Prelim 3 Flashcards
(246 cards)
1
Q
Where are B and T cells derived from?
A
- Multipotent (pluripotent) hematopoietic stem cell
- Common lymphoid progenitor
CAN EITHER:
Bone marrow- commit and develop into B cell
Thymus- commit and develop into T cell
2
Q
What are the different phases of B cell/T cell development and action
A
- Commit (to B or T lineage)
- Become useful: antigen receptor repertoire assembly, + and - Selection
- Search for infection
- Find infection (activate)
- Attack infection (differentiate and act)
3
Q
Commitment
A
In reference to B cells and T cells, is the irreversible change in gene expression by a common lymphoid progenitor cell (CLP) that sends it down a B cell lineage or a T cell lineage
4
Q
Where do B cells and T cells arise (commit)?
A
They commit in primary lymphoid tissues (thymus for T cells; bone marrow for B cells)
5
Q
What are other places according to a B or T cell?
A
- Secondary lymphoid tissues (spleen, lymph nodes, Peyer’s Patches, etc.) to activate and differentiate
- Periphery - everywhere else in the body, to defend or instruct tissue cells
6
Q
Where do the different stages occur in B cells?
A
- Commit : Bone marrow
- Become useful : Bone marrow and periphery (secondary lymphoid tissues)
- Search for infection : Secondary lymphoid tissues
- Find infection : Secondary lymphoid tissues
- Attack infection : Bone marrow and Secondary lymph nodes and periphery
7
Q
How do bone marrow stromal cells help differentiation
A
Bone marrow stromal cells provide signals that induce master transcription factors (TFs) and proliferation of developing B cells
1. cell adhesion molecules (CAMs, and VCAM-1 (VLA-4 on CLP binds to this)
2. Growth factors (cytokines) SCF (Stem-Cell Factor) and IL-7 (to induce gene expression)
8
Q
What does the CLP have that binds VCAM-1?
A
VLA-4
9
Q
What does the CLP have that binds SCF?
A
Kit
10
Q
What does the CLP have that binds IL-7?
A
IL-7 Receptor
11
Q
Stromal cell
A
A supportive cell in a tissue that provides surface molecules and cytokines for developing an immune cell. In this example, it’s a bone marrow stromal cell of long bones, like the femur, that support immature B cells
12
Q
Commit
A
Use master transcription factors and sets of genes that lock a stem cell into a particular lineage, like B or T lineage
13
Q
Master Transcription Factor (MTF)
A
Binds a common gene promoter on a related set of genes. The genes are expressed and confer certain capabilities to the cell
14
Q
What are the TF that commit the cell to the B lineage?
A
- Ikaros, PU.1 (From lymphoid progenitor–> immature B cell)
- E2A (later Lymphoid progenitor –> Immature B cell)
- FOX01, EBF (mid Early pro-B cell –> Immature B cell)
- PAX5/BSAP (Late pro-B cell –> Immature B cell)
15
Q
B cell CLP
A
- Ikarus, PU.1 bc of adhesion molecules
16
Q
Early pro-B cell
A
- Ikarus, PU.1 , E2A, FOX01, EBF
because of adhesion molecules but now you also have SCF with Kit
17
Q
Late pro-B cell
A
- Ikarus PU.1, E2A, FOX01/EBF, PAX5/BSAP
because of adhesion molecules, SCF with Kit, adn now IL-7 with the receptor
18
Q
Pre-B cell
A
Has all the TF and all the adhesion molecules, IL-7, and SCFz
19
Q
Marker for all stem cells (pluripotent hematopoietic stem cell)
A
CD34
20
Q
Marker for Common lymphoid Progenitor
A
- CD34
- CD10*
21
Q
Marker for B cell precursor
A
- CD34
- CD10
- CD127*
22
Q
Marker for Pro-B cell
A
- CD34
- CD10
- CD127
- CD19*
23
Q
What is the marker for all B cells?
