Lecture 4: Applied Innate Immunity Flashcards
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
how can we measure if T cells are being activated by RNA vaccine candidate?
- culture DCs with the diff RNAs
- add the DCs to the T cells with antigen that can be presented by DCs
- T cell will be activated if lots of cytokines and costimulatory molecules
what are 3 factors that dictate whether cells produce inflammatory cytokines to DNA/RNA?
- RNA modifications
- receptors on cells
- types of DCs bc each express diff TLRs
define physiological inflammation and example
an active immune response to maintain homeostasis
ex. macrophages phagocytosing dead cells but not enough to cause pathological inflammation
what type of cytokines are induced by helminths? what is their role?
Th2 –> allows expulsion of helminths
what happens to the expulsion of worms in:
- WT mice
- IL13 KO mice
- IL13/IL4 KO mice
WT mice –> expel the worm quickly
IL13 KO mice –> delayed expulsion
IL13/IL4 KO mice –> even further delayed expulsion
what happens to worm expulsion in IL25 KO mice?
IL25 KO mice had delayed worm expulsion similar to IL13 and IL4
what happens to the expulsion of worms in:
- WT mice
- rag1 KO mice
- WT mice + recombinant IL25
- rag1 KO mice + recombinant IL25
WT mice –> expel the worm quickly
rag1 KO mice –> maintain the worms
WT mice + recombinant IL25 –> expel the worm even faster than WT alone
rag1 KO mice + recombinant IL25 –> expel the worm as quickly as WT mice + recombinant IL25
what is rag1?
recombination activating gene –> required by lymphocytes to recombine TCR and BCR and be selected
why are rag1 KO mice unable to expel the worm?
without recombined TCR, T cells cannot be activated to help expel the worm
what is the significance of rag1 KO mice with recombinant IL25 being able to clear the worm very quickly? what does this mean for IL13?
rag1KO without IL25 could not expel the worm but with IL25 it was very efficient
therefore, IL25 is not acting thru T cell pathway –> IL25 alone is enough to expel the worm
sine IL13 is critical for expulsion, there must be another source of IL13 that is NOT activated T cells
what are FALCs and where are they located?
FALC = Fat Associated Lymphoid Clusters
found in mesenteric area of intestine
what is forward scatter in flow cytometry?
indicates the size of a cell
more light reflected = larger cell = higher on X axis
what is side scatter in flow cytometry?
indicates granularity of a cell
less light going thru = more granular = higher on Y axis
describe forward and side scatter of lymphocytes
on bottom right of graph –> lymphocytes are smaller and less granular
to study FALCs, why did they gate on the flow cytometry results that were smaller and less granular?
FALCs had lymphocyte-like cells so they wanted to focus on these
what did they do after the lymphocyte gate on FALC cells?
once they gated for the lymphocyte-like cells, looked at markers on cell surface of cell types that we already know (lineage positive cells)
i.e. find lineage NEGATIVE cells –> these are unknown cell types
what was unique about the unknown lineage negative cells?
they expressed markers of hematopoietic stem cells
what was the phenotype of the unknown lineage negative cells?
stained their nuclei and found the cells had huge nuclei with little cytoplasm –> similar to lymphocyte
describe the markers expressed on the unknown cells
the cells expressed markers that look like lymphoid origin cells
if the unknown cells look like lymphocytes, do they require the same signaling for their development as other lymphocytes?
yes –> KO the common gamma chain required as growth factor in all lymphocytes and the cells die
why are these new lymphoid-like cells considered to be unconventional lymphoid cells?
they are unconventional lymphoid origin cells –> unconventional bc no TCR or BCR
