Lecture 4: Applied Innate Immunity Flashcards
(45 cards)
describe experimental setup to look at bacterial RNA vs human RNA stimulating immune system
cultured human DCs and transfected with diff types of RNA in lipofectin to see how they trigger TNF-alpha
how does mammalian RNA stimulate TNF-alpha? why?
only mitochondrial RNA stimulated TNF-alpha bc it evolved from prokaryotes and is therefore similar to bacterial RNA so it stimulates immune response similarly
bacterial RNA + TNF-alpha stimulation
bacterial RNA stimulates TNF-alpha
are all naturally occurring RNA equally potent activators of DCs?
no –> mammalian RNA cannot trigger innate activity but bacterial RNA can
why does bacterial RNA trigger innate activity but mammalian RNA does not?
mammalian RNA has many modifications which prevent innate activity
bacterial RNA has fewer modifications to trigger more innate activity
how do we know that RNA is the active component that triggers TNF-alpha secretion?
Adding an RNAse eliminates all TNF-alpha signal
if mitochondrial RNA is part of the mammal, how does it signal the innate system?
mtRNA acts as a DAMP bc it is part of our cells –> when cells lyse, the cells think there is bacterial RNA and mount innate response
could the size of RNA be affecting whether RNA triggers TLRs?
no –> modifications don’t change the size of RNA
Are all RNA modifications equal?
no –> in TLR-expressing cell line, some modifications are more potent at reducing TLR response to RNA, i.e. RNA activates TLR7/8 and TLR3 differently
does TLR9 respond to modification?
no –> recognizes unmethylated RNA
what does TNF-alpha do?
- PRO-inflammatory
- anti-tumour
- causes acute phase response to induce vasodilation for inflammation
what does IL12 do? what type of cytokine is it?
made by DCs to stimulate IFNgamma production from T cells
Th1 cytokine
how do modifications affect TNF-alpha and IL-12 secretion?
RNA modifications reduce the ability for RNA to induce TNF-alpha and IL-12
why is it important to look at stimulation of different types of DCs?
diff DCs have diff receptors, diff combos of receptors, diff cytokines, etc. which means they respond differently to diff things and allows for some specificity in innate response
Production of TNF-alpha under diff conditions:
a) Methylation of adenosine
b) Psi modification
c) Methylation of adenosine and psi modification on same RNA
d) Methylation of adenosine and psi modification both present but on diff RNA
a) Methylation of adenosine
- DCs make TNF-alpha
b) Psi modification
- DCs cannot make TNF-alpha
c) Methylation of adenosine and psi modification on same RNA
- DCs cannot make TNF-alpha
d) Methylation of adenosine and psi modification both present but on diff RNA
- Methylation of adenosine rescues ability for DCs to make TNF-alpha
significance of RIG-I-like receptors?
antiviral PRR that recognizes certain RNA modifications but not others
OVERALL: 2 factors that allow RNA to induce DCs to mature and secrete cytokines
- DC subtype
- characteristics of the RNA modifications
describe the type 1 IFN system
DCs sense viral RNA thru TLR7/8 which stimulates type 1 IFN production by DC or macrophage
type 1 IFN can then act on the same cells OR act on epithelial cells
the epithelial cells will secrete ISG (IFN stimulating genes) that are directly antiviral/antibacterial which allows for SPECIFICITY
what does T cell activation by DCs require?
requires MHC, TCR, and co-stimulatory molecules
what is the role of co-stimulatory molecules?
amplify TCR signaling
describe the flow cytometry experiment used to see how DCs were being activated by RNA with modifications
- what specific markers were looked at?
- results with unmodified RNA
- results with modified RNA
looked at HLA-DR (MHCII on DC) and CD83 (costimulatory molecule for DC)
unmodified RNA: lots of MHCII and CD83 –> immune response activated
modified RNA: lower CD83 –> immune response inactive
**MHCII stays same bc always expressed on DCs
do RNA modifications need to be large modifications to inhibit the immune response or can they by single nucleotide modifications? how do we know?
single nucleotide modification is enough to prevent immune response
just one psi modification can completely inhibit the TNF response
do modifications affect TRANSLATION in order to block the immune response? how do we know?
yes, translation of modified mRNA is required to inhibit immune response
if you add translation inhibitor (cyclohexamide), TNF-alpha expression is restored –> therefore need translation to inhibit the immune response
what are 4 experimental tests/readouts that one could perform in vitro to assess the immunogenicity of a vaccine candidate?
- measure expression of co-stimulatory molecules
- measure expression/transcription of cytokines
- measure translation/secretion of cytokines
- measure T cell activation