A
CD19
24
Q
Pro-B Cell
A
Earliest identifiable cell of the B cell lineage. Has pan-B marker, CD19, but still has the CD34 stem cell marker. It does not have a B cell receptor yet
25
Germline
The Ig gene is not rearranged yet
26
Somatic recombination
Recombination of DNA segments of genes encoding antigen receptors in immature B and T cells
27
Different types of B cell
Stem Cell
Early pro-B cell
Late pro-B cell
Large pre-B cell
Small pre-B cell
Immature B cell
Mature B cell
28
Stem Cell (recombination)
H-chain genes: germline
L-chain genes: germline
Surface Ig: Absent
29
Early pro-B cell
H-chain genes: D-J rearranging
L-chain genes: germline
Surface Ig: Absent
30
Late pro-B cell
H-chain genes: V-DJ rearranging
L-chain genes: germline
Surface Ig: Absent
31
Large pre-B cell
H-chain genes: VDJ rearranged
L-chain genes: germline
Surface Ig: IgM chain transiently at surface as part of a pre-B cell receptor. Mainly intracellular
(the reason why is because we have to see if the heavy chain is successful in sending a signal)
(* there is a picture you should remember for this point)
32
Small pre-B cell
H-chain genes: VDJ rearranged
L-chain genes: V-J rearranging
Surface Ig: Intracellular IgM chain
33
Immature B cell
H-chain genes: VDJ rearranged
L-chain genes: VJ rearranged
Surface Ig: IgM expressed on cell surface
34
Mature B cell
H-chain genes: VDJ rearranged
L-chain genes: VJ rearranged
Surface Ig: IgD and IgM made from alternatively spliced H-chain transcripts
35
What are the different stages for a T cell? (same as B cell)
- Commit (to T lineage)
- Become useful, antigen receptor repertore assembly (+ and - Selection)
- Search for infection
- Find infection (activate)
- Attack infection (differentiate and act)
36
Where do the different stages for a T cell occur
- Commit: Thymus
- Become useful: Thymus
- Search for infection: Secondary lymph node
- Find infection: Secondary lymph nodes
- Attack infection: periphery, secondary lymph nodes
*the only places this differs from a B cell is the become useful bc this happens in the thymus but for the B cell it is in the thymus and the periphery and the attack infection occurs in periphery and secondary lymph node not in the bone marrow, periphery, and secondary lymph node
37
Where do certain lymphoid progenitors go to become T cells?
Common lymphoid progenitors (CLPs) travel through the bloodstream and enter the thymus. This is the only site that holds the necessary cues to commit to the T lineage
(there are no cues for B cell development in teh thymus, so, no B cells develop here).
38
(T) Pluripotent hematopoietic stem cell --> common lymphoid progenitor -->
DN1 T cell progenitor
DN2 pro-T cell
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(B) Pluripotent hematopoietic stem cell --> common lymphoid progenitor -->
B cell precursor
Pro-B cell
40
What is the thymus?
- A network of stromal cells to support developing T cells
- THe thymus is a T-cell making factory
41
T cell stromal cells example
In this example, the non-immune stromal cells are epithelial cells that supply Notch Ligand and present self-peptides for selection of immature T cells. Immune cells that help at some steps are macrophages and DCs that present self-peptides, too
42
What supplies Notch ligand?
Thymic epithelial cells
- Notch ligand is only supplied in the thymus
- It liberates a ready-made transcription factor, Notch 1, in CLPs. Then, Notch 1 drives genes that commit the cell to the T lineage
43
Which cells have Notch1?
Common Lymphoid Progenitors. The notch gets cleaved by Notch Ligand on thymic epithelial cells
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Thymocyte
Another name for developing T cells in the thymus
45
Thymic epithelial cell
Stromal support cell for many stages of developing T cells. Provides adhesion molecules (not shown), notch ligand, and presented MHC and self-peptides during selection
46
What are the different T cells during development
- ETP or DN1 --> DN
- Pro-T (DN2) --> DN
- Small pre-T (DN3) --> DN
- Large pre-T (DN4) --> DN
- CD4 and CD8 --> DP
- CD4, CD8, and TCR --> DP
- Single positive`
47
DP
Late-stage pre-T cells express both CD4 and CD8 co-receptors
48
Single positive
T cells that express CD4 or CD8, but not both. They are the final stage of immature T cells in the thymus
49
Transcription Factors for T cell & when they are expressed
- TCF1 (expressed the whole time)
- GATA3 (expressed the whole time)
- Bcl11b (Pro-T/DN2 cell to the end)
- KLF2 (Single positive)
- ThPOK* (Single positive encodes CD4)
- Runx3* (Single positive encodes CD8)
50
Thymus disappears over a lifetime
- Most mature T cells are made in a person by the time they have reached their twenties
- The mature T cells patrol and slowly renew themselves by homeostatic proliferation
51
3 Properties of a useful B cell or T cell
1. Express functional antigen receptor complexes that transmit intracellular signals
2. Respond to only ONE antigen (functionally monospecific)
3. Be self-tolerant (not react destructively to self-antigens)
A useful T cell has a 4th property
4. MHC-restricted: only bind antigens that are presented by MHC molecules
52
1. Express functional antigen receptor complexes that transmit signals
What are the two functions of antigen receptors?
Antigen receptors bind antigen and transmit intracellular signals
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Antigen receptor complex
The antigen receptor plus the invariant chains that transmit intracellular signals. For a T cell this includes CD3 chains. For a B cell, this includes Ig-alpha and beta chains
54
Is the coreceptor of a T cell part of the antigen receptor complex?
NOT PART OF THE TCR complex. But the co-receptors is brought close to the antigen receptor complex to bring kinases for stronger intracellular singals
55
How many molecules make up the B cell coreceptor
It is made up of 3 molecules: CD19, CD21, and CD81. NOT part of the BCR but brings signaling kinases (very important you remember what they bring)
56
1. Bind to ONE antigen only (functionally monospecific)
- If an antigen is repeated on a surface (like a multivalent Ag), it will cluster BCR complexes tht have the same specificity. (Pathogens often have repeating antigens on their cell wall)
- Clustering BCRs gather intracellular signaling enzymes and transmits a VERY STRONG activation signal
57
How to be monospecific?
ONLY rearrange and express 1 chromosomal copy of heavy chain and one copy of light chain (remember there are many possibilities and combinations that can exist). For a T cell its 1 copy of an alpha gene and 1 copy of a beta gene. This important control is called allelic exclusion.
58
3. Be self-tolerant (don't attack self antigens)
- Many immature B cells and immature T cells have antigen receptors that bind to self-antigens. The reason that antigen binding regions are generated by random somatic recombination and mutation
- To prevent self-reactive cells from attacking the body, they're eliminated or r-programmed before they are allowed to mature. This is negative selection
59
What is negative selection essentially?
Negative selection is the removal of cells that bind to self antigen. Positive selection is reinforcing/keeping cells alive that are useful.
60
How many cells does negative selection eliminate
About 75% of immature B cells and T cells
61
4. MHC-restricted T cell receptor will only bind antigens presented by MHC molecules
T cells only interact with host cells...not directly with the microbes
So, useful T cells must be able to bind the classical MHC molecules that present peptides.
62
An early pro-B cell is rearranging...
D and J segments
63
A late pro-B cell is rearranging...
V to DJ segments
64
A large pre-B cell has...
A pre-BCR complex and tests it!
Successful onces become many identical small pre-B cells
*remember L-chain is still not being touched here
65
What does the pre-BCR use to bind to the recombined heavy chain?
It uses the surrogate chain (L-chain) which is comprised of VpreB and lambda5
66
Is somatic recombination often successful on its first try?
No
67
How does the B cell go about rearranging the heavy chain loci? (diagram)
- There are 2 chromosomal copies of heavy chain loci to rearrange. A pro-B cell tries both simultaneously
- D-J rearrangements on both chromosomes
+ V-DJ rearrangement on first chromosme -->
+ Signaled to survive and become pre-B cell (50%
of cells)
- V-DJ rearrangement on second chromosome
-->
+ Signaled and become pre-B cells (50% of cells)
- Signaled to die by apoptosis (50% of all cells)
Essentially, there are two chromosomal copies of the heavy chain. They both have their D-J chain arranged at the start. The b cell will choose one chromosome and try to attach the V-DJ. If this is successful and it is transmits a signal it will receive signals to survive. If this is unsuccessful, the B cell will use the second chromosomal copy to attach teh V-DJ. If this is successful, then the cell survives. If this isn't successful , then the whole B cell is signaled to kill itself by apoptosis.
68
Non-productive DNA rearrangement at both loci=death
Adding and deleting nucleotides has many errors that create DNA that can't be transcribed due to nonsense. codons, premature stop codons or codons in the wrong reading frame (CPR)
69
Quality Checkpoint 1
Check heavy chain function
- Can the heavy chain assembly into a pre-B cell complex that transmits a signal?
70
If quality checkpoint 1 is successful, what are the three signals that happen to the cell?
1) NOT die by programmed cell death
2) Pause somatic recombination (of the other VDJ segment)
3) Proliferate
NPP
71
What does a working heavy chain do?
The new mu heavy chain hinds to Ig-alpha and a surrogate light chain (VpreB and lamdba5)
The fingers of bound VpreB will grab each other and cluster BCR complexes to transmit signals
72
What is the first instruction and what determines of these cells will die even if they do work?
The first instruction is to not die!
If immature lymphocytes don't earn timely survival signals using working antigen receptors, they automatically die
73
What is the second instruction and what happens as a result of it?
The second instruction is to pause somatic recombination
- STOP expressing recombinase (RAG) genes
- DEGRADE any existing RAG protein
- SILENCE the other heavy chain locus with chromatin to make it permanently inaccessible
--> this causes allelic exclusion and is why B cells are monospecific.
74
What is the third instruction and what staged-B cell does that end up making?
The 3rd signal sent by a pre-BCR complex is to make many copies of this successful cell!
Makes ~100 identical daughter cells (small pre-B cells) that have identical mu heavy chains
75
Somatic recombination of kappa light chain locus
- Each small pre-B cell rearranges V and J of k light chain gene
- Successful onces become different immature B cells
76
Which light chain will the B cell start with?
It will always start by creating one of the two kappa light chains
77
Successive rearrangement and why most pre-B cells successfully express a light chain
Most small pre-B cells successfully express a light chain. WHY? The segments are arranged for successive rearrangements tries and there are 4 light chain loci to use.
It will try all the different ways of rearranging that kappa chain. If it doesn't work, it will try the next kappa chain loci. If that doesn't work, it will try the lambda chain loci. If that doesn't work, it will try the second lambda chain loci. Usually, it works from rearranging the first kappa a few times
78
2nd Quality Check
Does the heavy and light chain work?
The light chain is assembled into a BCR complex and transported to the cell surface with Ig-alpha and B
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What signals does the B cell recieve if it succeeds in the 2nd Quality Check?
1) NOT to die
2) STOP somatic recombination
3) MIGRATE towards the exit of the bone marrow
80
At the stage of fulfilling the second quality check, what kind of B cell is this?
An immature B cell
81
3rd Quality Checkpoint
NEGATIVE SELECTION
At this point, there's a very diverse pool of immature B cells moving through the bone marrow on their way out. They can bind antigen.
Negative selection will eliminate immature B cells that bind self-antigens (about 75% of immature B cells which is very wastefu)
82
Selection of B cells happens in _____ ___ for many ___
Selection of B cells happens in multiple places for many days
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Where does negative selection of B cells occur?
- Bone marrow: Self antigens are shown and any immature B cell that binds have 2 options (rearrange a new light chain or die)
- Circulation: transitional B cells traveling in blood that bind soluble self-antigens are deactivated (anergized)
- Secondary lymphoid tissues: final chance for transitional B cells to bind self-antigen and be deactivated (anergized) or die from lack of survival signals. Stromal cells in follicles provide cytokines for survival/maturation (positive signal)
84
Can transitional B cells also die from a lack of survival signal?
Yes, even if they aren't anergized because they didn't bind antigen, if they don't receive the positive signal in time, they will die.
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Where does positive selection of B cells occur?
In the Secondary Lymphoid tissue. They recieve signals/cytokines from stromal cells
86
What is the direction of migration for a transitional B cell?
Out of the bone marrow, through the blood, to the secondary lymphoid tissue
87
What happens to B cells that bind antigen in the bone marrow
They are held there and they are given two options. Die by apoptosis, or change their light chain by receptor editing
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Receptor Editing
(recombining more V and J segments to create a different antigen receptor) is triggered by multivalent antigens that send strong signals to B cells in an immature state via cross-linked B cell receptors.
A light chain with try 2k and 2lambda chain until all the segments are used up.
Can rearrange as may times as possible until time runs out and it dies
89
Central tolerance
Is due to receptor editing or deletion of self-reactive, immature B cells in the bone marrow which is a central lymphoid organ (not binding antigen in the bone marrow)
*comparable to self tolerance
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Why does negative selection of transitional B cells continue for a while in the blood and spleen?
To test self antigens that aren't in teh bone marrow like thyroid hormones
There is no option for receptor editing outside of the bone marrow. Those recombinases are silences in most tissues because they are so dangerous. Therefore, self-reactive, immature transitional B cells are deleted
91
Peripheral tolerance (THERE IS A VERY IMPORTANT DISTINCTION HERE)
Elimination or inactivation of new self-reactive immature (transitional) B cells in the bloodstream and secondary lymphoid organs like the spleen. These are considered the periphery of the body as opposed to the central lymphoid organs
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4th Quality Checkpoint
Immature transitional B cells are still on a stopwatch of death. They must touch certain cells within secondary lymphoid organs or die in a matter of days. If they can, they will mature and patrol secondary lymphoid tissues.
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Transitional B cell
Immature B cells newly emerged from the bone marrow and have days to live
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What is the 4th signal the transitional B cell must get to survive?
They must enter follicles in the spleen or lymph nodes where STROMAL follicular dendritic cells provide the survival cytokine, BAFF
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Mature B cell
B cell that has gained access to BAFF in follicles as a final survivor and maturation signal (positively selected)
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Does every B cell touch BAFF?
No, even if they are perfectly good
97
What are the two classes of T cells that can develop in the thymus?
- Alpha beta T cells
- Gamma delta T cells
For the sake of this class, we will be focusing on alpha beta T cells
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Are the majority of cells alpha beta or gamma delta T cells?
the vast majority, 70-80% are alpha beta
99
Where are gamma-delta T cells typically located?
In the epithelial layers like the gut
100
What is the earliest marker for a committed double-negative T-cell progenitor
CD2
101
How is it possible to get gamma delta T cells?
If gamma and delta TCR chains are rearranged first (before the beta chain is made first and given the signal), a thymocyte becomes a gamma-delta T cell and leaves the thymus WITHOUT selection
102
Which T cells usually rearrange first?
Alpha beta TCR chains rearrange first and the immature aB T cell undergoes positive and negative selection in the thymus
103
What is rearranging when you have a DN2 T cell?
Beta, gamma, and delta chains
If the gamma delta chains are developed first, they will assemble and leave the cell as a committed gamma delta T cell. If the B cell is made first then it will become an UNCOMMITTED DOUBLE NEGATIVE THYMOCYTE
104
When you have a DN3 what is happening?
You are testing the Beta chain to see if it can send a signal with its surrogate chain which is quality check one
105
Beta and gamma chains are analogous to...
The heavy chain
106
Where is the delta chain located?
Inside the alpha chain
107
If a DN3 small pre-T cell passes the first quality checkpoint, what happens?
You will have MANY identical large DN4 pre-T cells which proliferate. After they become DN4 they will become DP and resume somatic recombination
108
Do gamma delta T cells express CD4 and CD8
No
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1st Quality Checkpoint for T cells
Check the pre-TCR complex
Does the beta chain work?
110
What is the surrogate chain for T cells?
pT-alpha is the surrogate Beta chains use for the alpha chain.
111
What are the signals of the 1st Quality Checkpoint and how does the T cell get them?
pT-alpha bound to the Beta chain will use its "sticky" fingers to bind to the neighboring beta chain (BUT NOT THE ONE DIRECTLY ADJACENT TO IT). This will test to see if the CD3 chains will produce a signal
1) NOT to die
2) PAUSE somatic recombination (allelic exclusion)
3) Express CD4 and CD8 co-receptors
4) Proliferate
112
What does step 2 in the 1st Quality Checkpoint entail for T cells?
- STOP expressing RAG genes
- Destroy RAG proteins
- COVER beta chain loci in chromatin
113
How is the 1st quality check signals in B cells different from T cells?
The only extra signal T cells get is to express their CD4 and CD8 co-receptors
114
T cell resuming recombination after making large DN 4 pre- T cells
DN4 will become Double positive cells. These cells have their Alpha, gamma, and delta chains recombining to make a fully functioning chain that can send a signal and work with the Beta chain (if you have an alpha chain)
2nd/3rd quality check: whether aB TCR complex can bind to self/
115
What are the different quality checkpoints for a T cell?
1st quality checkpoint: Test Beta chain
2nd/3rd quality checkpoint: test aB chain and see if it could bind to self-MHC (+)
4th quality checkpoint: test for self-tolerance
116
When do you have somatic recombination of alpha, gamma, and delta?
During DP cells. The only role of DN4 cells is to proliferate with the working beta chain. It quickly turns into DP for the somatic recombination at step 3 of signaling
117
What type of cell is created after the 2nd/3rd quality check?
A Single Positive cell (SP) immature T cell undergoes negative selection
118
2nd/third quality checkpoint for T cell
- Do alpha beta chains form a TCR receptor complex that binds self-peptide: MHC and transmits a signal?
If the alpha chain is not functional, the cell continues successive rearrangements of alpha chain for a few days
- This is also positive selection for T cells that bind well enough (moderately) to self-peptide held in MHC I or MHC II. This leads to MHC-restriction to self-
119
Successive rearrangements
Like light chains, alpha chains have MANY V and J segments for successive rearrangement
120
Where do checkpoints 1 and 2 occur in the thymus?
In the subcapsular zone and then the DP cells migrate towards the cortex
121
Where does checkpoint three occur in the thymus?
In the cortex, cortical thymic epithelial cells present self peptides on self MHC class I and class II
122
Where does checkpoint 4 occur in the thymus?
Immature T cells that bind with moderate strength receive survival signals. They are positively selected for their ability to bind self-peptide:MHC
123
How does positive selection work?
Cortical thymic epithelial cells express MHC I and MHC II
Double positive thymocytes must bind moderately or strong to peptide:MHC to recieve enough survival signals or they die. Weak or no binding dies
124
When is one co-receptor chosen
During positive selection, one co-receptor is chosen. Whichever co-recetor binds to the MHC will be kept on the T cell. The co-receptor will transmit singals to stop the production of the other co-receptor
... CD4 singals for ThPOK TF
... CD8 signals for Runx3 TF
After positive selection we have a signle-positive, self MHC-restricted immature T cell
125
4th Quality Checkpoint for T cells
To delete T cells that bind TOO STRONGLY to self peptide:MHC or re-program them to be suppressive regulatory cells when they bind host antigen
* the postiive selection DOES allow things to bind strongly so it is the negative selection that gets rid of those or reprograms them to keeo the moderate cells
126
Why is the thymus a good location for negative selection?
The thymus expressed MANY tissue-specific self-antigens
- A special transcription factor called AIRE turns on tissue specific genes in thymic epithelial cells and in professional APCs in the thymus (so the epithelial given signals for expressing antigens from tissue specific antigens like eye antigen even though they can't normally do so). They process the proteins into peptides and show them to developing T cells during negative selection. T cells that bind strongly are deleted or re-programmed
127
AIRE
Transcription factor drives expression of genes that are usually restricted to few tissues, like retina proteins or ovary proteins. This allows thymic epithelial cells and professional APCs in the thymus to present a WIDE selection of self-peptides for negative selection
128
How exactly does negative selection for T cells work?
Immature SP T cells migrate past thymic epithelial cells, myeloid dendritic cells, and macrophages in teh medulla on their way to the exit
Along the way, T cells can bind peptide: MHC I and peptide: MHCII. T cells that bind very strongly die
129
Central tolerance for T cells
Negative selection confers central tolerance of T cells. It is called central because this form of tolerance is generated in central lymphoid tissues, like the thymus
130
Do T cells that bind during negative selection need to bind moderately or strongly?
- Moderately allows signaling and survival which allows the T cell to exit the thymus and become mature and naive
- Prolonged strong signaling means death by apoptosis due to prolonged MAP kinase signaling
- Weak signal = death by neglect
- Brief, strong signaling = reprogramming into T reg
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Affinity Model of T Selection
The strength or duration of TCR binding to peptide:MHC determines a cell's fate
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Important info about regulatory T cells
- They are always CD4
- They are always CD25 hui (use lots of IL-2)
- They recognize self-peptide:MHC II
They use a specific master transcription factor: FoxP3 which programs them to express
- Down-regulatory cytokines, IL-10 and TGFb
- Co-inhibitory molecule called CTLA-4
133
Immature T cell
T cell that has not passed the positive and negative selection checkpoints in the thymus
134
Mature T Cell
MHC restricted T cell that bears functional TCR. There are many types of mature T cells, but they first appear at the exit of the thymus
135
S1P Receptor 1 (S1PR1)
Mature T cells express this chemotactic to exit the thymus which lead them for higher levels of sphingosine-1 phosphate in the blood streamS
136
Should the 4th quality checkpoint ever come across TCRs that can't bind well?
Not really, the 3rd quality checkpoint was made to select thing that bound tightly to MHC and peptide. So at this point, you should either have moderate or tight binding but not any loose binding
137
Effector T Cell
A mature T cell that has been activated and has acquired capabilities (differentiated) to carry out an immune response (effector function)
138
Do vaccines activate T cells?
Yes
139
What is the role of Naive T cells?
Naive T cells patrol the body. They are searching for antigen that binds their TCR. They patrol secondary lymphoid tissues, like lymph nodes
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Draining lymph nodes
The lymph nodes near an infection or vaccination site collect antigens and activated dendritic cells.
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How do naive T cells/lymphocytes enter lymph nodes?
From the blood
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How do lymphocytes and lymph return to the blood?
Via the thoracic duct
But lymphocytes like DCs use lymphatic vessels
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What do antigens use to enter the lymph nodes?
The lymphatic vessels
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What are the doorways of the lymph nodes for B and T cells?
High Endothelial Venules (HEV)
HEVs are "doorways" from veins into T cell areas of lymph nodes. Naive T and B cells have the proper cell adhesion molecules (CD62L) and chemokine receptors (CCR7) to enter via HEV
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How do Naive T cells enter the lymph nodes?
By extravasation
146
How does extravasation of Naive T cells work?
- Circulating lymphocyte enters a high endothelial venule in the lymph node. In the bloodstream, the Naive T cell is expressing LFA-1, CCR7, and L-selectin. On HEV endothelial cells, they are expressing CD34 and GlyCAM-1 which are Vascular addressins.
- ROLL: Binding of L-selectin to GlyCam-1 and CD34 allows rolling interaction
- LFA-1 is activated by CCR7 signaling in response to CCL21 or CCL19 bound to endothelial surface
- TIGHT ADHESION: Activated LFA-1 binds tightly to ICAM-1
- DIAPEDESIS: lymphocyte crosses the endothelium and enters the lymph node via diapedesis
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What is another name for L-selectin?
CD62L
148
What are the vascular addressins?
CD34
Gly-CAM-1
*only expressed on endothelial cell
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What is the cell adhesion molecule?
L-selectin or CD62L which is expressed on the naive cell
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What does L-selectin bind to during the rolling process?
It binds to CD34 and Gly-CAM1
151
What does the endothelial (HEV) cell have on its surface? What does the naive T cell have on its surface?
Naive T cell has L-selectin, LFA-1, and CCR7
Endothelial cell has CD34, Gly-CAM-1, CCL21/19, ICAM-1
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What do naive T cells do once they enter the lymph node?
Naive T cell spends a day in a lymph node and interrogates 100s of conventional myeloid dendritic cells in search of specific antigen
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Fibroblast reticular cells (FRCs)
They are stromal cells in the T cell zone that create the 3D structure of the tissue (they create these little islands for T and cDCs to meet) and secrete chemokines CCL19 and CCL21 to attract DCs and native T and B cells
154
What is the single exiting vessel of the lymph node?
ALL cells leave lymph nodes via efferent lymphatic vessels
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Cells flow through other downstream _____ _____ along the lymphatic vessel before they rejoin the ____ near the ____
Cells flow through other downstream lymph nodes along the lymphatic vessel before they rejoin the bloodstream near the heart
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do all secondary lymph nodes have an efferent lymphatic vessel
Yea
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How do DCs enter the lymphatics?
They enter via the affereent lymphatics.
- When a cDC recognizes a threat using its PRRs, it alters its expression to produce a lot of CCR7 so it can be attracted to CCL19 and CCL21 (that the FRCs produce).
- The cDC enters the neartest lymphatic vessel and uses CCR7 to migrate into the lymph nodes that have chemokines CCL19 and CCL21 in T cell
- The cDC processes antigen along the way so it can present it by the time it is in the lymph node. It also starts expressing co-stimulatory B7.
- The cDC meets up with the T cell that has the same antigen, forms an. immunological synapse, and uses B7 to activate the T cell
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What are the different signals for T cell activation?
Signal 1: Recognize antigen with receptor (and co-receptor)
Signal 2: Costimulation through its CD28. This causes it to make cytokines for the 3rd signal
Signal 3: Signal cytokine IL-2, tells it to activate into thousands of cells. Then, it will get further signals to determine what type (differentiation) of T cell it will go into
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How many activating signals does a T cell get from a cDC?
2 and gets one from itself
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When is B7 made?
Only when a cDC (or other APC) sense a threat
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Do the signals have to be long and simultaneous to activate T cells?
Yes, signals 1 and 2 must be on together, for multiple days, to keep the cell alive
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Immunological synapse
- Immunological synapse allows for the activation signals to be simultaneous and sustained
- It holds the cells together using adhesion molecules (LFA_1, ICAM-1 and Talin). They hold in the peripheral ring of the synapse (p-SMAC)
- Immnunological synapse gather the TCR, co-receptors, and CD-28 and their kinases in the central-region (c-SMAC) for strong, sustained signaling
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Can you have T-cell activation without an immunological synapse?
No
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Antigenic Signal 1 for a T cell
- TCR complex has ITAM tyrosine targets that must be phosphorylated
- Co-receptor has the Lck tyrosine kinase that can do the job (Lck is analogous of Blk, Lyn and Fyn)
- ZAP-70 kinase can dock on p-ITAMS and Lck phosphorylates it, too
- p-ZAP-70 kinase phosphorylates adaptors LAT, SLP-76, and GADS, builds a scafooled of actions of ITk and phospholipase C-y
- HOWEVER FOR THIS TO BE EFFICIENT, it needs simultaneous signals from co-stimulatory receptor, CD28 to make PIP3
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Antigenic Signal 2 for a T cell
- CD28 costimulatory receptor is gathered in the immune synapse. It's phosphorylated by Lck, too
- PI3 Kinase docks on p-CD28 and generates most of the PIP3 which is a dock for Itk and PLC-y = efficient scaffoled formation
- p-PLC-y cleaves cell membrane lipid into potent second messengers, IP3 and DAG - then transcription factors are activated
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What is signal 1 and 2 for T cells essentially making then?
Signal 1 is making the scaffold
Signal 2 is attaching the scaffold and having it held by PLC-y
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What does activating signals 1 and 2 do?
Generates many transcription factors for expressing many genes, including making the IL-2 cytokine downstream of its other transcription factors
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What is the T cell tyrosine kinase?
Lck
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What does IL-2 tell the T -cell?
To proliferate and survive
It is AUTOCRINE ACTING IL-2 and induced high-affinity IL-2 Receptor (CD25)
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What is the IL-2 receptor?
CD25
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How many chains does the IL-2 (CD25) receptor have?
Three chains. Two are already present on a naive T cell. When the T-cell gets the first two signals and get transcription factors, one of them activates the gene to make IL2Ralpha which is the missing chain. That gets made as well as the IL-2 so it is autocrine acting
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Clonal proliferation of activated T cell
Generates 100's of clones of useful T cells that have the same TCR (from one rare naive T cell). This occurs in the LYMPH NODE
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Why is the activation of T cells tightly regulated?
- To prevent destructive T activation when no threat is present
- To automatically slow T activation
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What are the 3 ways to regulate T activation?
1. Activation signals must be simultaneous and sustained
2. Co-stimulatory signal (B7) is very limited
3. Activation is automatically slowed down by co-inhibitory CTLA-4
ACA
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1. Activation signals must be simultaneous and sustained
Activation is not due to a brief chance encounter wiht a single peptide:MHC molecule on a DC
Requires many surface molecules
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c-SMAC
TCR
CD2
CD4
CD8
CD28
PKC-theta
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2. Co-stimulatory signals, like B7 are very limited
It is ONLY available when APCs detect a threat via their PRRs. Must go to the lymph node to make B7 as well
- If a naive T cell binds antigen, but not B7, it will become anergic (unresponsive to IL-2)
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What happens to a naive B7 that binds antigen but no B7?
Becomes anergic
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3. Lymphocyte activation is automatically slowed by co-inhibitory receptors, like CTLA-4
- Activated T cells automatically start to express CTLA-4 on their surface which slows down their activation, like breaks on a car
- CTLA-4 binds B7 so well that it takes it away from CD28. Therefore, the cell that binds can no longer get signal 2. IT ALSO, transmits inhibitory signals in T cells to slow activation and proliferation
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What are the 3 ways CTLA-4 stops T cell activation?
1. It outcompetes CD28 for binding with B7, preventing the T cell from getting the activation signal
2. CTLA-4 can bind to the B7 and engulf it into a phagosome to remove it from the surface
3. CTLA-4 may transmit a negative signal that inhibits T-cell receptor signaling
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What is analogous of the ZAP-70 for B cells?
Sck
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What is analogous of the Lck for B cells?
Blk, Lyn or Fyn (the Src family)
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How does signal 1 for a B cell create the scaffold?
- Src family phosphorylate the ITAMs on the Iga and Igb and CD19 tails
- Activated ITAMs recruit Syk which binds to the ITAMs on the Iga and b
- Syk gets phosphorylated by the Src family again
- Phosphorulated CD19 recruits PI3K which makes PIP3
- Phosphorylated Syk recruits molecules for the scaffold
- PIP3 recruits and activates (by phosphorylation) PLC-y and BTK so they can bind the scaffold
- The scaffodl binds to the PLC-y
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What are the different effector T cells?
- CD8 Cytotoxic T cells
- CD4 Helper T cells:
- Th1
- Th2
- Th17
- Tfh
- Treg
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What do CD8 T cells do?
Kill virus-infected cells
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What do Th1 cells do?
- Help macrophages to suppress intracellular infections
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What do Th2 cells do?
- Help basophils, mast cells, eosinophils, and B cells respond to parasite infections
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What do Th17 cells do?-
- Enhance the neutrophil response to fungal and extracellular bacterial infections
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What do Tfh cells do?
Help B cells become activated, switch isptype, and increase antibody affinity
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What do Treg cells do?
- Suppress the activities of other effector-T Cell populations
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What are the 4 properties of aB TCR effector T cells?
1. Programmed to act in specialized ways
2. Effector cell no longer requires B7 co-stimulation to act
3. Effector cells express different migratory adhesion molecules and chemokine receptors than naive T cells
4. All effector T cells secrete cytokines to direct the immune response
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1. