsequen engagemen o chemokine ecep o s leads o in eg in ac i a ion (S ep 2) and i m adhesion o leukocy es o endo helium (S ep 3), allowing hei ansmig a ion (S ep 4). 4 Sil a e al. E-Selec in Ligands in he Human MPS F on ie s in Immunology | www. on ie sin.o g Janua y 2018 | Volume 8 | A icle 1878 To ini ia e he ex a asa ion p ocess, ci cula ing phagocy es es ablish low-a ini y and e e sible in e ac ions ( e he ing) on a ge endo helial cells, achie ing low eloci y “ olling” adhesi e in e ac ions (S ep 1, Figu e2). Rolling exposes hese cells o chemokines ha a e immobilized by glycosaminoglycans on he endo helial su ace, and, in u n, acili a es engagemen o G-p o ein-coupled chemokine ecep o s (GPCRs) exp essed on he mononuclea phagocy e cell su ace (S ep 2, Figu e2), wi h esul an G-p o ein-d i en in eg in ac i a ion (3, 57). Ac i a ed in eg ins on phagocy es, p incipally e y la e ac i a ion p o ein 4 and LFA-1, bind o hei espec i e endo helial ecep o s ascula cell adhesion molecule-1 and in e cellula adhesion molecule-1 (ICAM-1), leading o i m adhesion o MPS cells on he endo helium (S ep 3, Figu e2) (3, 57). The binding o ac i a ed in eg ins hen allows diapedesis in o he issue (S ep 4, Figu e2). Two dis inc mechanisms enable diapedesis: (1) ansien disman ling o endo helial junc ions (pa acellula mig a ion) o (2) mig a ion h ough indi idual endo helial cells ( anscellula mig a ion) (58, 59). Al hough se e al cell-associa ed p o eins a e specialized a media ing he i s s ep o cell mig a ion, he selec ins and hei ligands a e he mos po en e ec o s o e he ing and olling adhesi e in e ac ions. These molecules a e esponsible o he ini ial low-a ini y binding in e ac ions o leukocy es on endo helial laye (60), a p ope y ela ed o he unique biophysics o lec in–ca bohyd a e in e ac ions unde luid shea condi ions. SeLeCTiNS AND THeiR gLYCOCONJUgATe LigANDS The Selec in Family The selec ins a e a amily o h ee ca bohyd a e-binding p o- eins ha can be exp essed on endo helial cells, leukocy es and pla ele s (Figu e3). Due o hei equi emen o calcium ions o binding, all h ee selec ins, E-selec in (CD62E), P-selec in (CD62P), and L-selec in (CD62L), belong o he C- ype lec in amily (61). Selec ins sha e a common s uc u e o i e di e - en domains: an N- e minal ca bohyd a e ecogni ion domain (CRD), an epide mal g ow h ac o -like domain (EGF), a a ying numbe o sho consensus epea s ha ha e homol- ogy o complemen egula o y domains (“CRs” o which he e a e 2, 6, and 9 wi hin L-, E-, and P-selec in, espec i ely), a ansmemb ane egion, and a C- e minal cy oplasma ic domain (Figu e 3) (62–64). While he CRD and EGF domains a e highly homologous be ween he h ee selec ins, he s uc u e 5 Sil a e al. E-Selec in Ligands in he Human MPS F on ie s in Immunology | www. on ie sin.o g Janua y 2018 | Volume 8 | A icle 1878 o he ansmemb ane and cy oplasmic po ions, as well as he ex acellula CR domains a e no conse ed ac oss he selec ins, esul ing in s uc u al di e si y and a ying molecula weigh s be ween selec ins (61, 65). Despi e sha ing common elemen s, he h ee selec ins ha e di e en unc ions in di e se pa hological and physiological p ocesses and a y in hei dis ibu ion and binding kine ics. The biology o L-selec in was i s elucida ed by use o an in i o assay in which suspensions o lymphocy es we e o e laid on o lymph node sec ions (66). This assay hen allowed o c ea ion o mAb ha could in e up his binding, such as he mAb known as “MEL-14” desc ibed by Galla in and cowo ke s (67) in 1983, and he ea e led in es iga o s o cloning o his s uc u e (62). L-selec in is highly exp essed on hema opoie ic s em cells and ma u e leukocy es, including all myeloid cells, subse s