Glycan modification of antigen alters its intracellular routing in dendritic cells, promoting priming of T cells
The induction of T cell immunity to viruses or tumors involves the presentation of viral or tumor antigens by antigen-presenting cells ( APC ) in the context of major histocompatibility complex ( MHC ) molecules . Loading of exogenously-derived antigens onto MHC class I molecules , a process known as cross-presentation ( Carbone and Bevan , 1990 ) is required to activate antigen-specific CD8+ T cells . Furthermore , cognate CD4+ T cell help is important for licensing the APC , which is essential for effective CD8+ T cell priming and induction of long-lasting memory ( Bennett et al . , 1997; Schoenberger et al . , 1998 ) . The molecular mechanisms underlying cross-presentation have been studied intensively , however , little is still known on the nature of the antigens and stimuli that are required for APC to route exogenous antigens efficiently into the MHC class I presentation pathway . Most exogenous antigens that originate from tumor cells or viruses are glycosylated in their native form ( Apweiler et al . , 1999 ) . The relative composition of the glycosylation machinery , which includes all the necessary glycosylation-related enzymes and co-factors , determines the final configuration of the glycan structures that decorate N- or O-linked glycosylation sites present in glycoproteins . The glycosylation machinery can be affected by multiple physio-pathological cues , including proliferation , activation , and the transformation status of the cell ( Ohtsubo and Marth , 2006 ) , and depends on environmental factors . APC , such as dendritic cells ( DCs ) and macrophages are able to sense glycans exposed on either self- or pathogen-derived antigens via glycan-binding proteins . Amongst these glycan-binding proteins are C-type lectin receptors ( CLRs ) that recognize defined carbohydrate-structures through their carbohydrate recognition domain ( CRD ) . Depending on the amino acid sequence , the CRD bears specificity for mannose , fucose , galactose , sialylated- , or sulfated structures . CLRs function both as antigen uptake and/or signaling receptors that modify DC-induced cytokine responses thereby influencing T cell differentiation ( van and Rabinovich , 2008 ) . The specialized internalization motifs in the cytoplasmic domains of CLRs allow the rapid internalization of antigens upon interaction ( Engering et al . , 2002; Herre et al . , 2004 ) . This suggests that DCs use CLRs to 'sense' the natural glycan composition of tissues and invading pathogens and , in response to this recognition , are able to modulate immune responses ( Geijtenbeek and Gringhuis , 2009 ) . The conjugation of antigens to CLRs-specific antibodies , such as DEC205 , mannose receptor ( MR ) , Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin ( DC-SIGN ) or CLEC9A , has proven to be an effective way to direct antigens to DCs , resulting in enhanced antigen uptake and presentation on MHC molecules ( Bonifaz et al . , 2004; Hawiger et al . , 2001; Caminschi et al . , 2008; Chatterjee et al . , 2012 ) . Also glycans specific for CLRs have shown their targeting specificity and potential to improve antigen uptake and presentation in MHC class I and II molecules when coupled to antigen formulations ( Singh et al . , 2009a; Garcia-Vallejo et al . , 2013; Unger et al . , 2012; Unger , 2014; Aarnoudse et al . , 2008; Yang et al . , 2008; Singh et al . , 2011 ) . To achieve immunity rather than tolerance inclusion of a strong adjuvant is necessary . Little is known on how naturally glycosylated antigens or alterations in the glycosylation of antigens may change and re-direct antigen internalization via CLRs , in addition to subsequent processing and presentation . MR was shown to mediate cross-presentation of the model-antigen ovalbumin ( OVA ) in a Toll-like receptor ( TLR ) -dependent manner and to recruit TAP-1 to endocytic organelles ( Burgdorf et al . , 2007; Burgdorf et al . , 2008 ) . Importantly , cross-presentation of OVA was only effective using high amounts of antigen ( Burgdorf et al . , 2007; Burgdorf et al . , 2008 ) . The interaction of OVA with MR , which has specificity for mannose ( Taylor et al . , 1992 ) , was speculated to be dependent on the presence of mannose glycans on OVA ( Burgdorf et al . , 2007 ) . In the current study , we investigated the effect of modifying the glycan composition of OVA on the efficacy of cross-presentation , priming and differentiation of T cells . We hypothesized that modification of the antigen with specific glycans would re-direct antigens to other CLRs resulting in altered intracellular routing and presentation of antigen and Th differentiation . We have chosen to conjugate the carbohydrate structure LewisX ( LeX ) to 2 free cysteine residues within the native OVA glycoprotein . LeX is a ligand of the murine C-type lectin macrophage galactose-type lectin ( MGL ) -1 ( Singh et al . , 2009b ) , which is expressed on murine plasmacytoid DC , CD8+ and CD8- splenic DC , DC in the small intestines , the sub-capsular and intra-follicular sinuses of T cell areas in lymph nodes , and on DC and macrophages in the dermis of skin ( Dupasquier et al . , 2006; Tsuiji et al . , 2002; Denda-Nagai et al . , 2010; Segura et al . , 2010 ) . Murine MGL1 is one of two homologues ( MGL1 and MGL2 ) of human MGL ( huMGL ) , which has been shown to interact with tumor cells through glycans exposed on MUC1 as well as with various pathogens ( van Vliet et al . , 2008 ) . However , MGL1 and MGL2 bind different glycan structures: while MGL1 binds LeX and Lea glycans , MGL2 binds N-acetylgalactosamine ( GalNAc ) and galactose glycan structures ( Singh et al . , 2009b ) . The YENF motif in the cytoplasmic tail of huMGL is essential for uptake of soluble antigens , which are subsequently presented to CD4+ T cells ( van Vliet et al . , 2007 ) . Murine MGL1 contains a similar motif ( YENL ) in its cytoplasmic tail , which likely plays a similar and important role in antigen uptake ( Yuita et al . , 2005 ) . Glycan binding to the CLRs DC-SIGN , Dectin-1 and Dectin-2 has been demonstrated to trigger the signaling capacity of these receptors , modulating DC-mediated T helper cell ( Th ) differentiation and cytokine production by DCs ( Geijtenbeek and Gringhuis , 2009 ) . This illustrates that glycan epitopes may not only improve antigen presentation , but may also affect Th differentiation and shape specific adaptive immune responses . This is underlined by the observation that DC-SIGN signaling induced upon sensing of specific mannose or fucose structures on pathogens differentially directed Th differentiation ( Gringhuis et al . , 2014 ) . Here , we demonstrate that conjugation of the LeX carbohydrate to the model antigen OVA alters its routing from a MR- and TAP1-dependent cross-presentation pathway into a TAP1- and Cathepsin-S-independent pathway , devoid of any TLR-signaling dependence , which simultaneously enhanced CD8+ T cell priming and Th1 skewing of CD4+ T cells . Cross-presentation was associated with prolonged intracellular storage of antigen in Rab11+LAMP1+ vesicles . Our results illustrate that small changes in the glycosylation profile of protein antigens can have a great impact on antigen routing within DCs , affecting cross-presentation and Th cell differentiation .
The well-known model antigen OVA carries one N-glycosylation site at N293 and , although the variety of OVA glycans have been previously described , their relative abundance has never been characterized in detail . We determined the glycan profile of OVA by normal-phase HPLC coupled with electrospray ionization mass spectrometry with an intercalated fluorescence detector . We found that the majority of glycan species on OVA corresponded to the complex-type ( 54 . 9% ) , while mannose-rich glycans ( mainly Man5 and Man6 , potential ligands of the MR ( Taylor and Drickamer , 1993 ) ) represented only 23% of all glycoforms ( Figure 1A ) . The remaining glycoforms were of the hybrid ( 16 . 2% ) and oligomannose ( Man3 , 3% ) type . The presence of mannose rich glycans on OVA correlate with the earlier reports that in particular MR interacted with OVA ( Burgdorf et al . , 2006 ) and mediated the TLR dependent and TAP-1 dependent cross-presentation of OVA ( Burgdorf et al . , 2008 ) . We reasoned that the conjugation of additional glycans to OVA would alter its CLR-dependent effects . To do this , we conjugated the tri-saccharide glycan structure LeX ( Galβ1–4 ( Fucα1–3 ) GlcNAc ) to the free cysteins of OVA using standard thiol-maleimide chemistry via the bifunctional crosslinker MPBH . LeX is a well characterized carbohydrate ligand of the C-type lectin MGL1 ( Hawiger et al . , 2001; Singh et al . , 2009b ) . Using mass spectrometry ( MALDI-TOF/TOF ) , we confirmed that OVA-LeX increases to 1 . 2 KDa in mass , indicating that at least 40% of the total OVA-LeX preparation contained the LeX ( Figure 1—figure supplement 1 ) . This increase corresponded to the addition of two LeX molecules per OVA molecule . 10 . 7554/eLife . 11765 . 003Figure 1 . Generation of OVA-neo-glycoconjugates with LeX that confers binding of OVA to MGL1 . ( A ) A glycan profile of OVA was generated using a multidimensional normal phase nano-HPLC coupled with an electrospray ionization interface mass spectrometer with an intercalated nanofluorescence detector . The different glycan species , indicated by numbers , are shown on the right; their relative proportion is represented in a pie chart . ( B ) ELISA showing functional modification of OVA with LeX glycans , as detected with anti-LeX antibodies and resulting in binding of MGL1-Fc . Unconjugated OVA does not carry any ligands for MGL1 . Modification of OVA with LeX did not alter the ability to bind to MR as illustrated by equal binding kinetics of MR-Fc to native OVA and OVA-LeX . OVA and OVA-LeX preparations contain similar amounts of OVA as detected with anti-OVA antibodies . DOI: http://dx . doi . org/10 . 7554/eLife . 11765 . 00310 . 7554/eLife . 11765 . 004Figure 1—figure supplement 1 . The MALDI-TOF/TOF mass spectrum of OVA-LeX ( Red ) shows an increase of 1 , 2 KDa compared to unconjugated OVA ( Blue ) , corresponding to addition of two LeX molecules per OVA molecule . DOI: http://dx . doi . org/10 . 7554/eLife . 11765 . 004 Furthermore , using anti-LeX antibodies the presence of LeX was detected on OVA-LeX , whereas absent on native glycosylated OVA , indicating that the glycan was successfully conjugated to OVA ( Figure 1B ) . While native OVA interacted with the MR ( MR-Fc , Figure 1B ) and as earlier described ( Burgdorf et al . , 2007 ) , the conjugation of LeX to OVA conferred high-avidity binding to MGL1 as revealed using the soluble recombinant form of MGL1 ( MGL1-Fc , Figure 1B ) . Moreover , the addition of LeX to OVA did not result in increased binding to the MR binding , indicating that LeX selectively binds MGL1 . Similar binding with an anti-OVA antibody was determined , which indicates that similar protein concentrations in the preparations of OVA and OVA-LeX used in our study are present ( Figure 1B , right panel ) . In conclusion , through glycan profiling we identified 23% mannose-rich glycans on native OVA that may facilitate MR binding . Addition of the glycan LeX , next to these mannose glycans , on cysteine residues ( two LeX moieties per molecule ) , conveyed MGL1 specificity to OVA . We explored the potency of OVA-LeX to enhance T cell priming in vivo . Since DC are crucial for priming of naive T cells , we first established the presence of MGL on both splenic and bone marrow-derived DC ( spDCs and BM-DCs ) of C57BL/6 mice by staining with the anti-MGL antibody ER-MP23 ( Figure 2A ) . Since the ER-MP23 antibody does not discriminate between the two murine MGL homologues MGL1 and MGL2 , MGL1 expression in wild type ( WT ) CD11c+ DCs was confirmed using qRT-PCR ( Figure 2B ) . Immunization of mice with OVA-LeX mixed with agonistic anti-CD40 antibodies resulted in the enhanced priming of OVA-specific CD8+ T cells as revealed from both higher numbers of OVA/H-2Kb tetramer binding CD8+ T cells and OVA-specific IFN-γ and TNF-producing CD8+ T cells than obtained with native OVA ( Figure 2C , Figure 2—figure supplement 1 ) . Using MGL1KO mice , that lack MGL1 expression on DC ( Figure 2B ) , we ascertained that MGL1 is the prime lectin involved in boosting the generation of antigen-specific effector T cells as upon immunization of these mice with LeX-conjugated OVA no enhanced frequencies of OVA-specific IFN-γ and TNFα-double-producing T cells were detected ( Figure 2D , Figure 2—figure supplement 1 ) . In fact , the OVA-LeX immunized MGL1 KO mice displayed comparable numbers of effector T cells as WT mice immunized with native OVA . Together , these data show that the conjugation of two LeX glycans on OVA re-directs OVA to the CLR MGL1 and thereby enhances CD8+ T cell priming in vivo . 10 . 7554/eLife . 11765 . 005Figure 2 . Immunization with OVA-LeX induces increased CD8+ T cell responses in vivo . ( A ) Expression of murine MGL on BM-DCs and CD11c+ spDCs was analyzed by flow cytometry . ( B ) MGL1 mRNA expression by BM-DC and splenic DC from WT and MGL1 KO mice was determined using qRT-PCR . GAPDH was used as a reference gene and the results are representative of three independent experiments . C57BL/6 mice were immunized s . c . with either OVA-LeX or native OVA mixed with anti-CD40 using a prime-boost protocol . Spleens were analyzed by flow cytometry to determine the frequency of ( C ) H2-Kb/SIINFEKL-tetramer-binding CD8+ T cells and IFN-γ or TNF production by activated CD8+ T cells was determined by intracellular staining after OVA-specific re-stimulation ex vivo . Dots represent individual mice ( n=4–5 mice/group; **p<0 . 01 ) . Bars indicate median of each group . Graphs shown are representative of two independent experiments . ( D ) C57BL/6 and MGL1 KO mice were prime-boosted with either OVA-LeX or native OVA mixed with anti-CD40 . Frequencies of IFN-γ and TNF-double-producing CD8+ T cells were determined by intracellular staining after OVA-specific re-stimulation of splenocytes ex vivo . Dots represent individual mice ( n=4–5 mice/group; *p<0 . 05 ***p<0 . 001 ) . Bars indicate median of each group . Data are representative of 2 independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 11765 . 00510 . 7554/eLife . 11765 . 006Figure 2—figure supplement 1 . Representative flow cytometry plots of ( A ) IFN-γ and ( B ) TNF- producing CD8+ T cells in spleens of C57BL/6 mice that were immunized with either OVA-LeX or native OVA mixed with anti-CD40 using a prime-boost protocol; numbers above the gates designate the percentage of IFN-γ+ or TNF+ CD8+ T cells . DOI: http://dx . doi . org/10 . 