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MIMM 314 > Lecture 4: Applied Innate Immunity > Flashcards

Lecture 4: Applied Innate Immunity Flashcards

1
Q

describe experimental setup to look at bacterial RNA vs human RNA stimulating immune system

A

cultured human DCs and transfected with diff types of RNA in lipofectin to see how they trigger TNF-alpha

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2
Q

how does mammalian RNA stimulate TNF-alpha? why?

A

only mitochondrial RNA stimulated TNF-alpha bc it evolved from prokaryotes and is therefore similar to bacterial RNA so it stimulates immune response similarly

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3
Q

bacterial RNA + TNF-alpha stimulation

A

bacterial RNA stimulates TNF-alpha

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4
Q

are all naturally occurring RNA equally potent activators of DCs?

A

no –> mammalian RNA cannot trigger innate activity but bacterial RNA can

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5
Q

why does bacterial RNA trigger innate activity but mammalian RNA does not?

A

mammalian RNA has many modifications which prevent innate activity

bacterial RNA has fewer modifications to trigger more innate activity

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6
Q

how do we know that RNA is the active component that triggers TNF-alpha secretion?

A

Adding an RNAse eliminates all TNF-alpha signal

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7
Q

if mitochondrial RNA is part of the mammal, how does it signal the innate system?

A

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

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8
Q

could the size of RNA be affecting whether RNA triggers TLRs?

A

no –> modifications don’t change the size of RNA

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9
Q

Are all RNA modifications equal?

A

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

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10
Q

does TLR9 respond to modification?

A

no –> recognizes unmethylated RNA

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11
Q

what does TNF-alpha do?

A
  1. PRO-inflammatory
  2. anti-tumour
  3. causes acute phase response to induce vasodilation for inflammation
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12
Q

what does IL12 do? what type of cytokine is it?

A

made by DCs to stimulate IFNgamma production from T cells

Th1 cytokine

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13
Q

how do modifications affect TNF-alpha and IL-12 secretion?

A

RNA modifications reduce the ability for RNA to induce TNF-alpha and IL-12

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14
Q

why is it important to look at stimulation of different types of DCs?

A

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

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15
Q

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

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

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16
Q

significance of RIG-I-like receptors?

A

antiviral PRR that recognizes certain RNA modifications but not others

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17
Q

OVERALL: 2 factors that allow RNA to induce DCs to mature and secrete cytokines

A
  1. DC subtype
  2. characteristics of the RNA modifications
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18
Q

describe the type 1 IFN system

A

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

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19
Q

what does T cell activation by DCs require?

A

requires MHC, TCR, and co-stimulatory molecules

20
Q

what is the role of co-stimulatory molecules?

A

amplify TCR signaling

21
Q

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

A

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

22
Q

do RNA modifications need to be large modifications to inhibit the immune response or can they by single nucleotide modifications? how do we know?

A

single nucleotide modification is enough to prevent immune response

just one psi modification can completely inhibit the TNF response

23
Q

do modifications affect TRANSLATION in order to block the immune response? how do we know?

A

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

24
Q

what are 4 experimental tests/readouts that one could perform in vitro to assess the immunogenicity of a vaccine candidate?

A
  1. measure expression of co-stimulatory molecules
  2. measure expression/transcription of cytokines
  3. measure translation/secretion of cytokines
  4. measure T cell activation
25
Q

how can we measure if T cells are being activated by RNA vaccine candidate?

A
  • 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
26
Q

what are 3 factors that dictate whether cells produce inflammatory cytokines to DNA/RNA?

A
  1. RNA modifications
  2. receptors on cells
  3. types of DCs bc each express diff TLRs
27
Q

define physiological inflammation and example

A

an active immune response to maintain homeostasis

ex. macrophages phagocytosing dead cells but not enough to cause pathological inflammation

28
Q

what type of cytokines are induced by helminths? what is their role?

A

Th2 –> allows expulsion of helminths

29
Q

what happens to the expulsion of worms in:
- WT mice
- IL13 KO mice
- IL13/IL4 KO mice

A

WT mice –> expel the worm quickly

IL13 KO mice –> delayed expulsion

IL13/IL4 KO mice –> even further delayed expulsion

30
Q

what happens to worm expulsion in IL25 KO mice?

A

IL25 KO mice had delayed worm expulsion similar to IL13 and IL4

31
Q

what happens to the expulsion of worms in:
- WT mice
- rag1 KO mice
- WT mice + recombinant IL25
- rag1 KO mice + recombinant IL25

A

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

32
Q

what is rag1?

A

recombination activating gene –> required by lymphocytes to recombine TCR and BCR and be selected

33
Q

why are rag1 KO mice unable to expel the worm?

A

without recombined TCR, T cells cannot be activated to help expel the worm

34
Q

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?

A

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

35
Q

what are FALCs and where are they located?

A

FALC = Fat Associated Lymphoid Clusters

found in mesenteric area of intestine

36
Q

what is forward scatter in flow cytometry?

A

indicates the size of a cell

more light reflected = larger cell = higher on X axis

37
Q

what is side scatter in flow cytometry?

A

indicates granularity of a cell

less light going thru = more granular = higher on Y axis

38
Q

describe forward and side scatter of lymphocytes

A

on bottom right of graph –> lymphocytes are smaller and less granular

39
Q

to study FALCs, why did they gate on the flow cytometry results that were smaller and less granular?

A

FALCs had lymphocyte-like cells so they wanted to focus on these

40
Q

what did they do after the lymphocyte gate on FALC cells?

A

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

41
Q

what was unique about the unknown lineage negative cells?

A

they expressed markers of hematopoietic stem cells

42
Q

what was the phenotype of the unknown lineage negative cells?

A

stained their nuclei and found the cells had huge nuclei with little cytoplasm –> similar to lymphocyte

43
Q

describe the markers expressed on the unknown cells

A

the cells expressed markers that look like lymphoid origin cells

44
Q

if the unknown cells look like lymphocytes, do they require the same signaling for their development as other lymphocytes?

A

yes –> KO the common gamma chain required as growth factor in all lymphocytes and the cells die

45
Q

why are these new lymphoid-like cells considered to be unconventional lymphoid cells?

A

they are unconventional lymphoid origin cells –> unconventional bc no TCR or BCR

MIMM 314 (23 decks)

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