o na u al kille cells, naï e T and Bcells, and cen al memo y Tcells. When leukocy es a e ac i a ed, cell su ace le els o L-selec in a e down- egula ed by p o eoly ic clea age ia me allop o ease-dependen shedding o he ex acellula domain (61, 68, 69). P-selec in was desc ibed in 1984 by McE e and cowo k- e s (70, 71) and Fu ie and cowo ke s (72) as a glycop o ein exp essed on he cell su ace o ac i a ed pla ele s. P-selec in is cons i u i ely exp essed by ci cula ing pla ele s and endo helial cells, whe e i is s o ed in α-g anules and Weibel–Palade bodies, espec i ely. Because i can be exp essed on endo helial cells, P-selec in oge he wi h E-selec in (desc ibed below) a e known as he “ ascula selec ins.” Following p o-in lamma o y s imulus by molecules such as h ombin o his amine, P-selec in is apidly ansloca ed om he g anules o he cell su ace by usion o in acellula s o age compa men s wi h he plasma memb ane. In mu ine endo helial cells, in lamma o y media o s, such as TNF-α, IL-1β, and LPS, induce P-selec in mRNA ansc ip ion, which equi es he coope a i e binding o he nuclea ac o κ-ligh chain-enhance o ac i a ed Bcells (NF-κB) and ac i a - ing ansc ip ion ac o -2 (ATF-2) o hei esponse elemen s wi hin he P-selec in p omo e (73–75). Howe e , impo an ly, he p omo e o P-selec in in humans and o he p ima es lacks binding si es o NF-κB and ATF-2 (76). Fo his eason, in human endo helial cells, he only ascula selec in inducibly exp essed by TNF-α, LPS, and IL-1β is E-selec in (77). E-selec in was i s epo ed by Be ilacqua and cowo ke s (63, 78) in 1980s as a leukocy e adhesion molecule on ac i- a ed endo helial cells. Skin and bone ma ow mic o essels exp ess E-selec in cons i u i ely (79), howe e , in o he is- sues, endo helial cells do no cons i u i ely exp ess E-selec in bu i s exp ession is s ongly up egula ed by in lamma o y cy okines, such as TNF-α and IL-1β. These cy okines po en ly induce ansien ansc ip ion (wi hin hou s o exposu e) o E-selec in mRNA in bo h human and mouse endo helial cells (80). Cy okine-dependen ac i a ion o E-selec in is media ed by NF-κB binding o egula o y domains in he E-selec in p o- mo e (81). Func ionally, E-selec in slows leukocy e olling o much lowe eloci ies han do ei he L- o P-selec in, a o ing subsequen leukocy e a es (4, 82). This capaci y, along wi h he inabili y o human endo helial cells o up egula e P-selec in in he p esence o IL-1β and TNF-α, is why E-selec in is con- side ed o be he mos impo an selec in o cell a icking o si es o in lamma ion in humans, and i plays a c i ical ole in he ec ui men o immune e ec o s o a ge in lamma o y si es. The Ca bohyd a e e-Selec in Ligands E-selec in ecognizes a ange o s uc u ally di e se glycan epi opes exp essed by human leukocy es ha ypically con ain α(1,3)- ucose (Fuc) and α(2,3)-sialic acid (Sia) modi ica ion(s) on a lac osamine backbone [consis ing o galac ose (Gal) linked o N-ace ylglucosamine (GlcNAc)], as shown in Figu e4 (79). The e minal e asaccha ide known as sialyl Lewis X (sLex— Siaα2-3Galβ1-4(Fucα1-3)GlcNAc) is he p o o ypical E-selec in- binding de e minan (83–85). Some sLex- a ian s uc u es can also exhibi E-selec in binding ac i i y, namely an in e nally ucosyla ed sLex- a ian (VIM-2) and o he polylac osamine s uc u es, in which Fuc modi ica ions occu a mo e han one GlcNAc esidue along he polylac osamine chain ( i- ucosyl-sia- lyl Lewisx and di- ucosyl-sialyl Lewisx) (86–88). In addi ion, o he glycan s uc u es ha a e no na i ely exp essed on leukocy es exhibi E-selec in binding ac i i y, namely he sLex isome , sialyl Lewis a (sLea—Siaα2-3Galβ1-3(Fucα1-4)GlcNAc) (89), some sul a ed de i a i es o Lex and Lea (3′-sul o-Lex and 3′-sul o-Lea, espec i ely) (90, 91), and a ucosyla ed glyco o m o LacdiNac ha displays a e minal N-ace ylgalac osamine (GalNAc) ins ead o Sia (GalNAc-Lewis x) (92). glycosyl ans e ases in ol ed in he Biosyn hesis o Selec in–Ca bohyd a e- Binding De e minan s E-selec in binding de e minan s a e ypically displayed a he end o O-glycans, N-glycans, o glycolipid p ecu so s uc u es, and equi e he coo dina ed and sequen ial ac ion o speci ic glycosyl ans e ases localized wi hin he lumen o he Golgi appa a us. Assembly o sLex is d i en by he e minal addi ion o Sia ( o Gal) and o Fuc ( o GlcNAc) h ough he ac ion o α(2,3)-sialyl ans e ases and α(1,3)- ucosyl ans e ases (FTs), espec i ely, on ype 2 lac osamine (LacNAc) chains (i.e., Gal connec ed o GlcNAc h ough a β(1,4)-linkage) (Figu e4) (93, 94). The sialyla ed o ms o Lewis an igens a e syn hesized by he ac ion o he α(2,3)-sialyl ans e ases (ST3Gal isoenzymes). These enzymes ans e Sia esidues o he Gal on he LacNAc chain, exclusi ely ac ing p io o ucosyla ion (95, 96). The e a e six membe s o he α(2,3)-sialyl ans e ase amily (ST3Gal-I– ST3Gal-VI), bu only ST3Gal-III, ST3Gal-IV, and ST3Gal-VI a e epo ed o sialyla e lac osamine chains (97). Impo an ly, ST3Gal-III exhibi s p e e ence o ype 1 lac osamine chain accep o s (whe ein Gal is connec ed o GlcNAc h ough a β(1,3)- linkage), whe eas ST3Gal-IV and ST3Gal-VI p e e en ially ac on ype 2 polylac osamine chains (98–100). When Type 1 lac osa- mines a e deco a ed wi h Sia in α(2,3)-linkage o Gal and wi h Fuc in β(1,3)-linkage o GlcNAc, his e asaccha ide is known as sialyl Lewis A (sLeA). So a , six human FTs ha e been ound o ca alyze he addi ion o Fuc a α(1,3) linkage o GlcNAc wi h a ype 2 lac osamine—FTIII, FTIV, FTV, FTVI, FTVII, and FTIX. Each enzyme exhibi s speci ici y o accep o subs a es and, FigURe 4 | Schema ic ep esen a ion o biosyn hesis o he E-selec in ligand de e minan s. The α(2,3)-sialyl ans e ases, ST3Gal-III, -IV, and -VI, e mina e he elonga ion o bo h O- and N-glycans by c ea ing sialyla ed ype 2 lac osamine chains. These can be u he ucosyla ed by he ac ion o he speci ic ucosyl ans e ases, yielding di e en Lewis- ela ed s uc u es ha display E-selec in binding ac i i y. 6 Sil a e al. E-Selec in Ligands in he Human MPS F on ie s in Immunology | www. on ie sin.o g Janua y 2018 | Volume 8 | A icle 1878 he e o e, has he abili y o gene a e dis inc ucosyla ed s uc u es (101, 102). Pa icula ly, FTIII and FTV a e unique in ha hey exhibi bo h α(1,3) and α(1,4) FT ac i i y on bo h sialyla ed and unsialyla ed ype 2 and ype 1 lac osamines he eby c ea ing (s)Lex and (s)Lea epi opes, espec i ely (103, 104). On he o he hand, FTIV and FTVI ucosyla e bo h sialyla ed and unsialyla ed ype 2 lac osamine chains, wi h FTIV c ea ing VIM-2 and Lex (105, 106) and, modes ly, sLex de e minan s (107, 108), and FTVI c ea ing hese s uc u es as well as di- ucosyl-sLex (108–110). Uniquely, FTVII can only ac on sialyla ed ype 2 lac osamines, yielding sLex and di/ i- ucosyl-sLex-s uc u es (111, 112), whe eas FTIX is known o syn hesize mos ly Lex (101, 106). Mos o he epo s ha assess he ole o he di e en glyco- syl ans e ases in ol ed in selec in ligand biosyn hesis in leu- kocy es ha e been pe o med using knock-ou mouse models, wi h a small p opo ion o hese s udies using human leukocy es o human hema opoie ic cell lines. Conce ning he ole o he α(2,3)-sialyl ans e ases, mu ine s udies sugges ha ST3Gal-IV and ST3Gal-VI collabo a e oge he in mu ine E-selec in ligand biosyn hesis, wi h ST3Gal-IV ha ing an impo an ole in he egula ion o E-selec in-dependen olling eloci y (113, 114). In e es ingly, ST3Gal-III does no seem o con ibu e o he syn hesis o mu ine E-selec in