7554/eLife . 11765 . 00610 . 7554/eLife . 11765 . 007Figure 2—figure supplement 2 . C57BL/6 and MGL1 KO mice were prime-boosted with either OVA-LeX or native OVA mixed with anti-CD40 . Frequencies of IFN-γ and TNF-double-producing CD8+ T cells were determined by intracellular staining after re-stimulation of splenocytes ex vivo . Representative facs plots of indicated mice are shown; numbers designate the percentage of IFN-γ and TNF-double positive CD8+ T cells . DOI: http://dx . doi . org/10 . 7554/eLife . 11765 . 007 Since we observed that LeX-modified OVA increased priming of antigen-specific CD8+ T cells we examined whether this also enhanced antigen-presentation to CD4+ T cells . Both OVA-LeX-loaded and native OVA-loaded spDCs induced CD4+ OT-II T cell proliferation to a similar extent ( Figure 3A ) , illustrating that the altered antigen uptake mediated by LeX did not affect loading on MHC class II molecules . Similar results were obtained using BM-DCs ( Figure 3A ) . Although we did not observe any differential effect of LeX on CD4+ T cell expansion , neoglycosylation of antigens could induce signaling via CLRs and herewith potentially influence Th cell differentiation ( Gringhuis et al . , 2014 ) . We therefore investigated whether OVA-LeX affected the differentiation of naive CD4+ T cells . Hereto BM-DCs and spDCs of C57BL/6 mice were pulsed with OVA-LeX and subsequently co-cultured with naive CD4+CD62Lhi OT-II cells . Co-cultures containing OVA-LeX loaded BM-DCs or spDCs contained significantly more IFN-γ-producing T cells than those containing OVA-loaded DCs ( Figure 3B ) . Neither induction of IL-4- nor IL-17A-producing CD4+ T cells was observed ( Figure 3B , upper and middle panel and data not shown ) . In addition , induction of Foxp3+ T cells was not detected ( data not shown ) . To exclude that the Th1 skewing by OVA-LeX loaded DCs was attributed to the more Th1 prone status of C57BL/6 ( Gervais et al . , 1984 ) , we also performed the Th-differentiation assay with cells derived from Th2 prone BALB/c mice ( Hsieh et al . , 1995 ) . We observed that naive OVA-specific CD4+ T cells from DO11 . 10 Tg mice that were stimulated with OVA-loaded BM-DCs differentiated into IL-4 secreting T cells ( Figure 3B , lower panels ) . However , the generation of IL-4-producing T cells was not influenced by loading DCs with OVA-LeX as these cultures contained comparable percentages of IL-4-producing DO11 . 10 T cells . Using these Th2-prone T cells , OVA-LeX-pulsed DCs still induced considerably more IFN-γ-producing CD4+ T cells than native OVA-pulsed DCs ( Figure 3B , lower panel ) . Since this assay takes three days longer than the antigen-presentation assay , it is possible that the higher frequency of IFN-γ-producing CD4+ T cells is due to increased division of OVA-specific CD4+ T cells . However we found that the amount of proliferation of OVA-specific CD4+ T cells induced by stimulation with OVA-LeX-loaded DCs after 6 days is similar to that induced by OVA-loaded DCs ( Figure 3—figure supplement 1 ) . The augmented induction of CD4+ Th1 cells was also observed in vivo as revealed from the higher frequencies of IFN-γ-producing OVA-specific CD4+ T cells in the spleens of OVA-LeX immunized mice than in mice immunized with native OVA ( Figure 3C , Figure 3—figure supplement 2 ) . These data indicate that the increased numbers of Th1 cells induced by OVA-LeX-loaded DC are not due to increased proliferation of OT-II cells , but probably due to MGL1-mediated signaling . 10 . 7554/eLife . 11765 . 008Figure 3 . Modification of OVA with LeX structures skews naive CD4+ T cells towards the Th1-effector lineage . ( A ) Pulsing of CD11c+ spDCs or BM-DCs with OVA-LeX results in equal OT-II proliferation as native OVA . Expansion of OVA-specific T cells was determined using 3H-thymidine incorporation . Data are shown as mean ± SD of triplicate cultures , representative of three independent experiments . ( B ) Flow cytometric analysis of OT-II or DO11 . 10 T cells differentiated by OVA-LeX or OVA-loaded spDCs or BM-DCs . Cells were gated on CD4+ T cells . Numbers in dot plots indicate the percentage of IFN-γ+ or IL-4+ of CD4+ T cells . Dot plots are representative of five independent experiments . ( C ) C57BL/6 mice were immunized s . c . with either OVA-LeX or native OVA mixed with anti-CD40 using a prime-boost protocol and the frequency of IFN-γ-producing activated CD4+ T cells in spleen was determined by intracellular staining after OVA-specific re-stimulation ex vivo . Dots represent individual mice , bars indicate median of each group ( n=5 mice/group , **p<0 . 01 ) . Graphs shown are representative of two independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 11765 . 00810 . 7554/eLife . 11765 . 009Figure 3—figure supplement 1 . No enhanced expansion of OT-II T cells when co-cultured for six days with OVA-LeX pulse-loaded DCs . WT BM-DC were loaded with OVA-LeX or native OVA for 4h and subsequently co-cultured with OT-II T cells for six days . Proliferation of OT-II T cells was determined by [3H]-thymidine uptake and presented as mean ± SD of triplicate cultures . Data shown are representative of two independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 11765 . 00910 . 7554/eLife . 11765 . 010Figure 3—figure supplement 2 . C57BL/6 mice were immunized s . c . with either OVA-LeX or native OVA mixed with anti-CD40 using a prime-boost protocol and the frequency of IFN-γ-producing activated CD4+ T cells in spleen was determined by intracellular staining after OVA-specific re-stimulation ex vivo . Representative facs plots of indicated mice are shown; numbers above the gates designate the percentage of IFN-γ+ CD4+ T cells . DOI: http://dx . doi . org/10 . 7554/eLife . 11765 . 010 Since we observed a great enhancement of antigen-specific CD8+ T cell priming in vivo when immunizing with OVA-LeX to target MGL1 , we wanted to reveal the mechanism that regulates this augmented cross-priming . The internalization of OVA by BM-DC was significantly increased when OVA was modified with LeX ( Figure 4A—figure supplement 1 ) . To investigate whether the addition of LeX glycans affected the efficiency of cross-presentation of OVA by DC , we loaded murine BM-DC or spDC with OVA-LeX and measured their potency to present OVA-derived peptides in MHC class I by measuring the proliferation of OVA-specific OT-I T cells . Strikingly , both BM-DCs as well as spDCs induced substantially more proliferation of OT-I T cells when they were pulsed with OVA-LeX compared to native OVA ( Figure 4B—figure supplement 2 ) . Even at low concentrations of antigen ( i . e . 7 . 5 µg/ml ) , the proliferating OT-I T cells were doubled ( 26 . 7% to 51 . 7% ) using OVA-LeX compared to using OVA ( Figure 4B ) , indicating that the modification of OVA with LeX greatly affected the cross-presentation of OVA . Moreover , detection of SIINFEKL/H-2Kb complexes on the cell membrane of OVA-LeX-loaded DCs by staining with the 25 . 1D1 antibody confirmed enhanced antigen loading on MHC-I molecules and transportation to the cell-surface of internalized OVA-LeX compared to native OVA ( Figure 4C + Figure supplement 3 ) . Cross-presentation of OVA-LeX was clearly mediated by MGL1 as demonstrated using MGL1 KO BM-DCs or steady-state spDCs ( Figure 4D ) . 