HC1: Immunological interactions and B-cell Research Flashcards
HC1
1
Q
Infection starts with inflammation > how is it induced?
A
Infection
> Phagocytosis by macrophages
> Cytokine and chemokine production
> Reaction and activation of endothelium and immune cells
> Inflammation
2
Q
Inflammation meaning
A
Influx of plasma with complement factors, neutrophils and monocytes, but also antibodies later on.
3
Q
Reaction upon inflammation
A
Increased lymph drainage to lymph nodes (LNs) + activation antigen presenting cells (APCs)
> induction specific immune response
> influx antibodies and T-cells
4
Q
Inflammation symptoms are result of:
A
Cytokines that activate endothelial cells to open up / increase permeability and vasodilation
> redness, heat, swelling
5
Q
Signals from infection site (cytokines etc) are received by:
A
everything in the circulation > neutrophils, T-cells and monocytes > adhere to endothelium
6
Q
Chemokine main functions
A
Upregulate receptors (integrins) to adhere to endothelium and cluster and create movement of immune cells against gradient when correct receptor expressed
7
Q
Plasma contains this immune components
A
Complement factors and antibodies
8
Q
How is pain generated in inflammation?
A
Inflammatory cells migrate into tissue upon activation and release inflammatory mediators that cause pain
9
Q
Mast cells: localization and function
A
Mast cells are present in periphery
> secrete histamines and other toxins to kill parasites
> tissue reacts to secretions: to excrete the parasite
» make more fluids and contraction of smooth muscle in GI-tract
10
Q
Allergy type of reaction
A
Anti-parasital reaction against non-pathogens
11
Q
Which granulocytes are attracted to infection site?
A
Neutrophils, (mast cells), basophils, eosinophils.
12
Q
Differentiation/maturation of dendritic cells
A
When recognition pathogens
> iDC to mDC
> Lymph drainage to LNs > activate adaptive immune cells
13
Q
Antigen presenting cells (APCs) cross-presentation
A
Present the phagocytosed pathogen in two ways
> Linear peptide of antigen presented on MHC-I for CD8+ T-cells or MHC-II for CD4+ T-cells
14
Q
T-cells use … to recognize other cells and potential pathogens upon activation
A
TCR and co-stimulation
15
Q
Types of infected cells
A
By viruses, some bacteria and cancer cells
> recognized by T-cells to kill (CTLs)
16
Q
B-cell function
A
Aided by some T-cells, produce antibodies to aid killing of infected cells and pathogens
17
Q
Which receptors are used to recognize pathogen first?
A
PRRs (Pattern Recognition Receptors) including TLRs (Toll-like Receptors) to recognize PAMPs (Pathogen Associated Molecular Pattern) like LPS of bacterial cell wall
> tails receptor intracellularly come together
> signalling to activate transcription factors like NFkB to upregulate secretory cytokines etc.
> gene programs activated
18
Q
Gene programs activated upon TLR signalling
A
For macrophages (innate)
> Cyto/chemokine secretion > inflammation
For Dendritic cells (adaptive)
> migration, antigen presentation, co-stimulation: naive T-cell priming
19
Q
Co-stimulatory signal from mDCs to naive T-cells
A
CD80/86 on DC
CD28 on T-cell
20
Q
T-cell cycling through lymph nodes
A
- T-cell enters LN cortec via high endothelial venules (HEVs)
- when not activated, leave within hours through HEVs
- when activated by APC DCs, lose ability to exit T-cell zone and begin proliferation and differentiation into effector T-cells and exit via cortical sinuses
21
Q
What do all T-cells do in LNs (naive)
A
Bind the DC with integrins and ‘try’ their TCR with the epitope
> not recognition: leave towards other LN
> recognition: stay
22
Q
What happens upon TCR - MHC/peptide binding
A
- Make very intimate synapse: immunological synapse
- Interact via co-stimulation and costimulatory cytokines
> once synapse formed: signal 2: CD80/86 (DC) - CD28 (T-cell)
23
Q
When is CD80/86 upregulated on DC?