ligand moie ies, since de iciency o his enzyme did no a ec E-selec in ligand exp ession o ac i i y on mu ine leukocy es (113). Su p isingly, ST3Gal-IV is epo edly he only human α(2,3)-sialyl ans e ase in ol ed in he biosyn hesis o E-selec in ligands in human myeloid leukocy es, since ST3Gal-IV-silenced HL-60 cells (a human p omyelocy ic cell line), and neu ophils de i ed om s able ST3Gal-IV knockdown hema opoie ic s em cells ail o engage in e he ing and olling in e ac ions on E-selec in-bea ing FigURe 5 | Schema ic ep esen a ion o he biosyn he ic pa hways leading o glycop o ein syn hesis: O-linked and N-linked glycosyla ion. The O-glycosyla ion p ocess is cha ac e ized by a s epwise suga addi ion ha occu s in he Golgi appa a us and in ol es a b oad a ay o enzymes. This syn hesis is ini ia ed by one o he N-ace ylgalac osaminyl ans e ase (ppGalNAcTs) amily membe s, o ming he Tn an igen. A e he i s suga [N-ace ylgalac osamine (GalNAc)] addi ion, Tn is ypically elonga ed by Co e 1 β(1,3)galac osyl ans e ase (Co e1GalT o T syn hase, whose Golgi exp ession equi es he ac i i y o i s chape one COSMC), c ea ing he “Co e 1” O-glycan (also known as “T an igen”). Co e 1 is hen u he leng hened by C2GnT-I, which adds an N-ace ylglucosamine (GlcNAc) o he GalNAc, o ming he “Co e 2” O-glycan s uc u e. Al e na i ely, Co e 1 sialyla ion, by ST3Gal-I o ST6GalNAc-II [ o ming sialyl-T (sT) o sialyl-6T (s6T) an igens, espec i ely] s ops Co e 2 o ma ion. In con as o O-glycosyla ion, he N-glycosyla ion p ocess equi es he p oduc ion o an oligosaccha ide p ecu so (GlcNAc2Man5) in he cy oplasmic ace o he endoplasmic e iculum (ER) memb ane. This glycan lipped in he ER lumen and is hen ans e ed en block om a lipid dono o he Asn esidue o a newly syn hesized p o ein wi hin he ER lumen, and hen u he p ocessed in he Golgi compa men . Biosyn hesis o hyb id and complex glycans is ini ia ed by he ac ion o MGAT-I, which adds a GlcNAc esidue o he mannose (Man) p esen on he α(1,3)-a m o he Man5GlcNAc2 s uc u e. Repe i i e addi ions o galac ose (Gal) and GlcNAc by β(1/4)GalT and β(1,3)GnT enzymes, espec i ely, can u he elonga e Co e 2 O-glycan and hyb id- and complex- ype N-glycan s uc u es, c ea ing he lac osaminyl ype 2 chains ha se e as accep o s o e minal sialo ucosyla ion eac ions. 7 Sil a e al. E-Selec in Ligands in he Human MPS F on ie s in Immunology | www. on ie sin.o g Janua y 2018 | Volume 8 | A icle 1878 subs a es (115). In case o α(1,3)-FTs, s udies demons a e ha mos ly FTVII, and o a lesse ex en FTIV, a e he key mu ine α(1,3) FTs ha media e leukocy e selec in ligand biosyn hesis. In ac , mu ine leukocy es lacking FTVII show poo adhesi e con ac s wi h E- and P-selec in, indica ing ha his FT plays a p ominen ole in mu ine E-selec in ligand biosyn hesis (116, 117). Howe e , o he s epo ed ha FTIV is c ucial o slow mu ine leukocy e olling eloci y (118, 119). Impo an ly, E-selec in binding ac i i y con e ed by mu ine FTIV, bu no by FTVII, appa en ly occu s mainly on glycolipids a he han glycop o eins (120). Con e sely, in human leukocy es, he e is e idence ha FTVII, FTIV, and FTIX could each ac in syn hesis o E-selec in ligand de e minan s (121). No ably, he mouse genome encodes only FTIV, FTVII, and FTIX (122), whe eas p ima es possess an addi ional h ee FT gene p oduc s—FTIII, FTV, and FTIV. These addi ional FTs p o ide o a much wide capaci y o c ea e sLeX; in addi ion, he exp ession o FTIII and FTV in p ima es uniquely d i es c ea ion o sLeA de e minan s. Human ci cula ing monocy es exp ess all he α(1,3)-FTs, wi h he excep ion o FTV, heigh ening he po en ial o a iabili y in glycoconjuga es bea ing sLex among human and mouse cells (123). No ably, sLeA