10 . 7554/eLife . 11765 . 011Figure 4 . MGL1 mediates cross-presentation of OVA-LeX independently of TLR signaling . ( A ) Uptake of fluorescent-labeled OVA-LeX or native OVA ( 30 µg/ml ) by WT BM-DCs was analyzed by flow cytometry after 90 min . Graphs indicate the mean ± SD of triplicates and are representative of three independent experiments . ( B ) CFSE-labeled OT-I T cells were incubated with BM-DCs pulse-loaded with indicated concentrations of OVA-LeX or OVA for 4h . Un-loaded DC served as controls . Proliferation of OT-I T cells was analyzed after 3 days by flow cytometry . Percentages of divided OT-I cells are indicated . ( C ) OVA-LeX induces more OVA257-264/H2-Kb I complexes at the cell-surface of DCs than native OVA , as shown by 25 . 1D1 staining 18h after pulse loading of BM-DCs with OVA-LeX or native OVA . *p<0 . 05 . ( D ) WT or MGL1 KO BM-DCs or CD11c+ spDCs are pulsed with OVA-LeX ( black bars ) or native OVA and OT-I proliferation was determined on day 3 by [3H]-thymidine uptake . Data are presented as mean ± SD of triplicates , representative of three independent experiments . ***p<0 . 001 , ns not significant . ( E ) Cross-presentation of OVA-LeX is independent of MyD88 and/or TRIF signaling . BM-DC from WT or MyD88/TRIF DKO mice were pulsed with indicated concentrations of antigen and co-cultured with OT-I T cells . DCs pulsed with the nominal epitope SIINFEKL served as controls ( right panel ) . Proliferation was determined by [3H]-thymidine uptake . Data are representative of two experiments and indicated as mean ± SD of triplicates . DOI: http://dx . doi . org/10 . 7554/eLife . 11765 . 01110 . 7554/eLife . 11765 . 012Figure 4—figure supplement 1 . Uptake of fluorescent-labeled OVA-LeX or native OVA ( 30 µg/ml ) by WT BM-DCs was analyzed by flow cytometry after 90 min ( upper panel ) . Control cells were incubated with medium . The percentage of gated antigen-positive DCs are indicated . Lower panel: histograms indicating the mean uptake of OVA ( left , black line ) or OVA-LeX ( right , black line ) versus medium ( grey filled histograms ) and EGTA ( dashed lines ) controls . Numbers indicate the MFI of OVA-positive DCs . DOI: http://dx . doi . org/10 . 7554/eLife . 11765 . 01210 . 7554/eLife . 11765 . 013Figure 4—figure supplement 2 . Enhanced cross-presentation of OVA-LeX by DCs as measured by 3H-Thymidine incorporation . BM-DCs and CD11c+ spDCs loaded with OVA-LeX enhanced OT-I proliferation compared to native OVA loaded DCs . Proliferation was determined on day 3 by [3H]-Thymidine uptake and presented as mean ± SD of triplicate cultures . Data are representative of four independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 11765 . 01310 . 7554/eLife . 11765 . 014Figure 4—figure supplement 3 . Representative flow cytometry plots of 25 . 1D1 staining of BM-DCs 18h after pulse loading with OVA-LeX or native OVA ( 750 ug/ml ) . DOI: http://dx . doi . org/10 . 7554/eLife . 11765 . 01410 . 7554/eLife . 11765 . 015Figure 4—figure supplement 4 . Cross-presentation of OVA requires TLR4 triggering and is TAP-dependent . WT or TAP1-deficient BM-DC were loaded with OVA ( 0 . 5 mg/ml ) in the presence or absence of 100 ng/ml LPS and subsequently co-cultured with purified OT-I T cells for 3 days . [3H]-Thymidine is incorporated during the last 18h and is presented as mean ± SD of triplicate cultures . Data are representative of two independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 11765 . 01510 . 7554/eLife . 11765 . 016Figure 4—figure supplement 5 . OVA-LeX formulations are free of endotoxins . Both OVA and OVA-LeX were tested for endotoxin levels . Human embryonic kidney ( HEK ) 293-TLR4/MyD88 transfectants ( kind gift of Dr . D . Golenbock ) were cultured in the presence of either antigen preparation ( 30 µg/ml ) or indicated amounts of E . coli-derived LPS ( Sigma Aldrich ) . The HEK transfectants respond to LPS by secreting IL-8 . In both preparations , LPS was below detection limits ( dashed line ) . Results are representative of two independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 11765 . 016 Cross-presentation of OVA via the MR was shown previously to be dependent on TLR signaling and the presence of high amounts of antigen ( [Burgdorf et al . , 2006; Burgdorf et al . , 2008; Blander and Medzhitov , 2006] and Figure 4—figure supplement 4 , left panel ) . The observed differences in cross-presentation between OVA and OVA-LeX were not due to any potential contamination with the TLR4 ligand LPS , as both protein preparations did not trigger IL-8 production by TLR4-transfected HEK293 cells ( Figure 4—figure supplement 5 ) . In addition , both OVA preparations neither induced maturation of BM-DCs nor altered their cytokine production ( data not shown ) . To exclude any potential role of TLR signaling on the MGL1-mediated cross-presentation of OVA-LeX , we made use of BM-DCs from mice that lack both MyD88 and TRIF ( i . e . MyD88/TRIF DKO ) . However , MyD88/TRIF DKO BM-DCs still induced more OT-I proliferation when targeted with OVA-LeX than with OVA ( Figure 4E ) and only a slight reduction of cross-presentation was observed compared to that induced by WT BM-DCs , suggesting a minor role for MyD88- or TRIF-signaling in MGL1-induced cross-presentation . In line with previous findings , neither exogenous loading of MHC-I molecules with OVA257-264 peptides ( Figure 4E ) nor MHC class II presentation of OVA-LeX and OVA was dependent on MyD88- or TRIF- signaling and resulted in comparable expansion of OVA-specific T cells ( data not shown ) . Several cross-presentation pathways have been described , one of which is dependent on the transport of peptides from the cytosol into MHC-class I loading compartments via TAP-molecules ( Amigorena and Savina , 2010; Adiko et al . , 2015 ) , whereas another cross-presentation pathway depends on endosomal degradation by the cysteine protease Cathepsin-S ( Shen et al . , 2004 ) . To study a role for TAP transporters in our model , BM-DCs of TAP1 KO and WT control mice were pulsed with OVA-LeX followed by incubation with OT-I T cells . Surprisingly , cross-presentation induced by OVA-LeX was not reduced by the absence of TAP as OT-I proliferation induced by OVA-LeX-loaded TAP1 KO BM-DCs was not decreased compared to OVA-LeX-loaded WT BM-DC ( Figure 5A ) . In accordance with previous publications ( Burgdorf et al . , 2008 ) , we showed that the administration of OVA with LPS is cross-presented in a TAP-dependent manner ( Figure 4—figure supplement 2 ) . Furthermore , the possibility that the results are confounded by reduced levels of MHC-class I on TAP1 KOBM-DCs were excluded as the presentation of exogenously loaded OVA257-264 peptide is equal by both WT and TAP1 KOBM-DCs ( Figure 5A ) . In addition , we excluded the involvement of the Cathepsin-S pathway for cross-presentation of OVA-LeX as cross-presentation of OVA-LeX by BM-DCs from Cathepsin-S KO mice ( Cat-S KO ) was not reduced compared to WT BM-DCs ( Figure 5B ) . As expected , the MHC-class II-restricted CD4+ T cell proliferation was compromised in the Cat-S KO BM-DCs ( data not shown ) , illustrating the involvement of Cathepsin-S in cleaving the invariant chain of the MHC-class II molecule ( Nakagawa et al . , 1999 ) . 10 . 7554/eLife . 11765 . 