Julia E. BabenseeWallace H. Coulter Department of Biomedical Engineering,

Georgia Institute of Technology and Emory University, Atlanta, GA

Biomaterial/GlycanCell Interactions

Dendritic Cell (DC) and Immunity

Medzhitov R., Nature Rev Immunology (2001)

DCs BridgeInnate and Adaptive Immunity

DCs Maintain ImmunologicalHomeostasis

http://www.dkfz.de/en/immungenetik/Heiko_group/Heiko-2.jpg

Key roles in:Maintaining self toleranceImmunity to pathogensCancerAutoimmunity

P.M. Kou

Distinct DC PhenotypesandImmunologicalOutcomes

Biomaterial Immunomodulatory Effect

M. M. Matzelle & J.E. Babensee, Biomaterials 25:295-304 (2004).

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PBS CFA 75/25 PLGA MP 75/25 PLGA SC

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+ + ++

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PBS CFA 75/25 PLGA MP 75/25 PLGA SCPBSPBS CFACFA 75/25 PLGA MP75/25 PLGA MP 75/25 PLGA SC

Biomaterial Immunomodulatory Effect

Combination Product

Activated APCsMφ, dendritic cells

Activated T cells

Adaptive Immunity

(Immune Response)

Innate Immunity

(Inflammation)

biomaterialcomponent

biologicalcomponent

PLGA Adjuvant Effect

Prime Mechanism By Which AdjuvantsEnhance Immune Responses

Maturation Of Dendritic Cells (DCs)

Dendritic Cells InfiltratePLGA Scaffolds

Plain PLGA PLGA with Adsorbed OVA

A. Paranjpe

Anti-DEC-205 labelingDay 7Original Magnification, 40X

Derivation of DCs forBiomaterial Treatment

1. Flow cytometry forexpression of surfacemarkers

2. Cytokine quantification3. Allostimulatory MLR4. Morphology5. Viability

Day 0 Day 5 Day 6

Human peripheralblood mononuclear cells

Biomaterial or controltreatment

DC Phenotype UponBiomaterial Contact

CD86

Agarose

Hyaluronic Acid

Alginate

Chitosan

PLGA

mDC

iDC

Supports DC maturation

Does not supportDC maturation

Supports DC Maturation

InhibitsDC maturation

Does not supportDC maturation

Fold increases over the immature DCs are shown to compare between different donors.★: p < 0.05, statistically higher than immature DC (=1); : p < 0.05, statistically lower than immature DC (=1); Bracket: p < 0.05, statistically different between two biomaterial treatments;‘┴’ indicates ‘or’.mean±SD, n=6 donors

Original magnification: 40×.

J. Park

Cytokine Profiles forBiomaterial-Treated DCs

(a) Pro-inflammatory cytokines(b) Chemokines(c) Anti-inflammatory cytokines

Proteins released into supernatanthave been measured using Bio-Pleximmunoassay (mean±SD, n=6 donors).

(a) (b)

(c)

J. Park

Red – Isotype Green – Foxp3

CD4+CD25+Quadrantonly

Untreated MNCs

MNCstreated with TGF-beta

iDC

mDC

PLGA

Agarose

CD4

CD25

J. Park

Autologous T cell Polarization by Biomaterial-Treated DCs

No

T ce

llC

o-cu

lture

with

T c

ells

Co-culture with Autologous CD3+ T cells

PLGAmDC iDC Chitosan Alginate HA Agarose

DCs collected

Inducer of Th1 (IFN-γ)with or without antigens

Inducer of Th1(IL-12p70) &Th2 (IL-10)withoutantigens

Inducer of Th1(IL-12p70) &Th2 (IL-10) withantigens

Inducer of IL-12p70without antigens

Inducer of IL-10with or withoutantigens

Inducer of IL-10with antigens

Inducer of IL-12p70with antigens

CD4+CD25+,Foxp3+Inducedwith antigentreatment

CD4+Inhibited with antigentreatment

Autologous T Cell Phenotype And Polarization By Biomaterial-Treated DCs

J. Park

Foxp3+

In Vivo Biomaterial Adjuvant EffectCorrelates with DC Response In Vitro

n = 6-7. Data are shown as replicates with mean represented as a line.

* p<0.05. ND = none detected

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Norton, L., Park, J., Babensee, J.E., J. Control. Rel., in press

Babensee, J.E. and Paranjpe A., JBMR (2005). Bennewitz, N.L. and Babensee J.E., Biomaterials (2005).

Agarose PLGAChitosan

Hyaluronic acid

Biomaterial Adjuvant Effect

• Biomaterials induced differential levels of DCmaturation

• PLGA, but not agarose, enhanced humoralresponse to a model antigen (ovalbumin) in mice

• Control biomaterial systems are needed tocorrelate DC response to biomaterial properties

matureimmature

DC Recognition of Biomaterials

P. Kou

Role of CLRs in Immunity and Tolerance

S. J. van Vliet, J. J. Garcia-Vallejo, Y. van Kooyk, Immunology and Cell Biology (2008).

C-Type Lectin Receptors (CLRs)

T.B.H. Geijtenbeek and S.I. Gringhuis, Nature Rev Immunol (2009).

CLRs and Homeostatic and Pathologic Functions

J.J. Garcia-Vallejo, Y. van Kooyk, ImmunologicalReviews (2009).

CLRs, Recognition Sites and Signaling

• Classified as mannose or galactose binding• Recognize pathogen-specific glycans and endogenous

glycans (e.g. mediate cell interactions, tumor immuneevasion, autoimmunity)

• Differential expression on cells (e.g. DC subsets) allowfor specific targeting with then different types of immuneresponses induced

• Immuno-stimulatory and immuno-inhibitory• Some CLRs with signaling ability due to motifs• Collaboration (+ve and –ve) with other receptors eg.

TLRs

Hypothesis• Glycans and glycoproteins have functional

immuno-stimulatory or -inhibitory effects on DCs– The form, context, and/or density of glycan presentation

will affect DC phenotype– Glycan moieties from serum glycoproteins can

functionally modulate DC response– Glycan modification of biomaterials have the ability to

modulate the in vivo host response to a material orvaccine delivery vehicle

Differential carbohydrate profileson SAMs of Alkanethiols

X axis: carbohydrate ligand (lectin probe)

10% serum

Y ax

is: a

bsor

banc

e at

405

nm

0

0.5

1

1.5

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4

Mannose (NPA) Sialyl (SNA-1) GlcNAc (UEA-II) mannose alpha(1,3)

and alpha(1,6)

mannose, D-

mannose (HHA)

CH3

OH

COOH

NH2

Mannose Family Galactose family

All SAM > CH3

NH2 > All SAM

OH > CH3 SAM

0

0.05

0.1

0.15

0.2

_-galactose(AIA) GalNAc (BPA) _-galactose(PNA)

CH3

OH

COOH

NH2

COOH >CH3 SAM

COOH >NH2 SAM

mean±SD, n=6 trials

S. Shankar, I.I. Chen, B. Keselowsky, A.J. Garcia, J.E. Babensee JBMR (2010).

N.D.

Avidin/alkaline phosphatase

Plant Lectin Probe

Biotin conjugate

CarbohydrateAu/Ti surface with alkanethiol

HTP Methodology

Day 0

FluorescentMicroplate

Reader

Day 5 Day 6

Incubate withanti-CD86-PEand anti-DC-SIGN-FITC

for 1 hr at 4oC

Fix in 0.03%FA for 30 – 45

min at RT

Cytotoxicitytest & Store forcytokineprofiling

FITC and PEfluorescencemeasured by

microplatereader

Zoom in the well

Day 6

Kou P. and Babensee JE. Acta Biomaterialia (2010)

Glycan Isolation and Biotinylation

HPLCforSepara-onofMan5‐9

HPLC&MALDI

FreeReducingGlycans(Man5‐9)

FreeReducingGlycans

Glycan‐AEAB‐LC‐Bio-n

2. 3.

4. 5.

1.

RibonucleaseBRibonucleaseB

6. 7.

N. Hotaling in collaboration with R. Cummings, Emory University

Glycan Purification and CharacterizationHPLC of Oligomannose 5-9 afterAEAB functionalization and afterseparation (before biotinylation)

Total amount of biotinylatedglycan-AEAB made

MALDI of biotinylatedglycans-AEAB

Man5-AEAB

Man6-AEAB

Man7-AEAB

Man8-AEAB

Man9-AEAB

Man5-AEAB-biotin

Man6-AEAB-biotin

Man7-AEAB-biotin

Man8-AEAB-biotin

Man9-AEAB-biotin

Enyme Link Lectin Assay on biotinylated glycans immobilized on TCPS by surface adsorbed SA. Asaturation assay was performed to determine what concentration of glycan was needed to saturate SAbinding sites for 4 different sizes of glycan A) a monosaccharaide, mannose, B) a trisaccharide, Lewisa,C) heptasaccaride, Oligomannose 5, and D) an undecasaccharide, Oligomannose 9. Brackets representstatistical difference from all indicated columns P ≤ 0.05.

Detection of Immobilized Glycans

N. Hotaling in collaboration with R. Cummings, Emory University

DC Response to 27 Biotinylated Sugars on SA Coated TCPS(n=7) TCPS iDC Normalized

TCPS

mD

C

SA iD

CSA

mD

C

a-G

lca-

Neu

5Ac

a-Fu

cb-

Gal

b-G

lcN

Ac

fuca

1-3G

lcN

Ac

Lact

ose

LN S'LN

Lex

DiL

exTr

iLex

SLex

Lec

SLec

Lea

Lea-

LC

SLea

Ley

Ley-

Lex

a-M

anTr

iMan

Man

5M

an6

Man

7M

an8

Man

9

0

2

4

6 **

#

#+ + + + + +

CD86

/DCS

IGN

Fold

Cha

nge

DC response to 27 biotinylated glycans (4000pmol/well).* represents statistical difference from iDC p≤ 0.05.# represents statistical difference from streptavidin iDC p≤ 0.05.+ represents no difference from TCPS mDC or streptavidin mDC.

DC Responses to Glycans

N. Hotaling in collaboration with R. Cummings, Emory University

n=6 independent determinations, mean ±S.D.* Different from iDCs on TCPS, p < 0.05; # Different from iDCs on SA coated TCPS, p<0.05.

DC Response to OVA and CationizedBSA Functionalized with Mannose or

Synthetic Oligomannose 5 (Syn-Man5)

N. Hotaling in collaboration with D. Ratner, University of Washington

Bio-Man5 fromR. Cummings

Syn-Man5 fromD. Ratner

Summary• DCs may use mechanisms analogous to

pathogen recognition to respond tobiomaterials (e.g. TLRs, CLRs)

• Glycans recognizable by DC CLRs aredetected associated with the adsorbed serumprotein layer, as directed by the underlyingsurface chemistry. This correlates with adifferential DC phenotypic response.

• Protein glycosylations are important forimmune cell (e.g. DC) interaction with abiomaterial

• Context of glycan presentation to DCs isimportant for an immunofunctional effect.

• Identify immunomodulatory glycans andoptimal mode of presentation for DC effects

• Modification of biomaterial surfaces andvaccine carriers can be used to direct hostimmune responses

T.B.H. Geijtenbeek and S.I. Gringhuis, Nature Rev Immunol (2009).

AcknowledgementsFunding1RO1EB004633-01A1, National Institutes of Health, NIBIB/NHLBIGeorgia Tech/Emory Center for the Engineering of Living Tissue(GTEC) grant, an NSF ERC, EEC-9731643CAREER Award, National Science FoundationArthritis Investigator Grant, The Arthritis FoundationJ&J – Georgia Institute of Technology Healthcare Innovation AwardConsortium for Functional Glycomics (NIH/NHLBI)Wallace H. Coulter GT/Emory-PKU BME Collaborative Seed Grant

Students/Postdocs

Melissa Stein Matzelle

Leah Moore

Mutsumi Yoshida

Saul Lee

Abhijit Paranjpe

Nancy Bennewitz

Sucharita Shankar

Peng Meng Kou

Jessica Mata

Jaehyung Park

Kate Lee

Todd Rogers

Christina Duden

Inn Inn Chen

Nathan Hotaling

Dr. Lori Norton

Collaborators

Judith Kapp, Christian Larsen, Bali Pulendran, R. Cummings,

D. Smith (EU)

Mary Marovich and Mike Eller (Walter Reed)

Polly Matzinger (NIAID, NIH)

John L. Brash and Rena Cornelius (McMaster University)

Andres Garcia, Ben Benjamin Keselowsky, Jeffrey Capadona,

Dr.Brent Carter, Johnafel Crowe (GT)

D. Ratner (University of Washington)