Week 15 Flashcards
Which innate immune cell populations are enhanced and coordinated by the three different subsets of ILCs/Th?
ILC1/Th1: Monocytes/Macrophages
ILC2/Th2: Mast cells, basophils and eosinophils
ILC3/Th17: Neutrophils
Main cytokine produced by ILC1s/NKs
IFNg, in response to IL-12 and IL-18 produced by pathogen activated DCs and macrophages.
Do ILC2s produce IL-4?
ILC2s do not appear too produce IL-4 in vivo, suggesting that they might not promote the differentiation of Th2 cells
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PSGL-1
P-selectin glycoprotein ligand-1 (PSGL-1), a homodimeric sialo- glycoprotein that is the major ligand for tethering and rolling on the P- and E-selectin expressed by activated endothelial cells at sites of inflammation
Upregulated by activated effector T cells after activation in the LN.
FucT-VII
a1,3-fucosyl- transferase VII, a key enzyme required for both P- and E-selectin ligand generation. Expressed constitutively by myelomonocytes and on effector T cell when activated.
cutaneous lymphocyte antigen (CLA)
adhesion molecule that is an isoform of PSGL-1 that differs in its pattern of glycosylation and binds to E-selectin on cutaneous vascular endothelium. Expressed by effector T cells that are primed in the draining LNs of skin to home back to the cutaneous tissue.
homeostatic chemokines
In contrast to inflammatory chemokines, homeostatic chemokines are produced at steady state for tissue-homing of lymphocytes.
Signals from Th1 cells required for M1 macrophage activation
IFNg and CD40L (or lymphotoxin)
Activated M1s produce TNFa, which is necessary for the maintainence of M1s,
Two ways that Th1 cells recruit more phagocytic cells
Activated Th1 cells produce IL-3 and GM-CSF, which stimulate the hematopoietic production of new monocytes in the BM.
TNFa and lymphotoxin produced by Th1 cells activate the endothelial cells to increase adherence of circulating monocytes, which extravasate and differentiate into Mqs.
Chemokine receptors expressed by Th2 cells, eosinophils, and basophils
CCR3, CCR4
How do mucosal mast cells differ from mast cells in other tissues?
Mucosal mast cells have significantly lower amounts of FcER, and make very little histamine. When activated, they produce inflammatory mediators that lead to increased permeability in the epithelium and vasculature and contribute to intestinal motility.
Chemokine receptors expressed by Th17 and ILC3s
CCR6.
Which cells (and what signal) are required for the development of M cells?
Subepithelial mesenchymal cells highly expressing the membrane-bound form of RANKL have been defined as M cell inducer cells.
How is IL-22 signaling unique in the FAE?
IL-22 dependent AMP production is downregulated in the FAE due to production of high levels of IL22BP in the FAE microenvironment.
How do CX3CR1+ MNPs navigate TED extension?
They produce occludins that allow them to navigate the tight junctions between epithelial cells.
What are manners in which access to antigens is regulated in the mucosa?
Goblet-Associated Passages (GAPs)
CX3CR1+ MNP TED luminal sampling
M cells
a4B7 receptor
MaDCAM-1 homing to the lamina propia
How do the DCs of PPs imprint lymphocytes:
They are better at imprinting a gut homing phenotype in lymphocytes, particularly the expression of a4B7. Better than splenic or other LN DCs.
How do PP DCs imprint gut homing on PP lymphocytes?
Vitamin A / Retinoic Acid
What are the two different types of IELs?
- (Type A) Traditional CD8+ CTLs (aB TCR with CD8a:B heterodimer)
- `(Type B) CD8a:a homodimer cells w/ a:B or g:d TCRs that respond to stress signals/ligands.
Where is RORgt expressed?
in differentiated cells it is expressed in ILC3s, Th17 cells, and iTregs but ALL T cells that pass through the thymus express it at some point (so it is not good for a cre model)
molecules required for the accumulation of CD8+ VACV-specific T cells in the skin
P- and E-selectin ligands.
CD4 and IFNg seems not to be required.