is no exp essed on any p ima e leukocy es as hese cells do no syn hesize Type 1 lac osamines (3). O he glycosyl ans e ases in ol ed in he biosyn hesis o sLex ha e also been s udied o hei ele ance in gene a - ing unc ional selec in ligands (Figu e 5). Rega ding sLex p esen a ion on O-glycans, one s udy epo ed ha leukocy es om mice de icien in he enzyme equi ed o ini ia ion o O-glycosyla ion, ppGalNAcT-1, showed impai ed ec ui men du ing in lamma ion due o a signi ican educ ion in E- and 8 Sil a e al. E-Selec in Ligands in he Human MPS F on ie s in Immunology | www. on ie sin.o g Janua y 2018 | Volume 8 | A icle 1878 P-selec in ligand le els (124). Mice lacking he O-glycan co e 1 β3galac osyl ans e ase (C1GalT-I) showed d ama ic loss o leukocy e olling on E-selec in and, consequen ly, hese leukocy es did no ansmig a e in o in lamed issues (125). T ansgenic mouse s udies, whe e he O-glycan co e 2 β6-N- ace ylglucosaminyl ans e ase-I (C2GnT-I) was knocked ou , also showed educed E-selec in and P-selec in binding ac i - i y o leukocy es unde s a ic and shea -based olling assays, wi h impai ed leukocy e ec ui men o si es o in lamma ion (126–128). In ag eemen , in human moDCs, he down egula- ion o C2GnT-I, wi h concu en up egula ion o ST3Gal-I and GalNAc α(2,6)sialyl ans e ase (ST6GalNAc)-II, esul s in a loss o he co e 2 s uc u es equi ed o O-glycan display o sLex (Figu e5) (129). Fu he mo e, s udies using HL-60 cells ha e e ealed ha he ST6GalNAc-II o e exp ession ab oga es sLex cell su ace display and educes he numbe o adhe en cells o E-selec in unde low condi ions, ein o cing he no ion ha he e exis s a compe i ion be ween ST6GalNAc-II and C2GnT-I o co e 1 accep o s, a ec ing he biosyn hesis o sLex-bea ing co e 2-O-glycan s uc u es (Figu e 5) (130). In e es ingly, in mice, knockou o one o he β(1,4)galac osyl ans e ases (o he amily o i e isoenzymes) in ol ed in Type 2 lac osamine syn hesis, β(1,4)galac osyl ans e ase-I (β(1,4)GalT-I), showed educed in lamma o y esponses and impai ed P-selec in binding ac i i y; howe e , he con ibu ion o his enzyme in he syn hesis o E-selec in coun e - ecep o s emains o be elucida ed (131). One s udy has ecen ly e alua ed he con ibu ions o N-glycans, O-glycans and glycosphingolipids (GSLs) o E-selec in binding by human myeloid cells unde physiological low condi ions (132). To add ess his issue, O-glycan and GSL syn hesis was abolished by, espec i ely, knocking-ou he co e 1 Gal ans e ase chape - one, i.e., he C1GalT-I-speci ic Molecula Chape one (COSMC), β1,2 GlcNAc- ans e ase (MGAT-I), and UDP-glucose ce amide glucosyl ans e ase (UGCG). No ably, hese s udies indica e ha while O-glycans a e indispensable o myeloid cell binding o L- and P-selec ins, N-glycans play he majo ole in he ini ial myeloid cell ec ui men in o E-selec in-bea ing subs a es, wi h O-glycans playing a mo e modes ole. In addi ion, bo h glycolip- ids and N-glycans a e esponsible o he slowing down o olling eloci ies ha p ecede i m a es (132). Mos s udies ha ha e assessed biologic modula o s o E-selec in ligands in leukocy es ha e been pe o med using human and mu ine T cells. An a ay o cy okines has been shown o egula e E-selec in ligand exp ession ia up egula ion o down egula ion o speci ic glycosyl as e ases ha con ol sLex exp ession. Speci ically, IL-2, IL-7, IL-15, and IL-12 inc ease he exp ession o glycosyl ans e ases in ol ed in he biosyn hesis o E-selec in ligand de e minan s, whe eas IL-4 has he oppo- si e e ec (133). This selec in ligand up egula ion in Tcells in esponse o cy okine signaling was shown o be dependen on Th1 ansc ip ion ac o T-be (134) and on STAT4-media ed pa hways (135). In e es ingly, human myeloid cells