A
Only when PAMP triggering of DC by PRR like TLR
> so, recognition of CD80/86 by nT-cell is to check that DC has seen a pathogen
24
Q
T-cell and DC
> signal 1: TLR-MHC/Peptide
> signal 2: costimulation
What happens when:
- Only signal 1
- signal 1 + 2
A
Only signal 1: inactivation of T-cell > anergy of T-cell (anergic T-cell)
Both: clonal expansion
25
Signal 3 in T-cell activation
Cytokines released by DC into immunological synapse
> for differentiation signal to make cell a CD8+ cytotoxic T-cell or to different CD4+ T helper cell subtypes
26
Cytokines (signal 3) for CD8+ T-cells drive:
Upregulation signals for perforins, granzymes and FAS-ligand > to induce apoptosis in target cells
27
Types of CD8+ T-cells
Naive, memory and effector
28
Cytotoxic T-cell (CTL) killing mechanism
- Perforines and granzymes secreted in immunological synapse between target cell and CTL
- Perforines make hole in cell membrane
- Granzymes induce apoptosis in cell when entering
- Or: upregulation FAS-ligand to recognize FASR on target cell (death receptors)
> Apoptosis rather than necrosis: no release intrinsic contents > little leakage
29
DAMPs
Danger Associated Molecular Patterns
> related to autoimmunity
> are inside cells, sometimes released by necrosis, and can induce immune response against healthy cells like PAMPs do
30
Immunological synapse (IS)
- Strong interaction after TCR recognition
- DC and T-cells make extensions > interfacial podosomes: for maximal interaction
31
IS: SMACs
On outside of synapse: adherence, and on inside: TCR and costimulation
> Most cytokines are secreted in synapse core (in autoimmunity also outside synapse)
> cSMAC: central supramolecular activation cluster: TCR, CD28, CD4, CD8 and cytokies
> pSMAC: peripheral supramolecular activation cluster: adherence
32
Signal 4 of APC-T-cell
Homing inducing signal
> upregulate some chemokine receptors to direct T-cells to a certain direction for certain chemokines
> most important homing: go through circulation and pass by chemokines nearby the infecion site
33
CD4+ effector T-helper cells and functions
Th1: help macrophage kill all phagocytosed contents better, for resistant microbes to innate response, and help in CTL maturation and response
Th2: promote eosinophils, neutrophils and mast cells in response against helminth parasites
Th17: promote neutrophils for killing of extracellular bacteria
Tfh: assist B-cell antibody response to create plasma cells which secrete antibodies for nearly all microbes
Treg: when little costimulation, create tolerance
34
Th1 cytokine released to macrophages with intracellular microbe
IFN-y, released in IS
35
Differentiation to Tregs
TCR- MHCII/Peptide
Little CD28-CD80/86 costimulation
TGFb and IL-10
36
Cancer and cytokines made
Upregulate TGFb and IL-10 to create tolerance and no attack to tumor
37
Targets of all CD4+ T-cell subtypes
Th1: bacteria and tumor
Th2: parasites
Tfh: help antibody production
Th17: neutrophul activation, fungi/bacteria
Treg: downmodulation/tolerance
38
Which signal regulates pathogen class-specific CD4+ Th differentiation
PRRs and PAMP signal regulates which cytokines produced by APC, which determines the differentiation
39
Costimulatory cytokines and T helper subtypes
Made by APC
Th1: IL-12
Th17: TGFb, IL-1, IL-6
Treg: low costimulation and TGFb and IL-10 (no TLR activation)
40
Which T-cells involved in response to extracellular pathogen: bacteria?