017Figure 5 . LeX-modified antigen is cross-presented in a TAP- and Cathepsin-S-independent fashion . To examine whether cross-presentation of OVA-LeX involves TAP or Cathepsin-S ( A ) TAP1 KO and ( B ) Cat-S KO BM-DCs and WT BM-DCs were pulsed with OVA-LeX or native OVA and co-cultured with OT-I T cells for 3 days . DCs exogenously loaded with SIINFEKL for 3h served as control . Proliferation was determined by [3H]-Thymidine uptake and data are presented as mean ± SD of triplicates ( representative of three experiments ) . DOI: http://dx . doi . org/10 . 7554/eLife . 11765 . 017 As the dominant cross-presentation of LeX-modified OVA was neither dependent on TAP nor required TLR signaling , we hypothesized that this may be due to the altered uptake and intracellular routing of OVA in DCs . We therefore used imaging flow cytometry , a method that allows high-throughput image analysis of cells in flow with near-confocal resolution to analyze the intracellular routing of fluorescent labeled OVA-LeX . Co-staining with markers for early endosomal ( EEA-1 ) , late endosomal/lysosomal ( LAMP1 ) and recycling endosomal ( Rab11 ) compartments illustrated a swift co-localization of OVA-LeX with EEA1 and Rab11 as shown by high co-localization scores at 15 min ( Figure 6A , Figure 6—figure supplement 1 ) . However , this co-localization score was strongly decreased at 60 min . At this time-point , higher co-localization scores were detected for OVA-LeX with LAMP1 and Rab11 ( Figure 6B , Figure 6—figure supplement 2 ) . We then further dissected the intracellular pathway of OVA-LeX using confocal laser scan microscopy ( CLSM ) and compared it to the intracellular routing of native OVA . We confirmed that native OVA , which internalized via MR , was routed to EEA1+Rab11+ compartments within 2h ( Figure 6C , upper-left panel and [Burgdorf et al . , 2008] ) . However , the neoglycosylation with LeX altered the intracellular routing of OVA , showing its presence predominantly within LAMP1+Rab11+ compartments ( Figure 6C , upper-right panel ) . Moreover , OVA-LeX internalized by BM-DC from MGL1 KO mice was routed to EEA1+Rab11+ compartments and did not end up in LAMP1+Rab11+ compartments , similar to OVA in WT BM-DC ( Figure 6C , lower panels + Figure 6—figure supplement 3 ) . Together , these data suggest that upon internalization , OVA-LeX is rapidly shuttled to Rab11+EEA1+ compartments from where it moves to Rab11+LAMP1+ compartments , where it persists for longer periods ( i . e . >24h ) . Thus , these results indicate that internalization via MGL1 allows the antigen to enter the endosomal/lysosomal pathway . 10 . 7554/eLife . 11765 . 018Figure 6 . OVA-LeX is routed to Rab11+LAMP1+ compartments where it is stored for presentation in MHC class I . ( A , B ) WT BM-DCs were pulsed with AlexaFluor 674-OVA-LeX ( 30 µg/ml ) and chased at the indicated time-points to assess triple co-localization scores of OVA-LeX with ( A ) EEA-1 and Rab11 or ( B ) LAMP1 and Rab11 using imaging flow cytometry . ( C ) WT ( upper panels ) and MGL1 KO ( lower panels ) BM-DCs were incubated with Dylight-633-OVA-LeX or native OVA ( 30 µg/ml ) and 2h later co-localization of OVA antigen ( Red ) with early endosomal ( EEA-1 , Green ) or endosomal/lysosomal ( LAMP1 , Green ) and recycling endosomal ( Rab11 , Blue ) compartments was analyzed using CSLM . From a z-stack , histograms were created for a selected area ( indicated by a line , upper part of each panel ) using the Leica confocal software . Histograms were created from each fluorochrome and overlays were made by the program . Arrows indicate co-localization of antigen ( Red ) with EEA1&Rab11 or LAMP1&Rab11 . ( D ) MGL1-Fc binding to LeX-PAA was determined at indicated pH by ELISA . DOI: http://dx . doi . org/10 . 7554/eLife . 11765 . 01810 . 7554/eLife . 11765 . 019Figure 6—figure supplement 1 . Examples of WT BM-DCs pulsed with AlexaFluor 674-OVA-LeX displaying high co-localization of OVA-LeX with EEA-1 and Rab11 as measured by imaging flow cytometry . DOI: http://dx . doi . org/10 . 7554/eLife . 11765 . 01910 . 7554/eLife . 11765 . 020Figure 6—figure supplement 2 . Examples of WT BM-DCs pulsed with AlexaFluor 674-OVA-LeX displaying high co-localization of OVA-LeX with LAMP1 and Rab11 as measured by imaging flow cytometry . DOI: http://dx . doi . org/10 . 7554/eLife . 11765 . 02010 . 7554/eLife . 11765 . 021Figure 6—figure supplement 3 . WT ( upper panels ) and MGL1 KO ( lower panels ) BM-DCs were incubated with Dylight-633-OVA-LeX or native OVA ( 30 µg/ml ) and 0 . 5 h and 2 h later co-localization of OVA antigen ( Red ) with early endosomal ( EEA-1 , Green ) or endosomal/lysosomal ( LAMP1 , Green ) and recycling endosomal ( Rab11 , Blue ) compartments was analyzed using CSLM . From a z-stack , histograms were created for a selected area ( indicated by a line ) using the Leica confocal software . Histograms were created from each fluorochrome and overlays were made by the program . Arrows indicate co-localization of antigen ( Red ) with EEA1&Rab11 or LAMP1&Rab11 . DOI: http://dx . doi . org/10 . 7554/eLife . 11765 . 021 The development of early endosome into late endosome/lysosome coincides with a decreasing pH gradient . The pH at which MGL1 dissociates from its ligand is indicative of the compartment in which the antigen becomes available for degradation and loading on MHC-molecules . We therefore analyzed MGL1-binding to its ligand LeX at different pH that resembled the pH of the intracellular compartments . MGL1 starts dissociating from LeX already at pH 6 . 5 ( Figure 6D ) . This indicates that OVA-LeX becomes available for degradation in the early and late endosomal compartments , compartments both associated with cross-presentation . The sustained presence of OVA-LeX in Rab11+LAMP1+ compartments ( Figure 6B+C ) prompted us to investigate whether these compartments facilitate prolonged cross-presentation , as shown by van Montfoort et al . for OVA-immune complexes ( van Montfoort et al . , 2009 ) . Indeed , even two days after antigen pulse , OVA-LeX-loaded DC induced strong expansion of OT-I T cells , suggesting that OVA-LeX-loaded DC have prolonged cross-presentation capacity ( Figure 7A ) . At the highest antigen concentration used ( i . e . 30 µg/ml ) , the percentage of proliferated OT-I T cells was only slightly reduced 48 h after pulse compared to that induced by DCs that were pulsed with OVA-LeX for 4 h ( Figure 7A ) . Expansion of OT-I T cells driven by 48 h pulse loaded DCs was even detectable at low antigen concentrations ( 3 . 75 µg/ml ) . OT-II proliferation induced by DCs pulsed with OVA-LeX for 48h was also still detectable although less pronounced than the OT-I induced proliferation ( Figure 7B ) , suggesting that the prolonged storage of antigen in these intracellular compartments predominantly favored cross-presentation . 10 . 7554/eLife . 11765 . 022Figure 7 . MGL1 targeting with OVA-LeX shows sustained antigen presentation in MHC-I . WT BM-DCs were pulsed for 4 h with titrated amounts of OVA-LeX and washed with culture medium . DCs were then chased for 48 h in antigen-free medium . ( A ) BM-DCs pulsed for 4h with OVA-LeX induced MHC-I antigen presentation as measured by CFSE-labeled OVA-specific OT-I cells ( upper panel ) . Sustained presentation is shown after 48 h ( lower panel ) . ( B ) MHC-II antigen presentation 4h and 48h after pulse-loading with OVA-LeX , analyzed by OT-II proliferation . Data are presented as percentage of proliferated T cells and representative of three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 11765 . 022 Taken together , these data suggest that antigen internalized by MGL1 is routed from RAb11+EEA1+ compartments towards Rab11+LAMP1+ compartments , which seem to associate with the extended antigen-processing and cross-presentation .
