Immunology Flashcards
Mechanisms of Leukocyte Migration
1
Q
What is an unique hallmark of cells of the immune system?
A
Motility -> most cells stay fixed through their lifetime
2
Q
True or false: entry into secondary lymphoid organs is random
A
False; this is a restricted and controlled process
3
Q
How do DCs enter lymph nodes? What happens after they have completed their function in the lymph node?
A
Entry: afferent lymphatics
DCs don’t leave the lymph nodes; they die there
4
Q
What is an important regulator of location and movement of lymphocytes in lymph nodes?
A
Lymphocytes are attached to stromal cells & ECM; these regulate movement and location
5
Q
Which two classes of signaling molecules are involved in cellular migration?
A
- Chemokines
- Integrins and integrin ligands
6
Q
What are chemokines?
A
Small secreted proteins that drive chemotasis
7
Q
How many structurally related families of chemokines are there?
A
Four
8
Q
Which two groups of chemokines can be distinguished based on their expression?
A
- Inflammatory chemokines -> expressed in inflammation
- Homeostatic cemokines -> constitutive expression in lymphoid organs
9
Q
Which cells are mainly attracted by inflammatory chemokines?
A
Activated cells (lymphocytes)
10
Q
Which cells are mainly attracted by homeostatic chemokines?
A
Naïve lymphocytes & DCs
11
Q
Which three mechanisms can be used for chemotaxis with chemokines?
A
- Leukocyte chemotaxis based on soluble gradient -> cells migrate towards higher concentration
- Leukocyte haptotaxis -> based on gradient of chemokines bound to sugar structures on cell membranes
- Leukocyte chemokinesis/haptokinesis -> migration without gradient
12
Q
What are integrins & integrin ligands involved in?
A
Cell adhesion
13
Q
What is the structural makeup of integrins?
A
Multimeric proteins consisting of α- and β-chains
14
Q
Which classes of integrin receptors are there? (4)
A
- Leukocyte-specific receptors
- Laminin receptors
- Collagen receptors
- RGD-receptors
15
Q
What are the ligands of integrin receptors?
A
ECM proteins or cell surface proteins (ICAM/VCAM)
16
Q
How do DCs enter the lymph nodes? How do lymphocytes enter?
A
DCs enter via afferent lymphatics
Lymphocytes enter via HEV
17
Q
What is special about the HEV?
A
It contains cuboidal epithelium with HEV-specific sugar structures: peripheral node adressins (PNAd)
18
Q
What are peripheral node adressins?
A
HEV-specific sugar structures that allow lymphocytes to leave circulation there
19
Q
What is the homing receptor used by lymphocytes to find the HEV?
A
L-selectin (CD62L)
20
Q
What does activation of L-selectin on lymphocytes in the HEV lead to?
A
Rolling of cells
21
Q
What happens after rolling adhesion using L-selectin?
A
Sticking, induced by inside-out chemokine signaling
22
Q
What happens to integrins when they encouter chemokines? How does this relate to the term of inside-out signaling?
A
Integrins are usually present in inactive configuration to prevent binding to random surfaces; they are activated by chemokines encountered during rolling adhesion, allowing them to make high affinity bonds to molecules expressed on endothelium in HEV (ICAM)
There is a signal from inside the blood vessel, allowing the cell to extravasate
23
Q
How does shear stress contribute to leukocyte extravasation?
A
Causes cytoskeletal rearrangements that cause clustering of integrins -> increases binding affinity
24
Q
What are the steps of extravasation in the HEV?
A
- Rolling induced by selectins and adressins
- Sticking/stopping induced by chemokine-mediated activation of integrins (inside-out signaling)
- Diapedesis under the influence of chemokines
25
What is outside-in signaling? What is inside-out signaling?
Outside-in = activation of integrins through chemokines
Inside-out = integrin binding causing adhesion
26
What are location-specific markers of trans-HEV migration? What are cell type specific markers?
Local:
1. PNAd = peripheral node adressions
2. MAdCAM1 = mucosal adression cell adhesion molecule
Cell-specific:
T-cell: CCL21, produced by HEV
B-cell: CXCL13, produced by lymph node stromal cells
27
Where do lymphocytes collect when they do not encounter antigen in the lymph node?