Good enough definition of herd immunity
When vaccine coverage is above the threshold for herd protection, infection cannot spread in the population and susceptible individuals are indirectly protected by vaccinated individuals.
Vacinia virus
Cowpox virus that has been passaged numerous times to render it apathogenic; used to inoculate against smallpox via skin scarification
FTY720
S1P agonist that prevents lymphocyte egress from the LNs
Why are so many vaccines administered intramuscularly?
I.m. administration is often preferred because it is easy to perform and generally well tolerated, with a low risk for adverse reactions at the site of injection
Skin vaccination has recently been shown to be a potentially superior method of inoculation, as it is more immunocompetent than muscle sites.
There were no differences in humoral immunity between i.m. and s.c. routes, but much higher adverse effects in s.c. in trials, so IM became the dominant method.
Why do adjuvants have to be given within three hours of a vaccine?
Adjuvants will often assist with the licensing of the DCs - without synchronous licensing, the immune response is blunted.
CD8a expression in DCs.
CD8a expression delineates DC populations in mice, but not in humans, who do not express CD8a on DCs.
CD8a+ DCs make up 20-30% of spleen and LN DCs at homeostasis, are semi-mature (CD86-mid), and reside within T cell zones.
CD8a- DCs (in mice) mostly reside in the MZ and the red pulp f the spleen and migrate to T cell zones after microbial stimulation.
What are the effects of using alum as an adjuvant?
alum enhances antigen uptake by DCs, cell recruitment to the injection site and stimulation of immune cells via the inflammasome, although there was some dispute regarding the specifics of the latter mechanism
However, taken together, some common themes emerge: alum affects antigen uptake, induces danger signals, recruits various types of immune cells and elicits TH2 responses.
Alum is TLR-independent (still works in MyD88/TRIF KO mice). Uric acid may be required for the activation of inflammasome.
What are some broad benefits of adjuvants?
Dose sparing (less antigen per vaccine increases the global supply)
fewer doses needed per person
More rapid response
More broad response to combat antigenic drift
Greater antibody titer magnitude
Alum
Classical adjuvant including a range of aluminium precipitated under basic conditions, usually aluminum sulfate mixed with sodium or potassium hydroxide plus a variable amount of phosphate
sensed by NLRP3 inflammasome
Roles of alum as an adjuvant
antigen depot
mild irritant (recruits leukocytes)
does not enhance CD8
triggers some necrosis, which contributes to inflammasome activation
Alum salts promote DNA release with consequent activation of IRF3 and enhanced migration of inflamatory monocytes to dLNs.
Depends on IL-1B, uric acid
Rubella vs HIV in terms of correlates of protection
Pregnant women are routinely screen for anti-rubella antibodies because it is important that they have immunity to this TORCH infection.
For HIV, the presence of antibodies is not a correlate of protection at all
Three main bacterial pathogens that cause bacteremic disease and their correlates of protection
Haemophilus influenzae type b
pneumococci
meningococci
Correlates of protection are opsonophagocytic or bactericidal antibodies
Why is alum an unsuitable adjuvant for vaccines against HIV malaria or tuberculosis?
Alum induces strong humoral immune responses, but is a poor inducer of cellular immunity, which is required for protection against these diseases.
alum nodules
Formed within 4h post-injection due to interactions between alum and fibrinogen. Are thought to serve as a ‘depot’ for slow release of antigen, although this has come under question, as removing the nodules after their formation does not appear to affect immune response. Additionally fibrinogen-deficient mice seem equally unaffected.
What is the one immunological pathway that alum is definitely dependent on as an adjuvant?
NLRP3-deficient mice have abrogated vaccine responses.
AS04
Adjuvant System 04 (AS04), which is composed of alum and the TLR4 ligand, monophosphoryl A
How might macrophages be involved in the etiology of SLE?
a role for macrophages in the pathogenesis of SLE was first proposed following the discovery that SLE macrophages were defective in their ability to clear apoptotic cell debris, thus prolonging exposure of potential auto-antigens to the adaptive immune cells
How might neutrophils be involved in the etiology of SLE?