ea ed wi h g anulocy e-colony s imula ing ac o (G-CSF) show inc eased cell su ace exp ession o E-selec in ligands associa ed wi h sig- ni ican inc eases in gene exp ession o he glycosyl ans e ases ST3Gal-IV, FTIV, and FTVII (136). e-SeLeCTiN LigAND ACTi iTY DiSPLAYeD BY CiRCULATiNg MPS SUBSeTS Among he cells o MPS, human ci cula ing monocy es and, o a lesse ex en , human blood cDCs and moDCs a e he mos com- p ehensi ely analyzed g oup in e ms o E-selec in ligand ac i - i y. In ou s udies, human classical monocy es (CD14++CD16−) showed signi ican ly highe le els o sLex de e minan s as compa ed o in e media e monocy es (CD14++CD16+), whe eas non-classical monocy es (CD14+CD16++) we e almos de oid o sLex exp ession (123). Ano he s udy compa ed he a icking capaci y o human monocy e subse s by analyz- ing hei abili y o bind o ac i a ed endo helial monolaye s, and commensu a ely, classical monocy es showed no iceably highe capabili y o adhe ing o eac i e endo helium han did non-classical/in e media e monocy es (137). In ag eemen wi h human s udies, mu ine classical monocy es (Ly-6Chi) exhibi g ea e binding o E-selec in unde low condi ions and exp ess highe le els o he sca olds ha bea sLex de e minan s compa ed o non-classical monocy es (Ly-6Clo) (138, 139). This di e en ial pa e n o E-selec in ligand display is in ag eemen wi h he speci ic mig a o y equi emen s among he monocy e subse s: classical monocy es a e ypically ec ui ed o in lamed lesions (138, 139), whe eas non-classical monocy es mig a e o non-in lamed endo helium (14) upon which hey pa ol heal hy issues in a LFA-1-dependen manne (19). Indeed, al hough he i s obse a ions o non-classical monocy es we e made in non-in lamed skin blood essels (19), hese cells we e u he desc ibed in he mic o ascula u e o kidney unde s eady-s a e condi ions (20). The pa olling p o ile ha hese cells exhibi is independen o he ac i a ion s a e o he endo helium, since non-classical monocy es cons i u i ely sca enge he luminal side o non- eac i e endo helium (18). The e o e, hei abili y o bind o endo helium seems o be independen o E-selec in ecep o / ligand in e ac ions, bu , ins ead, appea s c i ically egula ed by LFA-1 exp ession and i s in e ac ion wi h endo helial ICAM (19, 20). No ably, al hough selec ins play a majo ole in he ini ial adhesi e con ac s wi h endo helium su aces, in eg ins can also suppo e he ing and olling e en s unde low condi ions, albei wi h less po ency han do selec ins (140, 141). Mul iple adhesion molecules a e in ol ed in monocy e a achmen o endo helium. While E-selec in ecep o /ligand in e ac ions p ominen ly media e S ep 1 e en s in ansmig a ion o all leukocy es, L-selec in-dependen binding in e ac ion ha e also been obse ed o po en ly media e human pe iphe al blood monocy e binding o ac i a ed ascula endo helium unde shea s ess (142–144). Thus, e en hough he majo i y o he epo s indica es ha ini ial monocy e adhesion o ac i a ed endo helial cells is mos c i ically dependen on E-selec in ecep o /ligand in e ac ions (123, 145–150), dis inc in e ac ions we e also epo ed by o he au ho s. The di e ences ha e o do wi h di - e ences in he leukocy e popula ions unde s udy, a ia ions in he assay condi ions employed (i.e., shea s ess le els employed, o a o y shea e sus luid shea condi ions, empe a u e, e c.), di e ences in he adhesion me ics (i.e., numbe o adhe ed cells, numbe o olling cells, olling eloci y measu emen s, 9 Sil a e al. E-Selec in Ligands in he Human MPS F on ie s in Immunology | www. on ie sin.o g Janua y 2018 | Volume 8 | A icle 1878 e c) al oge he compounded by he inna e biologic di e ences be ween mice and human cells, could al e na i ely emphasize he con ibu ion(s) o o he adhesion molecules. Conce ning DCs, human blood cDCs exp ess high le els o sLex de e minan s, which allow hem o e he and oll on E-selec in unde low condi ions (151, 152). Impo an ly, in in i o in a i al mic oscopy s udies, human blood cDCs adop i ely ans e ed in o mice we e obse ed o oll along es ing mu ine skin endo helium and ex a asa e a si es o in lamma ion (151). No ably, human moDCs signi ican ly exp ess sLex, especially on O-glycan s uc u es. Upon ma u a ion o moDCs wi h he TLR4 ligand, LPS, sLex exp ession is down egula ed due o dec eased C2GnT-I exp ession and up egula ion o ST6GalNAc-II and ST3Gal-I (129). Biologically, hese obse a ions sugges ha sLex is less ele an o ansendo helial mig a ion o TLR4- induced-ma u e moDCs, o ha ma u a ion is a s ep ha ollows ansendo helial mig a ion. By con as , IFN-γ-induced ma u a- ion o moDCs leads o an up egula ion o C2GnT-I, esul ing in inc eased exp ession o co e 2 O-glycan subs a es o sLex deco a ion (153). These ea u es sugges ha sLex-bea ing co e 1-de i ed (o co e 2) O-glycans a e equi ed o human moDC mig a ion and a e modula ed acco ding o speci ic ma u a ion s imuli. Human pDCs also exp ess sLex, allowing hei ec ui - men o some in lamed issues (38, 39, 154) and o eac i e lymph nodes (43), a p ocess belie ed o be media ed by he exp ession o E-selec in in HEVs (45, 47). gLYCOCONJUgATe STRUCTUReS THAT DiSPLAY e-SeLeCTiN LigAND DeTeRMiNANTS iN CiRCULATiNg MPS CeLLS Se e al di e se and s uc u ally singula glycos uc u es wi h E-selec in binding ac i i y ha e been iden i ied on human classi- cal monocy es o human blood DCs. Human classical monocy es g ea ly display sLex deco a ions on an a ay o p o ein sca olds, consis ing o P-selec in glycop o ein ligand-1 (PSGL-1), CD43 and CD44, and GSLs (123), whe eas human ci cula ing DCs appea o display sLex solely on PSGL-1 (129, 155). Cu aneous Lymphocy e An igen The cu aneous lymphocy e an igen (CLA) is he E-selec in- eac i e glyco o m o PSGL-1. PSGL-1 is a ansmemb ane 240-kDa homodime ic, mucin-like glycop o ein exp essed on leukocy es (and, epo edly, on some ac i a ed endo helial cells) ha plays a c ucial ole in he homing o leukocy es in o in lamed issue (156, 157). E-selec in binding ac i i y o PSGL-1 is con e ed by sialyla ed and ucosyla ed co e 2-based-O-glycans ha a e clus e -dis ibu ed along he s alk egion o he PSGL-1 ex acellula domain (158). A numbe o s udies ha e iden i ied PSGL-1 as one o he se e al sca olds exp essed by human clas- sical monocy es displaying E-selec in-binding ac i i y (123, 155). On he o he hand, PSGL-1 is he only known sca old ha p e- sen s sLex de e minan s on human ci cula ing cDCs (151, 155) and moDCs (129). Ye , Sil a e al. obse ed ha al hough PSGL-1 is essen ial o P- and L-selec in ecogni ion by human moDCs unde luid shea condi ions, i is no manda o y o e he ing o E-selec in (153). Mo eo e , he e a e epo s ha ex a asa ion o mu ine imma u e DC o in lamed issues equi es bo h E- and P-selec in, bu no PSGL-1 (159). Toge he , hese da a sugges he exp ession o ligands o E-selec in in addi ion o CLA by human and mu ine DCs. S ill, PSGL-1 is he dominan ligand o P- and L- selec in and is he only known glycop o ein ha binds all h ee selec ins (160, 161). Acco dingly, ci cula ing monocy es ha ha e al eady bound o E-selec in on in lamed endo helium can also in e ac wi h L-selec in exp essed by o he ci cula ing monocy es/DCs ia PSGL-1 and suppo hei seconda y cap- u e, po en ia ing mononuclea phagocy e ec ui men o si es o in lamma ion (162). Hema opoie ic Cell e- and L-Selec in Ligand HCELL is a sialo ucosyla ed glyco o m o CD44 ha exhibi s po en E-selec in (and L-selec in) binding ac