We here demonstrate that the glycosylation-profile of antigens has a major influence on antigen uptake and intracellular compartmentalization , thereby affecting both antigen presentation and the type and strength of the induced immune response . Modification of the model-antigen OVA with LeX glycans directs OVA towards MGL1 , skewing naive CD4+ T cell differentiation towards IFNγ-producing Th1 cells . Moreover , targeting OVA to MGL1 through the conjugation of LeX , substantially enhanced cross-presentation as revealed by the increased frequency of OVA-specific CD8+ effector T cells in vitro and in vivo . Importantly , MGL1-dependent cross-presentation occurred at low antigen dose and independently of TLR-signaling . Moreover , this cross-presentation pathway did not involve TAP-transporters and Cathepsin-S . MGL1 targeting involved antigen routing to a Rab11+LAMP1+ compartment in which antigen was present for prolonged periods . Previous studies on CLR-mediated antigen uptake and cross-presentation , in particular by the MR , demonstrated a clear requirement for a TLR ligand ( Kovacsovics-Bankowski and Rock , 1995; Burgdorf et al . , 2008; Sancho et al . , 2008; Segura et al . , 2009 ) . A common denominator in some of these studies is that the use of antibody–antigen conjugates could potentially induce a different signal than the natural ligand , due to binding to different part of the receptor or through co-engagement of Fc-receptors . The fact that the addition of LeX glycans to OVA obviates the need for TLR signals for the induction of cross-presentation and Th1 priming in vitro , may indicate that MGL1 signaling is involved in these processes . Some CLR , like DC-SIGN , Clec9A and Dectin-1 are known to induce signaling after triggering by their natural ligands ( Geijtenbeek and Gringhuis , 2009; Sancho et al . , 2009 ) . Till now , no signaling pathway has been described for MGL1 . Triggering of huMGL resulted in ERK1/2 and NF-κB activation , and results in elevated levels of IL-10 and TNF ( Li et al . , 2012; van Vliet et al . , 2013 ) . In our studies , the uptake of LeX-modified OVA through MGL1 was not associated with any DC maturation or altered cytokine production by DCs . The fact that OVA-LeX induced an enhanced frequency of Th1 cells in vitro and of antigen-specific effector T cells in vivo when combined with agonistic anti-CD40 Abs illustrates that a yet to be determined costimulatory signal is essential for the induction of effector CD8+ and CD4+ T cells . This powerful function of MGL1 to establish antigen-specific immunity , stands opposite to its recently demonstrated anti-inflammatory function , which include induction of IL-10 production and altered adhesive function by APC ( Saba et al . , 2009; Westcott et al . , 2009 ) . Various models for cross-presentation of antigens have been contemplated . The 'cytosolic pathway' of cross-presentation allows receptor-mediated endocytosis or phagocytosis and antigen translocation into the cytosol , where they are degraded into antigenic peptides by the proteasome and transported into the lumen of the endoplasmic reticulum ( ER ) ( Kovacsovics-Bankowski and Rock , 1995; Shen et al . , 2004 ) or ER/phagosomal fusion compartments ( Guermonprez et al . , 2003; Houde et al . , 2003 ) by TAP-transporters . A variation to the cytosolic pathway was described in which soluble OVA is taken up via the MR and is supplied to stable early endosomal compartments ( Burgdorf et al . , 2007 ) . In these compartments , the MHC class I machinery as well as TAP-transporters are selectively recruited , facilitating direct loading of antigen-derived peptides onto MHC class I molecules without trafficking to the ER . The 'vacuolar pathway' allows antigens to be degraded into peptides in early endosomal compartments by endosomal proteases like Cathepsin-S , to be loaded on MHC class I molecules in the same compartment , thus is TAP-independent ( Shen et al . , 2004 ) . The current described models for cross-presentation depend on peptide-transport by TAP from the cytosol to either ER or ER/phagosomal fusion compartments or on endosomal degradation of antigens via Cathepsin-S ( reviewed in [Amigorena and Savina , 2010; Burgdorf and Kurts , 2008; Mintern et al . , 2015; Adiko et al . , 2015] ) . Although we observed localization of OVA-LeX in early endosomes , absence of either TAP molecules or Cathepsin-S did not influence the enhanced cross-presentation of OVA-LeX . Our data indicate that the MGL1 cross-presentation pathway is different than that of the MR and may occur via antigen storage compartments similar to those described for FcγR-targeted antigens ( van Montfoort et al . , 2009 ) . We observed prolonged presence in Rab11+LAMP1+ compartments in the absence of a maturation stimulus or apparent DC maturation . It is tempting to speculate that in these compartments cross-presentation of OVA is facilitated by Rab11 as it was recently shown that Rab11 activity mediates delivery of MHC-I molecules to phagosomes used for cross-presentation during infection ( Nair-Gupta et al . , 2014 ) . However , recruitment of additional components involved in antigen cross-presentation by the SNARE Sec22b40 complex cannot be excluded ( Cebrian et al . , 2011 ) . The prolonged antigen cross-presentation via MGL1 allows sustained cross-priming of CD8+ T cells in vivo and thus the efficacy of tumor vaccines that target MGL1 may be higher than of conventional vaccines containing non-targeted antigens . In the current study , we show that already at very low concentrations , soluble protein antigens are efficiently cross-presented upon uptake by MGL1 . DCs pulsed with a low dose of OVA-LeX ( 7 . 5 μg/ml ) induced significant CD8+ T cell proliferation , whereas the same concentration of native OVA was hardly cross-presented ( Figure 4 ) . Others have demonstrated that native OVA can only be cross-presented when a high dose was used ( 0 . 5–1 . 