Neutrophils from SLE patients have been shown to have increased NETosis and decreased clearance of NETs, providing additional genetic material for the formation of autoimmunity
Downstream signaling of type I IFNs
type I interferons bind to heterodimeric IFNARs and signal through JAK1 and TYK1 to form STAT1/STAT2 heterodimers that form a heterotrimer with IRF9. This heterotrimer is also called ISGF3, and binds DNA elements termed interferon-sensitive response element (ISREs)
Downstream signaling of IFNg (type II interferon)
IFN-γ binding to its receptor activates JAK1 and JAK2, and predominantly STAT1 homodimers. STAT1 binds a distinct DNA element termed a gamma- activated site (GAS) and directly activates a distinct set of ISGs, notably chemokines such as CXCL10 and transcription factors including IRFs
RIG-I like receptors (RLR)
RNA helicases that sense RNA from viruses in the cytoplasm and induce inflammatory cytokines and type I interferons
broad expression in tissues
Consist of mainly two sensors: RIG-I and MDA5
3 distinct domains of RIG-I like receptors
N-terminal domain - CARD domains that are essential for downstream signaling
A central DExD/H box RNA helicase domain that is required for ligand recognition or binding but also has ATPase activity
C-terminal repressor domain essential for autoregulation.
What are the adaptor proteins for nucleic acid sensing in the cytosol and which organelles are they associated with?
STING is downstream of cGAS, AIM2 and is associated with the ER
MAVs is downstream of RIG-I/MDA5 and is associated with the mitochondria
Why does TLR9 (and other TLRs) need to form homodimers for signaling?
The ligand for TLR9 (CpG) fits into the groove created by the endosomal domain of two receptors - they cannot recognize the ligand or be activated by it as monomers.
Cellular distribution and downstream cytokine production of TLR7, TLR8, and TLR9 in humans.
TLR7 and TLR9 are highly expressed in pDCs and B cells and lead to IFNa and antibody production.
TLR8 is highly expressed in monocytes, mDCs, and neutrophils and leads to the production of TNF, IL-6, IL-1 etc.
This is different in mice (very little TLR8 in mice, and more widespread TLR7 and TLR9 expression in myeloid cells), which makes murine research of these pathways complicated
What is the presumed distribution of evolutionary pressure on TLRs in humans?
A study looked at nonsense or damaging mutations in each of the TLRs from individuals across the world, and found that the majority of TLR mutations were in non-nucleic acid-sensing TLRs. Nucleic-acid sensing TLRs had nearly no mutations, suggesting a highly selective pressure on these receptors.
Why are humans deficient for MyD88 or IRAK4 not more susceptible to viral infections?
There is great redundancy in IFN signaling - even lacking nucleic acid TLR sensing, these humans can still make IFN in response to virus via multiple other cytosolic sensing pathways.
Kinetics of type I IFN response to viral infection
pDCs are typically the first cells to respond to a viral infection, through nucleic acid sensing via TLR7/9 that produces lots of IFN I.
This is followed later by other sources of type I IFN, including cDCs, macrophages, B cells, monocytes, stroma etc. through TLRs, RLRs, DNA sensors, etc.
Kinetics of pDC activation
Activated pDCs at 8-10h post-activation will have over 60% of their gene expression be Type I Interferon production. At this stage they express little to no costimulatory molecules (CD86)
This is more or less completely shut down after 48h. At this point, the morphology changes greatly to more of a DC-like morphology, and CD86 is highly upregulated.
How are pDCs able to produce so much type I IFN so quickly?
They make lots of pre-formed IRF7 and keep it in the cytoplasm. Upon activation, this transcription factor is translocated to the nucleus for rapid IFN-I production
How are pDCs like “Adjuvant cells”?
They recognize the antigen, produce lots of IFN-I, and this IFN-I makes everything work ‘better’
What is some evidence of a role for TLR7 and TLR8 in autoimmunity?
Overexpression of TLRs is enough to promote autoimmunity/break tolerance
TLR7 gene duplication is responsible for disease in yaa lupus model
WT mice that overexpress TLR7 or TLR8 spontaneously develop lupus or arthritis, respectively.
What aspect of pDC activation is likely responsible for pathology?
Chronic inflammation or activation of pDCs leads to pDC-dependent lesions. It is the length (not the magnitude) of pDC activation that leads to pathology.
Which cytokine heightens the pDC activation and type I IFN response to genetic material?
CXCL4 (also secreted by pDCs themselves) leads to higher IFNa secretion.
How does the stiffness of the local microenvironment affect pDC activation?
pDCs make less type I IFN with increasing stiffness of their surroundings - thus when fibrosis/wound-healing begins to happen the pDCs can sense this and begin to inhibit their IFN-I production.
Major cytokines leading to fibrosis in scleroderma
IL-4, TGF-B, and IL-6.
TLR8 in mice
Differs by five amino acids in mice, and this leads to an inability to recognize RNA appropriately. This makes it difficult to study TLR8 in mice.
Role of pDCs in SSc (Systemic scleroderma)
pDCs infiltrate the skin of SSc patients and produce large amounts of IFN-a and CXCL4, which leads to fibrosis. This was recently shown to be dependent on ‘ectopic’ expression of TLR8 in pDCs.
pDC-specific (mostly) markers
the C-type lectin BDCA2 (also known as CLEC4C)
and the immunoglobulin superfamily receptor immunoglobulin-like transcript 7 (ILT7; also known as LILRA4)
Extrafollicular Th cell phenotypes
CXCR4+ ICOS+ CD40L+
DNase1L3
DNase1L3 is an endogenously produced extracellular DNase targeting DNA from NETs and apoptotic cells. Mice deficient in this enzyme develop anti-dsDNA antibodies and SLE like disease.
TLR7 ligand
ssRNA
TLR7 and TLR9 expression
pDCs and B cells (mostly)
GP2
glycoprotein expressed by apical side of M cells that recognizes bacterial FimH protein found in type I pili and likely facilitates their transcytosis to the underlying follicle.
Chemokines expressed in the FAE that recruit gut-associated DCs to sample antigen
CCL20 (CCR6 is ligand)
CCL9 (CCR1 is ligand)
lymphoid compartment of the gut endothelium
mainly lymphocytes, nearly all of which are CD8 T cells in the SI
T cell chemokine receptor in the lamina propia
integrin a4B7 and CCR9
naive T cell chemokine receptors that allow entry into the HEVs of PPs in the SI
CCR7 and L-selectin
CD103
alpha-e:B7, which ligates E-cadherin on epithelial cells
MAdCAM-1
Addressin expressed on the endothelial cells of blood vessels of the gut wall; binds to a4B7 expressed by lymphocytes activated in PPs or MLN
CCL25
Chemokine expressed by the epithelial cells of the small intestine that ligates CCR9 expressed by SI-homing lymphocytes
CCL28
Chemokine expressed by the colon that ligates CCR10 expressed by LI-homing lymphocytes
How frequently do mucosal DCs migrate to the MLN?
5-10% of mucosal DC population migrates to the MLN to present antigen every day. In the absence of infection, this produces Tregs that home back to the gut wall
retinoic acid
Vitamin A derivative that is produced by stromal cells of MLNs and gut DCs and is required for the induction of IgA responses and Treg induction in mucosal immunity.
What is unique about macrophages in the LP of the gut?
They require constant replenishment from blood monocytes. They do not migrate back to the draining lymph nodes and cannot present antigen to T cells.
Their job is to engulf and degrade commensal products that breach the barrier, releasing anti-inflammatory cytokines all the while.
They may in turn pass antigen to DCs for presentation to T cells.
rheumatoid factor
anti-IgG autoantibody made by B cells whose BCR is cross-linked by immune complexes leading to rheumatoid arthritis
Three ways in which immune privileged sites are kept so
- physical barrier (blood/brain barrier, e.g.)
- abundant TGFB that promotes Tregs
- High levels of Fas ligand
myasthenia gravis
autoantibodies produced against the acetylcholine receptor block receptor function at the neuromuscular junction, resulting in a syndrome of muscle weakness