- CTLs
- Th1: help CTLs
- Tfh: help B-cells make antibodies for extracellular bacterium
- Th17: promote neutrophils in extracellular bacteria killing
> multiple TLRs activated > multiple CD4+ T-cell subtypes made. > one TLR for one cytokine program
41
IFN-y as confirmer
Confirms the Th1 phenotype by contributing to progeny to being Th1
42
Th1/2 plasticity
Can interchange when getting too much IFN-y (Th1) or IL-4
> little plasticity
43
Acquired B-cell response: B-cels differentiates to two antibody secreting cells:
- Plasmablasts
- Plasma cells
44
B-cell precursor to plasma cell or memory cell
- B-cell precursor rearranges its immunoglobulin genes in bone barrow
- Negative selection in bone marrow: immature B-cell bound to self-surface antigen in bone marrow is removed from repertoire by receptor editing or apoptosis
- Mature B-cell bound to foreign antigen is activated (in LNs or lymphoid organs like spleen
- Activated B-cells give rise to plasma cells or memory cells: antibody secretion and memory in lymphoid tissue and bone marrow
45
Signals for B-cell activation, survival and proliferation, in case of thymus-independent antigen
Signal 1: BCR signal
> Pro-apoptotic and activation signal
> Three dimensional antigen
Signal 2: TLR signal
> Anti-apoptotic signal
> Three dimensional epitope (PAMP)
Only signal 1 > apoptosis
46
BCR structure and Fc, Fab, Fab2
- Two heavy chains and two light chains
- Disulfide bonds
- Antigen binding fragment with three antigen specific variable loops on ends: Fab
- Fc: constant tail
- Fab2 fragment: two Fab fragments together: the variable Y form with disulfide bond between them
- both heavy chain and light chain have 3 loops: six loops per Fab fragment
47
What happens after naive B-cell activation? T cell independent
Differentiation to short-living plasmablast that makes IgM only
> always made
> early response, when no T-cells to assist
48
Affinity BCR
Low affinity > low binding energy per Fab
>> which is okay: when enough BCR bound to antigen then still activation: high total avidity (total binding force) of the B-cell
49
IgM structure
Secreted as multimer complex (pentamer) by crosslinking the pentamer with the J-chain (joining chain)
>> increase avidity
50
BCR is either ... or ...
IgM or IgD
51
IgM compared to other antibodies
- Produced immediately
- Low affinity
- Penta / hexamer
- Does not need T-cell help or GC reaction
52
Use of Penta/hexameric structure of IgM
IgM does not need somatic hypermutation and affinity maturation > low affinity. So the multimeric structure generates high avidity.
53
IgM and localization
It is big > cannot easily extravasate to infection site
54
IgM and opsonization
Is not very good, no free Fc tails avaiable where macrophages and other phagocytes can bind to with Fc receptors to phagocytose targeted pathogen
55
Where is IgM very good for?
Complement activation!
56
What is needed to make other antibodies than IgM?
T-cell help
57
Three signals for B-cell activation, survival and proliferation: Thymus-dependent antigen
Signal 1: BCR
Signal 2: MHC-II - TCR
> B-cell can phagocytose antigen upon binding to BCR and break down content and load on MHC-II to activate T-cell
>> B-cells can express CD40 that binds CD40L on T-cell as co-stimulation.
>> In the B-cell zones of the LNs
Signal 3: cytokines
58
Germinal centre creation
B-cells that encounter antigen in follicle of LN form primary focus, some proliferating B-cells migrate into follicle to form germinal centre
> after GC reactions: plasma cells migrate to medullary cords or leave via efferent lymphatics to bone marrow to stay as long living plasma cells
59
Plasma cells function
Make steady-state protecting antibodies
60
Memory cells made in which B-cell response?
Mainly in T-cell dependent B-cell response.
61
Signal 1 of T-cell dependent B-cell response
BCR
> upon activation: CD40 upregulation and phagocytosis and MHC-II loading with antigen linear peptide to present to T-cells.
