Immunotherapeutic action of extracellular vesicles and effects of TLR signaling to immune dysfunction of SCI patients
Author
Güçlüler, Gözde
Advisor
Gürsel, İhsan
Date
2017-03Publisher
Bilkent University
Language
English
Type
ThesisItem Usage Stats
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Show full item recordAbstract
The primary aim of this thesis is to extend the breadth of in vivo application of
externally loaded exosomes as prophylactic or therapeutic carriers against disease
treatment. Exosomes are secreted from all cells and could be purified from all bodily
fluids; however, engineering of exosomes to carry specific ligands post-purification
is a daunting task. Herein, we show that lyophilization of exosomes together with the
biological cargo alone or in combination of CpG ODN motifs, model protein antigen
ovalbumin or lipidic ligand alpha-galactosylceramide (αGC) followed by controlled
reconstitution successfully internalizes these cargos within exosomes. Furthermore,
the bioactivity of the loaded ligand(s) surpasses the unloaded free ligand activities.
When tested in vivo, exosome incorporated ligand(s) proved to be significantly
effective against model tumors such as E.G7 thymoma or established melanoma
models. The mechanism behind this elevated immune activity is the ability of
exosomes to be delivered to target cells and boost immune antigen dependent
immune activation. Our in vitro findings revealed that encapsulation of CpG ODN
into exosomes enhances immunostimulatory activity of CpG ODN than free form as
evidenced by superior levels of cytokines like IL6, IL12 and Type-I and II
interferons. This magnified immune activity might be partly due the increased APC
activation observed as elevated CD86/MHCII surface marker expression.
Immunization of C57/Bl6 mice with exosomal CpG ODN plus OVA induced strong
Th1-biased anti-OVA response. Following thymoma induction in naive and OVAimmunized
animals, >85% of exosomal vaccine treated mice cleared tumors whereas
almost all naive animals were positive for tumor. This data suggests that CpG ODN
encapsulation into exosomes improve immunostimulatory activity, provide better
anti-OVA immunity thereby contribute effective tumor clearance in mice. A second
aim of this thesis was to establish that it is feasible to load exosomes with more than
two ligands. Next, invariant natural killer T (iNKT) cell ligand αGC was included
within exosomes as the third element next to OVA and CpG ODN. Initial in vivo studies revealed that exosomes containing αGC were significantly more potent in
inducing antigen dependent immune responses in comparison to free form of CpG
ODN, OVA and αGC. In therapeutic tumor vaccine model, two exosome injections
(@d: 9 and d: 15) were done to B16-OVA tumor bearing animals and tumor
regression was followed. Mice that had triple exosomal ligands significantly reduced
tumors compared to mice treated with non-exosomal ligands. This study confirmed
that exosomes with triple ligands could be effectively control established tumor
development.
In this thesis, the elucidation of the involvement of extracellular vesicles (EVs) on
the pathogenesis of autoimmune/autoinflammatory diseases was studied. The
underlying mechanism in BD pathogenesis is still unclear. We found that one of the
human cathelicidin group members, antimicrobial peptide LL37 along with EVs
were elevated in active BD patients` plasmas. Strikingly, majority of plasma LL37
was associated with circulating EVs. We found that there was a strong correlation
between i) LL37 level, ii) EV #/ml plasma and iii) cytokine production.
In the last part of this thesis, one of the possible mechanisms of immune dysfunction
contributing to severe neurological deterioration of chronic spinal cord injured (SCI)
patients was unearthed. We aimed to investigate whether there is a correlation
between susceptibility to infections of chronic SCI patients within the context of
impaired innate recognition of pathogen associated molecular patterns (PAMPs). Our
data implicated that although there was no dysfunction of B cell, or CD4+ Treg
activity, but sensing TLR7 and TLR9 ligands by monocytes and pDCs were ablated
in patients with SCI, leading to lower IFNγ and IP10 production along with costimulatory
molecule expression, that could explain the immunological dysfunction
in patient with SCI contributing to persistent complications.
Keywords
Extracellular vesiclesMicrovesicles
Exosomes
Vaccine carrier
Antitumor therapy
Behçet’s Disease
LL37
Spinal cord injury