Medullary sinuses
28
Which signaling mechanism regulates lymphocytes exit out of lymph nodes?
Sphigosine-1-phosphatase (S1P)
29
How does S1P regulate exit of lymphocytes?
S1P = synthesized by endothelial cells & red blood cells --> high concentraiton in body fluids --> lymhocytes want to migrate to body fluids
S1P-receptor gets internalized when it is activated --> downregulated in high [S1P] environments --> cell able to move into tissue again
When cell moves into tissue: repopulation of surface with S1P receptors --> once again allows cells to leave tissue
30
What is CD69 used as a marker for?
Early activation marker of lymphocytes
31
What is the function of CD69?
Prevents re-expression of S1P-receptor -> prevents lymphocytes from leaving lymph nodes to soon, allowing them to become fully activated
32
How can drugs that mimic S1P be used to influence immune disease?
Cause downregulation of S1P --> lymphocytes are sequestered in lymphoid organs, preventing them from causing damage elsehwere
33
How do activated T-cells know where to head after being activated in the lymph node?
They are instructed by activating DCs for their location -> signals can be traced back to specific tissue
Tissue-specificity of T-cells ensured by specific configuration of sugars, chemokines & integrins
34
What is the structural makeup of IgG?
Heavy chain: 3 constant, 1 variable domain
Light chain: 1 constant, 1 variable domain
Chains are connected by disulfide bonds in the hinge region
35
What is the function of the variable domain of the antibody? What is the name of this fragment?
Antigen binding, Fab-fragment
36
What is the Fc-fragment of the antibody?
The constant tail of the Ig
37
How can the Fc-domain of antibodies be modified?
Carbohydrate structures
38
What are the functions of the Fc-domain? (3)
1. Fc-receptor binding
2. Complement activation
3. Transport, such as transport across epithelial surfaces
39
True or false: Fc-receptors are expressed on almost every immune cell
True
40
True or false: all antibody isotypes use the same FcR
False; every isotype has its own FcR
41
True or false: all antibodies of a certain isotype have the same affinity for their FcR
False; FcR's have different affinities for antibody subclasses
42
True or false: FcRs always activate immune processes
False; FcR's can also have an inhibitory function
43
What are the functions of FcR's? (8)
1. Clearance of immune complexes
2. Phagocytosis, leading to antigen presentation
3. Production of cytokines
4. Antibody-dependent cellular cytotoxicity (ADCC)
5. Modulation of cellular activation
6. Mast cell degranulation
7. Prevention of auto-immunity by forming a late auto-immune checkpoint for B-cells
8. Transportation/recycling of IgG
44
What is the function of FcRs for mast cells/basophils?
Degranulation
45
What is the effect of FcRs on neutrophils, macrophages & monocytes?
Enables phagocytosis & oxidative burst
46
What is the function of FcRs for DCs?
Modulation of antigen presentation & immune responses
47
What is the effect of FcRs on plasma cells? Why is this useful?
Apoptosis -> plasma cells are stopped when antibody concentrations become (too) high
48
What is the process of FcR-mediated ADCC?
1. Antibody binds antigens on surface on target cells
2. NK-cell recognizes Fc-tail of antibody
3. FcR's on NK-cell crosslink, leading to release of perforin/granzyme
4. Target cell dies by apoptosis
49
In which instances is FcR-mediated ADCC activated? (4)
1. Defence against microbes
2. Anti-tumour responses
3. Auto-immune responses, when auto-antibodies bind to self-cells
4. Depleting antibodies/plasma cells (when used with anti-CD20 Ab)
50
How does FcR modulate antigen presentation on MHCII?
Immune complexes are recognized by through their Fc-tail by FcR -> complex internalized and degraded for presentation on the MHCII complex
Presence of FcR in the endosome prevents degradation of the antigen so that it can still be presented
51
Why are FcR's important for the loading of MHCI in cross-presentation pathways?
TRIM21 is an intracellular FcR that is necessary for the loading of MHCI, as it directs captured antigen to the proteasome instead of the MHCII-loading compartment -> antigens end up in the intracellular pathway and are presented on MHCI, allowing for the activation of T-cells
52
Which Fc-γ receptors are present in humans, in order of high to low affinity? What is their CD number?
1. FcγRI = CD64
2. FcγRIIA/B/C = CD32
3. RcγRIIIA/B = CD16
53
What is unique about FcγRI?
