1. Sialic acid as a key modulator of DCs biology
Improved dendritic cell-based anti-cancer vaccines trough glycan engineering
T Ferro1,5, M Silva1, Z Silva1,5, G Marques1, H Crespo1, H Soares1, S van Vliet2, A Fernandes3,5, Y van Kooyk2, T Matos4, J A Ferreira6,7, P Videira1,5
Deciphering molecular aspects: the role of MHC-I in antigen presentation
Introduction
Dendritic cells (DCs) are the most potent antigen presenting cells
Originating either from the lymphoid or myeloid lineage, DCs interact and partipate in both innate and adaptive immune responses
To understand the molecular mechanisms behind this phenomena, sialylated proteins on DCs surface were identified by a sialic acid binding lectin (SNA) pull down, followed by mass spectrometry.
The MHC-I was identified and hypothesised as a possible candidate to be modulated by sialic acid content. In fact, sialidase treated human DCs show higher ability to activate anti-tumor activity on T cells and increased expression of MHC-I.
Maturation Signals
Higher levels of
sialylation
Immature DC
Lower levels of
Mature DC
Dendritic cells show increased expression of MHC-I after desialylation
MHC-I
SNA
α2,6 sialic acid binding lectin
DC contains high levels of the monossacharide sialic acid at the termini of glycans of certain cell surface glycoproteins
MHC-I is glicosylated and sialylated: target for sialidase enzymes
Cell lysates were treated with sialidase and probed for MHC-I by Western Blot.
MHC-I shows decreased molecular weight upon desialylation.
Glycoprotein
Sialic acid
N-acetyl glucosamine
Galactose
Manose
Fucose
Asn
DC maturation induces loss of sialic acid
Maturation can be also achieved by sialic acid shortage
Desialylation improves MHC-I stability on cell surface
CMVpp65 peptide matches HLA-A02*01 pocket. MHC-I is more stable after sialidase treatment. Extrinsic sialylation by ST6Gal1 counteracts this effect. Control cells in black.
A MHC-I decay assay was performed on T2 cells following sialidase treatment.
MHC-I is stable on the membrane for up to 3 hours after sialidase treatment, when Golgi export is blocked by brefeldin A.
Lack of sialic acid induces superior anti-tumoral immune responses
T2 cell line: experimental model to study MHC-I (HLA-A02*01) turnover upon desialylation
Sialic acid removal improves 
co-stimulatory molecules expression
Conclusions
Sialic acid as a key modulator of DCs biology
- Immune response is dependent of sialic acid content at cell surface
- Desialylated human DCs show increased hability to activate T cells and promote cytotoxic responses
T cells primed by desialylated DCs show higher degranulation
Increased secretion of cytokines
Maturation
TH1 phenotype
T cell activation
Improved IFN-γ secretion
DCs Tumor lysates
DCs
DCs Sialidase treated + Tumor lysates
T + DCs Tumor lysates T
T + DCs Sialidase treated + Tumor lysates
T + DCs
- MHC-I as a molecular effector of anti-tumoral responses
DCs were pulsed with MCF-7 lysates
Cytotoxic activity was evaluated by tumor cell viability following interaction with primed T cells
Degranulation evaluated by CD107 expression
T + DCs Tumor lysates
T + DCs Sialidase treated + Tumor lysates
- Sialic acid may influence MHC-I turnover at cell surface
Author affiliations: 1. CEDOC, NOVA Medical School / Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Portugal
2. Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands
3. CQE, Centro de Química Estrutural, Instituto Superior Técnico, ULisboa, Portugal
4. StemLab, Cantanhede, Portugal
5. UCIBIO, Departamento Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, Portugal
6. QOPNA, Mass Spectrometry Center, Department of Chemistry, University of Aveiro, Aveiro, Portugal
7. Experimental Pathology and Therapeutics Group, Portuguese Institute of Oncology, Porto, Portugal
References: 1. Oncotarget. 2016; 7: doi: /oncotarget.9419
2. Patent PCT/IB2016/ (A VIABLE CELL POPULATION, METHOD FOR PRODUCTION AND USES THEREOF)
This work was supported by the Applied Molecular Biosciences Unit - UCIBIO which is financed by national funds from FCT/MEC (UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI FEDER ).