> All activated B-cells get chemokine receptor to go to T-cell zone and some T-cells get chemokine receptor to go to B-cell zone
> meet on the edge of zones to interact
> only T-cell help when antigen is presented by the B-cell (to check: B-cell is directed to the specific pathogen / antigen)
> pro-apoptotic signal
62
Signal 2 of T-cell dependent B-cell response
MHC-II/ linear peptide to TCR
> anti-apoptotic signal
> Co-stimulation CD40
63
Signal 3 of T-cell dependent B-cell response
CD4+ T-cell (Tfh) cytokines: IL-21 for IgG generally!
> induce specific class-switching by IL-21 and CD40 signalling by CD40L of Tfh (to IgG/ IgE, somatic hypermutation and affinity maturation) > eliminate antigens with high-affinity antibodies
64
Difference lacking signals of B-cells and T-cells in activation
T-cells become anergic
B-cells undergo apoptosis and die
65
Presentation antigen on MHC by B-cell
Phagocytose whole pathogen, breaks it down to present it upon BCR recognition: phagosome fuses with the vesicle containing MHC-II
66
Results B and T-cell interaction
Both start dividing, and maturing of B-cells to make more IgM and IgG
67
Which three components essential for IgG response
Pathogen, T-cell (for CD40L and IL-21), B-cell
68
Tfh cells in B-cell help
Needed for the germinal centre reaction and therefore for long-lived immunity
>> B-cells which leave after first- T-cell interaction and do not enter GC reactions: become short-lived plasmablasts and early memory B-cells (IgM producing cells ready to make GCs after re-infection)
>> Tfh cells give TCR signal and CD40L costimulation with Il-21 for survival and class switch and further SHM
69
When are B-cells going to B-cell zone for GC reaction rather than leave to become plasmablast?
After receiving signals 2 and 3 from the Tfh cell
> CD40L to CD40
> IL-21 to IL-21R
70
GC reactions create specific memory B-cells, why needed
Higher affinity needed and class-switching
> create high affinity memory B-cells and short and long-living plasma cells > after re-infection directly to plasmablasts and plasma cells
71
Where do plasma cells go upon maturation?
To bone marrow to create high affinity antibodies for long time
72
Germinal centre reactions (name the zones as well)
Initial contact: border T-cell zone and GC light zone
> Then: B-cell to GC dark zone (because, many nuclei): proliferation, somatic hypermutation and class-switch recombination
> Then: Antigen acquisition in light zone by interacting with FDC (follicular DC) which presents antigen to BCR: take it up
> Then: Present antigen on MHC-II and co-stimulation with CD40 (via BCR signalling) to Tfh cell and receive cytokines
> Then: if high affinity: leave for differentiation and migration
> or if not high affinity enough: go back to dark zone for another cycle (affinity maturation)
73
Somatic Hypermutation
Induce mutations on antigen binding site loops when proliferating
74
What happens to all B-cells coming fro first GC reactions in dark zone when returning in light zone?
First: do they recognize the antigen of the FDC better with their BCR?
> Yes: capture it and present to T-cell (Tfh) with CD40 which is upregulated
>> receive survival and mitogenic (induce mitosis, proliferation) signals via CD40 and cytokines: go back to dark zone
> No: no antigen capture by interacting with FDC: death by neglect
75
Which form of cell death is death by neglect of B-cells?
Apoptosis
76
In affinity maturation, regular antigen check with FDCs is needed. What makes FDCs special dendrites?
They can hold antigens for a long time
77
Class-switch recombination
T-cell gives signals to induce this after interaction (CD40L+IL-21)
> Different switch regions before the genes for parts of different antibodies
> Switch regions before different gene regions can interact and loop out parts of DNA (is irreversible because DNA level)
> Switch to different antibody type
> most 5' is the C-mu part (determining IgM)
> switch to C-delta (IgD, rare), C-gamma (IgG), C-epsilon (IgE) or C-alpha (IgA) (in this order 5'>3', so all parts need to be looped out except C-alpha to get IgA, whereas only C-mu and C-delta out for IgG)
78
Why are there many C-gamma genes?
Multiple subclasses of IgG
79
How is class-switching, also called isotype switching, determined?