Due to its high affinity, it is able to bind monomeric IgG, whereas the other receptors require multimeric IgG
54
How do FcγR's signal into the cell? What are the exceptions? (2)
Most have an ITAM -> activating motif, activating the cell
Exceptions:
1. FcγRIIIB -> no intracellular domain
2. FcγRIIB -> ITIM
55
What is the effect of ITAM activation? What is the effect of ITIM activation?
ITAM = kinase -> activates cellular functions
ITIM = phosphatase -> inhibits cellular functions
56
What is the function of the FcγRIIB on B-cells? How does it affect plasma cells?
Inhibitory receptor on B-cells, affects signaling through the BCR. When FcγRIIB and BCR crosslink at the same time: inhibition of BCR singaling
Causes apoptosis in plasma cells
57
What does a lack of FcγRIIB result in?
Increased humoral responses
58
Which factors influence FcR-mediated signal output? (3)
1. Expression levels of receptors
2. Polymorphisms in the receptor, increasing/decreasing activity
3. Glycosylation of Fc-domain of the antibody -> affects binding strength of FcR and Ab
59
How can expression levels of FcR's vary?
1. Regular gene activity regulation
2. Copy number variations
60
How does glycosylation of Fc-domains of antibodies affect their effect? What is the effect of a lack of glycans?
It changes their binding affinity to the FcR
Lack of glycans prohibits binding to FcR
61
Which glycosylation strongly impacts binding affinity to FcγRIII?
Fucose -> 50x higher affinity
62
What determines whether an antibodies becomes fucosylated or not?
Encounter of soluble antigens leads to fucosylated IgG (=high affinity), whereas crosslinking of BCRs by membrane-bound antigen leads to unfucosylated IgG
63
Which factors are known to influence glycosylation of antibodies? (4)
1. Age
2. Following immunization
3. During pregnancy
4. Some auto-immune diseases
64
On which chromosome are the FcγII/FcγRIII's located? How does the gene locus impact FcR function?
Chromosome 1; can contain SNPs and CNVs that influence amount and/or activity of FcR's
65
With which disease are SNPs of FcγRIIB associated? Why?
SLE -> less inhibitory effect on BCR leads to more auto-immune features
66
Can lack of FcγRIIB alone lead to auto-immunity?
No; it can be a contributing factor, but more factors are needed
67
What is the effect of insufficient FcR-mediated clearence of immune complexes?
Sustained myeloid cell activation and induction of auto-immunity
68
What are the funtions of the neonatal FcR (FcRn)? (2)
1. Recycling of IgG, lengthening its half-life
2. Transport through mucosa and placenta
69
How does the FcRn mediate recycling of IgG?
Presence of FcRn in the endosome causes antibodies in the endosome to be transported back to the cell surface
70
True or false: all subclasses of IgG are recycled efficiently by the FcRn
False; the efficiency of recycling is subclass-dependent
71
What is the function of the polymeric IgR? Where can it be found?
Involved in transport of IgA/IgM through mucosal surfaces by binding to J-chains and allowing for transcytosis
Found in musocal surfaces
72
Where can the FcεR be found? What is its function?
Mast cells & basophils -> degranulation of FcεR causes degranulation
This can lead to allergies
73
What isotype does the FcαR bind?
Monomeric IgA
74
Which therapy makes use of FcRs?
IVIG
75
What are the described effector mechanisms of IVIG? (4)
1. Blocking of FcR's
2. Saturation of FcRn, causing for less effective recycling of pathogenic antibodies
3. Modulation of DCs
4. Modulation of FcR's on plasma cells
76
What is the difficulty in studying FcR's?
FcR's in mice differ from humans -> hard to find a good model system
77
What is the function of costimulation in T-cell activation? Is this process antigen-dependent or antigen-independent?
Ensures survival and proliferation of T-cell
Antigen-independent
78
What is the function of cytokines in T-cell activation?
Differentiation
79
What is the most important costimulatory molecule for T-cells? By which cells is it expressed?
CD28, expressed by naïve and some memory T-cells
80
What is the effect of CD28 activation on T-cells?
Marked increase of TCR-induced proliferation and survival
81
Which molecules activate CD28? Where can they be found?
CD80/CD86, found on professional APCs
82
Which signal is necessary to sustain the immune response after CD28 has started T-cell proliferation? Which cells express its ligand?
ICOS -> maintains activity of already differentiated cells
ICOS-L is expressed by activated APCs
83
Which T-cells express ICOS?
Effector and memory T-cells
84
Which negative costimulatory molecules turn off T-cell activation (2)?
1. PD-1
2. CTLA-4
85
What are the intracellular effects of costimulation? (2)
1. Upregulation of pro-survival genes (example: Bcl-xL)
2. Transcription of IL-2/IL-2R genes
86
What happens in the absence of costimulation?
Clonal anergy -> important mechanism in peripheral tolerance
87
What happens when anergic T-cells encounter costimulation at a later point?
They remain anergic
88
True or false: CD28 is essential for memory T-cell subset formation
True
89
What is 4-1BB? For which cell type is it especially important?
TNF-receptor family member that can replace CD28 to activate T-cells
Mianly important for CD8+ T-cells
90
Which cytokines induce a Th1 cell? (3) What is its transcription factor?
1. IL-12
2. IFN-γ
3. IL-18
Transcription factor = T-bet
91
Which cytokine induces a Th2 cell? What is its transcription factor?
IL-4
Transcription factor = GATA3
92
Which cytokines induce a Th17 cell? (4) What is its transcription factor?
1. IL-1
2. IL-6
3. IL-23
4. TGF-β
Transcription factor: RORγT
93
Which cytokines induce a Treg cell? (2) What is its transcription factor?
1. TGF-β
2. IL-2
Transcription factor: FoxP3
94
Which cytokines induce Tfh cells? (2) What is their transcription factor?
1. IL-6
2. IL-21
Transcription factor: BCL-6
95
How does CTLA-4 inhibit costimulation?
Competes with CD28 for CD80/CD86, preventing costimulation (higher binding affinity)
96
How quickly after activation is CTLA-4 induced?
~24 hours, peaking after 2-3 days
97
Which cells express PD-1, and which PD-1L?
T-cells express PD-1
APCs express PD-L1
98
What is presence of inhibitory receptors linked to?
Exhaustion of T-cells
99
When does T-cell exhaustion occur? (3)
Situations of chronic antigen persistence:
1. Chronic viral infections
2. Tumours
3. Auto-immune disease
100
What is the first exhaustion marker?
PD-1
101
What is the effect of stimulation on exhausted T-cells?
Apoptosis of T-cells
102
Which three signals need to be present for T-cell exhaustion to occur?
1. Persistent antigen
2. Negative costimulation
3. Chronic inflammation through cytokines
103
What are the two modes of action of inhibitory molecules? What are examples for each mode of action?
1. Phosphatases, dephosphorylating tyrosine kinases involved in stimulatory signaling; PD-1
2. Competition for costimulatory molecules; CTLA-4
104
What are characteristic transcription factor changes in T-cell exhaustion?
1. TCF1 = needed for T-cell proliferation -> drops after activation
2. TOX = increased upon activation, then decreases after some time -> does not drop in case of exhaustion
105
True or false: exhausted T-cells are completely inactive
False; they still reteain some activity
106
What are the characteristics of exhausted T-cells? (7)
1. Functional defects
2. Upregulation of inhibitory receptors
3. Proliferative effects (inhibited)
4. Survival defects (tendency for apoptosis)
5. Transcription factor changes: TOX+/TCF1-
6. Epigenetic changes
7. RNA transcriptome changes
107
What is a CAR T-cell?
A T-cell with a chimeric antigen receptor (CAR)
108
What is a CAR? How does it recognize antigen?
A chimeric combination of CD28 + 4-1BB + TCR ζ-chain that can robustly activate T-cells when encountering antigen
Recognizes antigen by Fab fragment of antibody as receptor unit
109
What are the steps in CAR T-cell therapy?
1. Remove blood from patient to obtain T-cells
2. Make CAR T-cells in the lab with specific CAR for target antigen and multiply these
3. Re-infuse T-cells, after which they will clear tumours
110
What are the current problems with immune checkpoint inhibitor therapy? (5)
1. Only minority of patients respond
2. Not all types of tumour respond
3. Responses may not be long-lasting
4. Emergence of resistance in patients that previously responded
5. Adverse effects