0 mg/ml ) and when accompanied with TLR-triggering ( Burgdorf et al . , 2008; Segura et al . , 2009 ) . Although the glycan-modification was associated with increased antigen uptake and enhanced cross-presentation , it did not result in enhanced presentation of antigen on MHC-class II indicating that increased uptake of antigen is not per se a requirement to facilitate MHC class I and II presentation . Therefore , it is most likely that the CLR ( and thus the glycosylation of the antigen ) dictates how efficiently an antigen is cross-presented . Based on our study , MGL1 cross-presents antigen much more efficiently than the MR . The activity of the glycosylation machinery is subjected to subtle regulation and depends on the cell-type or activation status of the cell . Upon malignant transformation glycan profiles may change dramatically . LeX carbohydrate structures are described to be expressed in the brain ( Lieberoth et al . , 2009 ) , and can be de novo expressed on cancer stem cells ( Read et al . , 2009 ) or pathogens , and the glycosylation pattern of in vivo accumulating antigens , being brain tissue , tumor tissue or pathogen structure , is crucial for directing specific CLR antigen uptake and cross-presentation ( Hittelet et al . , 2003; Ohana-Malka et al . , 2003 ) . Although in our studies only two LeX glycans were conjugated to each OVA molecule , it cannot be ruled out that multivalent presentation of LeX , such as often observed on tumors or pathogens ( Bergman et al . , 2004; Saeland and van , 2011 ) , may alter avidity-induced MGL-1 signaling and antigen presentation , and induce an anti-inflammatory immune repertoire . At this stage , we do not know whether high glycan valency further enhances or inhibits the MGL1-mediated ( cross- ) priming . The fact that pulsing of DCs with OVA-LeX resulted in improved cross-priming and Th1-skewing indicates that conjugation of these carbohydrates to tumor-antigens could be beneficial for the induction of potent anti-tumor responses . Therefore , the use of glycans for targeting CLR for anti-cancer immunotherapy could have several advantages . First , they mimic natural function of the receptors , inducing ‘natural’ signaling cascades in DCs . Furthermore , they are very small molecules , easy to use and relatively cheap to produce . Importantly , many glycans may be considered self-antigens , in contrast to recombinant antibodies that are often not completely of human origin . These properties make it possible to decorate any antigen of choice with glycans to target specific receptors . In conclusion , our studies indicate that the glycan composition of protein antigens is of fundamental importance in dictating the intracellular routing and Th-skewing , and should be considered as a major determinant in the design of therapeutic vaccines against cancer and infectious diseases .
C57BL/6 mice ( Charles River Laboratories ) were used at 8–12 weeks of age . MGL1KO mice , which have a null mutation within the Clec10a gene , are on the C56BL/6 background and were kindly provided through the Consortium for Functional Glycomics . TAP1 KO mice have a null mutation within the Tap1 gene . MGL1 KO , TAP1 KO , OT-I and OT-II TCR transgenic mice were bred in our animal facilities under specific pathogen-free conditions . All experiments were performed according to institutional , state and federal guidelines . Fluorochrome-conjugated antibodies used: anti-CD11c-APC , anti-IFNγ-APC , anti-TNFα-PE-cy7 , anti-IL-4-PE , anti-IL17-PE , anti-Foxp3-PE ( e-Bioscience , Vienna , Austria ) , and anti-LAMP-1-v450 ( BD-Pharmingen ) . Unconjugated mouse anti-OVA ( Sigma Aldrich ) , mouse anti-LeX ( Calbiochem ) , rat anti-mMGL ( ER-MP23; kind gift from Dr . P . Leenen , Erasmus MC , Rotterdam , The Netherlands ) , rat anti-LAMP1 ( BD-Pharmingen ) , rabbit anti-Rab11 ( Life Technologies ) , goat anti-EEA1 ( Santa Cruz Biotechnology ) and rabbit anti-EEA-1 ( Dianova ) . Secondary antibodies used: peroxidase-labeled F ( ab’ ) 2 fragment goat anti-human IgG , F ( ab’ ) 2 fragment goat anti-mouse IgG , ( Jackson ) , peroxidase-labeled goat anti-mouse IgM ( Nordic Immunology ) , goat anti-rat Alexa 448 , goat anti-rat Alexa 647 , donkey anti-goat Alexa 488 , donkey anti-goat Alexa 647 , donkey anti-rabbit Alexa 555 and donkey anti-rabbit Alexa 488 ( Molecular Probes ) . MGL-1-Fc was generated as described earlier ( Singh et al . , 2009b ) . MR-Fc was kindly provided by L . Martinez-Pomares ( University of Nottingham , Nottingham , UK ) . LeX ( lacto-N-fucopentose III; Dextra Labs , UK ) carbohydrate structures were conjugated to OVA ( Calbiochem ) as previously described ( Singh et al . , 2009a ) . In short; the bifunctional cross-linker ( 4-N-maleimidophenyl ) butyric acid hydrazide ( MPBH; Pierce ) was covalently linked to the reducing end of the LeX and the maleimide moiety of the linker was later used for coupling the LeX to OVA . Neo-glycoconjugates were separated from reaction by-products using PD-10 desalting columns ( Pierce ) . Additionally , a Dylight 549-N-hydroxysuccimide ( NHS ) label ( Thermo Scientific ) was covalently coupled with OVA or OVA-LeX ( Dylight-549-OVA ) . Free label was removed using a PD-10 column ( Pierce ) . The presence of LeX and CLR binding to OVA was measured by ELISA . In brief , OVA-conjugates were coated directly on ELISA plates ( NUNC ) and binding of MR-Fc , MGL1-Fc , anti-LeX and anti-OVA antibodies to OVA was determined as described ( Singh et al . , 2009a; Hawiger et al . , 2001 ) . The presence of endotoxin was measured using a LAL assay ( Lonza ) following manufacturer’s protocol . OVA was deglycosylated by incubation in 5 IU of PNGase F ( Roche Applied Sciences ) o/n at 37°C . Proteins were extracted by reverse phase chromatography using Sep-Pak Vac C18 disposable cartridges ( Waters ) . Glycans were further purified by reverse phase chromatography using Superclean ENVI-Carb cartridges disposable columns ( Supelco ) . Glycans were lyophilized and re-dissolved in 30 ml of 7-Amino-4-methylcoumarin ( 160 mM , Sigma Aldrich ) and 2-Picoline borane ( 270 mM , Sigma Aldrich ) in DMSO:acetic acid ( 4:1 , Riedel deHaën ) . 4-AMC-labelled glycans were purified by size exclusion chromatography using a Bio-Gel P2 ( Bio-Rad ) column with 50 mM ammonium formate ( Sigma Aldrich ) as running buffer . 4-AMC-labelled glycans were lyophilized and analyzed by multidimensional normal phase HPLC ( UltiMate 3000 nanoLC , Dionex ) using a Prevail Carbohydrate ES 0 . 075 x 200 mm column ( Grace ) coupled with an LCQ Deca XP with electrospray interface mass spectrometer ( Thermo Finnigan ) tuned with maltoheptoase ( Sigma Aldrich ) labeled with 4-AMC and with an intercalated fluorescence detector ( Jasco FP-2020 Plus , Jasco ) ( maximum excitation 350 nm , band width 40 nm; maximum emission 448 nm , band width 40 nm ) as previously described ( Kalay et al . , 2012 ) . Matrix-assisted laser desorption/ionization-time of flight ( MALDI-TOF ) mass spectrometry measurements were done using a 4800 MALDI-TOF/TOF Analyzer ( Applied Biosystems ) . Mass spectra were recorded in the range from to 19 , 000 to 155 , 000 m/z in the linear positive ion mode . The data were recorded using 4000 Series Explorer Software and processed with Data Explorer Software version 4 . 