i i y. CD44 is a ansmemb ane p o ein ha exis s in a wide a ie y o p o ein iso o ms due o al e na i e splicing and ex ensi e pos - ansla- ional modi ica ions (wi h molecula weigh anging om 80 o 220kDa). CD44 is exp essed by mos mammalian cells, whe e i se es as he p incipal ecep o o hyalo onic acid and pa ici- pa es in a b oad ange o cellula ac i i ies, including lymphocy e ac i a ion, leukocy e a icking, hema opoiesis, cell g ow h and su i al, and umo dissemina ion (163). Pos - ansla ional modi ica ions along wi h ex ensi e al e na i e splicing allow he o ma ion o mul iple p o ein iso o ms, exp essed in a issue-speci ic manne (164, 165). The s anda d p o ein iso o m o CD44 (CD44s o CD44H) is encoded by mRNA ansc ip s comp ising exons 1–5 and 16–20 (“s1–s5 and s6–s10”). CD44s is ubiqui ously exp essed by mammalian cells and is he o m mos o en displayed by hema opoie ic-lineage cells. In addi ion o CD44s, non-hema opoie ic cells cha ac e is ically display CD44 a ian iso o ms con ain pep ide p oduc s o a ian exons (exons “ 2– 10”) in addi ion o he s anda d exon pep ide p oduc s. CD44 pos - ansla ional modi ica ions include he addi ion o di e en glycan s uc u es, namely glycosaminoglycans, and N- and O-glycan subs i u ions (166). While p e ious s udies indica ed ha HCELL was only exp essed by human hema - opoie ic s em and p ogeni o cells (HSPCs) (167, 168), and some hema ologic (167, 169) and solid malignancies (170), HCELL was ecen ly epo ed o be exp essed by classical human monocy es (123). Impo an ly, o human HSPCs, he sLex de e minan is exclusi ely displayed on N-glycan lac osamines on CD44s, bu classical monocy es exp ess sLex on O-glycans o CD44s (123). Because o i s abili y o engage E- and L-selec in unde ela i ely high luid shea condi ions (i.e., in excess o 20dynes/cm2 shea s ess), HCELL is conside ed he mos po en L- and E-selec in ligand exp essed on mammalian cells (167, 171). CD43e CD43, also known as sialopho in o leukosialin, is a cell su ace glycop o ein exp essed by nea ly all hema opoie ic cells and is in ol ed in se e al impo an p ocesses, including cell de elop- men , ac i a ion, su i al, and mig a ion (172–176). Glycosyla ion 16 Sil a e al. E-Selec in Ligands in he Human MPS F on ie s in Immunology | www. on ie sin.o g Janua y 2018 | Volume 8 | A icle 1878 178. Me zaban JS, Bu dick MM, Gadhoum SZ, Dagia NM, Chu JT, Fuhlb igge RC, e  al. Analysis o glycop o ein E-selec in ligands on human and mouse ma ow cells en iched o hema opoie ic s em/p ogeni o cells. Blood (2011) 118(7):1774–83. doi:10.1182/blood-2010-11-320705 179. Tiemeye M, Swiedle SJ, Ishiha a M, Mo eland M, Schweing ube H, Hi ze P, e al. 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Glycobiology (2015) 25(12):1392–409. doi:10.1093/glycob/cw 046 Con lic o In e es S a emen : Acco ding o he Na ional Ins i u es o Heal h pol- icies and p ocedu es, he B igham and Women’s Hospi al has assigned in ellec ual p ope y igh s ega ding HCELL and GPS o he in en o (RS), who may bene i inancially i he echnology is licensed. RS’s owne ship in e es s we e e iewed and a e managed by he B igham and Women’s Hospi al and Pa ne s Heal hCa e in acco dance wi h hei con lic o in e es policy. All o he au ho s decla e no compe ing inancial in e es s. Copy igh © 2018 Sil a, Videi a and Sacks ein. This is an open-access a icle dis- ibu ed unde he e ms o he C ea i e Commons A ibu ion License (CC BY). The use, dis ibu ion o ep oduc ion in o he o ums is pe mi ed, p o ided he o iginal au ho (s) o licenso a e c edi ed and ha he o iginal publica ion in his jou nal is ci ed, in acco dance wi h accep ed academic p ac ice. 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