By costimulatory cytokines received from T-cells, IL-21 for IgG
80
IL-4 induces the subtype.... and IL-12 the subtype ...
IL-4 > IgG1 and IgE
IL-12 > IgG3, IgG1, IgA
81
Different isotypes of antibodies have different functionalities. Name those
Best opsonization: IgG
Best complement activation: IgM and IgG3
Best neutralization: IgG and IgA
Best sensitization of mast cells: IgE
(IgD is weird, worst at everything, function not clear)
82
Why is opsonization by IgM and IgA not very good
IgM is penta/hexamer, IgA is dimer
> no free Fc tails for Fc-receptors of phagocytes
83
Selection upon after GC reactions: results
Class-switched during GC reactions
Selected for affinity matured high-affinity cells to differentiate to plasma cells and B memory cells
84
Where does the affinity maturation selection step take place?
At FDC and B-cell interaction
> if antigen taken up and presented to Tfh cell > then further selection
85
IgA antibodies may lay ready on mucosae to bind pathogens: can viruses or other pathogens still bind receptors of human target cells for infection?
Yes
86
Transcription factor markers for differentiation for plasmablast vs plasma cell
Plasmablast: Blimp1
Plasma cell: Pax5
87
Big research question in B-cell research is how TFs for differentiation of maturing B-cells after affinity maturation for differentiation is acquired. Which markers are there for plasma cells?
CD38, CD27, CD138
88
Why matching HLA in blood transfusion? How to detect mistakes early? Why?
No antibody response wanted > inflammation and lung problems
> sickle cell anemia requires blood donation
> prevent plasma cells from going to bone marrow, then permanent autoimmune response
> research into selecting B-cell maturing for differentiation needed
> markers needed for maturing B-cells (before the plasma cells)
89
Markers for long living B memory cells
Not there yet
90
In vitro B-T cell interactions for differentiation
3T3 (epihelial) cells with induced CD40L expression in lab and added IL-4 and IL-12 in vitro to naive B-cells
>> BCR signal is to present peptides, but CD40 and cytokine signals (and how strong they are) determine how the B-cell differentiates
91
CD27 and CD38 in scatter plot (FACS): what are the double positives and double negatives?
Double positives: PB/PC: plasma blast or cell
Double negatives: naive B-cell
92
Why no PB/PC formed after B-T cell interaction in culture
The B-cell needs to get the signal multiple times
> adding new cytokines to replicae GC reactions
> PB/PC formed
93
How to analyze pre-stadia between naive B-cell and PB/PC
Single-cell RNA-seq and clustering with UMAP to show cell populations
> Different populations: one is high in Pax5 (PB) and one in Blimp1/Prdm1 (PC)
> quite dramatic transition state: no intermediated population shown
94
Analysis B-cell premature stages with SARS-CoV-2 research
rGCG (recent Germinal Centre Graduate) is precursor of short-lived plasmablast and long-lived plasma cells from GC reactions
> put biotin on Spike (S) antigen > streptavidin (binds biotin) bound to fluorophore > incubate > B-cells bind with fluorophore
> SARS-CoV-2 specific B-cells found with different fluorophores
> performed with other epitopes as well and controls like tetanus toxoid
> more severe disease: higher antibodies for Receptor binding domain (RBD) antigen and NC markers against SARS-CoV-2
95
Antibody titers over time:
Decrease
96
scRNAseq of SARS-CoV-2 incubated B-cells: which markers and which population
Resting memory and activated memory and active B-cells (plasma cells)
> CD73: for long-living memory B-cells (resting memory B-cells have less than active ones, long ago)
> CD71: decreasing gradient,: activated IgG B-cells (rGCGs) that have recently undergone GC reactions express CD71 and CD43
97
Which B-cells more enriched with more severe disease?
IgG+ memory B-cells and activated IgG B-cells (rGCG)
98
Activated IgG B-cells are precursors for:
Antibody secreting cells (ASCs) but also possibly B memory cell precursors.
> MBs (memory) go from active to resting