9 ( all from Applied Biosystems ) . C57BL/6 or Mgl1-/- mice were injected s . c . either with 100 μg OVA-LeX or OVA mixed with 25 µg anti-CD40 Ab ( 1C10 ) on day 0 and day 14 . Mice were sacrificed one week after boost and the amount of antigen-specific CD8+ T cells was analyzed in the spleen by staining with H2-Kb-SIINFEKL tetramers ( Sanquin ) . Additionally , frequencies of OVA-specific cytokine-secreting T cells were analyzed by flow cytometry . Hereto , spleen cells were re-stimulated overnight with either 2 µg/ml SIINFEKL or 200 µg/ml EKLTEWTSSNMEER OVA peptides in the presence of 5 µg/ml Brefeldin A , then IFN-γ and TNFα expression were assessed by intracellular staining using specific antibodies . BM-DC were cultured as previously described ( Singh et al . , 2009a ) . BM of Myd88/Ticam1 DKO ( referred to as MyD88/TRIF DKO ) and Ctss-/- ( referred to as Cat-S KO ) mice was kindly provided by Dr . T . Sparwasser ( Twincore , Hannover , Germany ) and Dr . K . Rock ( Massachusetts Medical School , Worcester , MA , USA ) , respectively . CD11c+ spDCs were isolated as previously described ( Singh et al . , 2009a ) . OVA-specific CD4+ and CD8+ T cells were isolated from spleen and lymph nodes cell suspensions from OT-II and OT-I mice , respectively , using the mouse CD4 and CD8 negative isolation kit ( Invitrogen , CA , USA ) according to manufacturer’s protocol . T cell proliferation assays were performed as described ( Singh et al . , 2009a ) . In short , DCs were pulsed with OVA-LeX or OVA for 4h before incubation with OVA-specific OT-I or OT-II T cells ( 2:1 DC:T ) . [3H]-Thymidine ( 1 µCi/well; Amersham Biosciences ) was present during the last 16h of a 72h culture . [3H]-Thymidine incorporation was measured using a Wallac microbeta counter ( Perkin-Elmer ) . Alternatively , OT-I or OT-II T cells were labeled with CFSE and after 3 days dilution of CFSE was analyzed by flow cytometry . Differentiation of naive OT-II T cells , induced by OVA-LeX or OVA -pulsed BM-DCs or spDCs , was measured by an in vitro Th differentiation assay described earlier ( Singh et al . , 2011 ) . mRNA was isolated by capturing poly ( A+ ) RNA in streptavidin-coated tubes using a mRNA Capture kit ( Roche , Basel , Switzerland ) . cDNA was synthesized using the Reverse Transcription System kit ( Promega , WI , USA ) following the manufacturer’s guidelines . Real time PCR reactions were performed using the SYBR Green method in an ABI 7900HT sequence detection system ( Applied Biosystems ) . BM-DCs were incubated with 30 μg/ml Dylight-633-OVA or OVA-LeX for 30min or 2h at 37°C , fixed and permeabilized for 20 min on ice , and stained with primary and secondary antibodies . Co-localization was analyzed using a confocal laser scanning microscope ( Leica SP5 STED ) system containing a 63x objective lens; images were acquired in 10x magnification and processed with Leica LAS AF software . For imaging flow cytometry , approximately 1x106 BM-DCs were incubated with OVA-LeX for 30 min at 4°C , washed twice in ice-cold PBS and then transferred to 37°C . At the indicated time-points , cells were washed twice and fixated in ice-cold 4% paraformaldehyde ( PFA , Electron Microscopy Sciences ) in PBS for 20 min . The cells were then permeabilized in 0 . 1% saponin ( Sigma ) in PBS for 30 min at RT and subsequently blocked using PBS containing 0 . 1% saponin and 2% BSA for 30 min at RT . Stainings were performed at room temperature ( RT ) in PBS supplemented with 0 . 1% saponin and 2% BSA . After staining , cells were washed twice in PBS , resuspended in PBS containing 1% BSA and 0 . 02% NaN3 and kept at 4°C until analysis . The cells were acquired on an ImageStream X100 ( Amnis ) imaging flow cytometer . A minimum of 15 , 000 cells was acquired per sample at a flow rate ranging between 50 and 100 cells/second at 60x magnification . At least 2000 cells were acquired from single-stained samples to allow for compensation . Analysis was performed using the IDEAS v6 . 1 software ( Amnis ) . The cells were first gated based on the Gradient RMS ( brightfield ) feature and then based on area vs aspect ratio intensity ( both on brightfield ) . The first gating identified the cells that appeared in focus , while the second excluded doublets and cells other than BM-DCs . 3-colour co-localization was calculated using the bright detail co-localization 3 feature . The pH dependency of MGL1 binding to LeX on antigens was determined by ELISA . Hereto , LeX-PAA ( Lectinity Holdings ) was coated onto NUNC Maxisorp plates o/n at RT . The plates were blocked with 1% BSA in TSM buffer ( 20 mM Tris-HCl; 150 mM NaCl; 2 mM CaCl2; 2 mM MgCl2 ) . After washing , MGL1-Fc was added in TSA with different pH and were kept in this buffer throughout the assay . Binding was detected using peroxidase-labeled F ( ab’ ) 2 fragment goat anti-human IgG . Graphpad prism 5 . 0 was used for statistical analysis . The Student’s t-test and one-way ANOVA with Bonferroni correction were used to determine statistical significance . Statistical significance was defined as p< 0 . 05 .
Antigen uptake by dendritic cells and intracellular routing of antigens to specific compartments is regulated by C-type lectin receptors that recognize glycan structures . We show that the modification of Ovalbumin ( OVA ) with the glycan-structure LewisX ( LeX ) re-directs OVA to the C-type lectin receptor MGL1 . LeX-modification of OVA favored Th1 skewing of CD4+ T cells and enhanced cross-priming of CD8+ T cells . While cross-presentation of native OVA requires high antigen dose and TLR stimuli , LeX modification reduces the required amount 100-fold and obviates its dependence on TLR signaling . The OVA-LeX-induced enhancement of T cell cross-priming is MGL1-dependent as shown by reduced CD8+ effector T cell frequencies in MGL1-deficient mice . Moreover , MGL1-mediated cross-presentation of OVA-LeX neither required TAP-transporters nor Cathepsin-S and was still observed after prolonged intracellular storage of antigen in Rab11+LAMP1+ compartments . We conclude that controlled neo-glycosylation of antigens can crucially influence intracellular routing of antigens , the nature and strength of immune responses and should be considered for optimizing current vaccination strategies .
Immune cells called dendritic cells play a crucial role in defending the body against tumor cells and invading viruses . The dendritic cells take up molecules called antigens from these threats and then display them on their surface . This enables the antigens to be identified by other immune cells that are capable of killing the viruses and the tumor cells . The dendritic cells recognize the antigens with the help of receptor proteins called C-type lectin receptors ( CLRs ) . These receptors can bind to sugar molecules that are naturally found on many antigens . For example , a C-type lectin receptor called MGL1 can bind to sugars known as LewisX and Lewisa on tumor and virus proteins . However , it is not clear how important these receptors are in triggering immune responses . An antigen called Ovalbumin – which is found in chicken egg white – can trigger immune responses in mammals and so researchers often use it to study the immune system . Although this antigen has several sugar molecules attached to it , quite a large amount of Ovalbumin is needed to trigger strong immune responses . Now , Streng-Ouwehand et al . examine whether attaching LewisX to Ovalbumin can results in stronger immune responses in mice . The experiments show that injecting mice with Ovalbumin-LewisX triggers a much stronger immune response than normal Ovalbumin does . This enhanced response was not observed in mice that lacked the MGL1 receptor , which suggests that this receptor is involved in detecting Ovalbumin-LewisX . Furthermore , the dendritic cells store the altered Ovalbumin for longer than they store normal Ovalbumin , which gives the cells more time to present the altered Ovalbumin to other immune cells . Vaccines and some other therapies help to boost immune responses to viruses and tumors by exposing the body to antigens . However , these therapies often use antigens that don’t have sugar molecules attached to them , or are missing the sugar molecules they would normally have . Streng-Ouwehand et al . ’s findings suggest that adding specific sugars to antigens in immune therapies might help to make these therapies more effective .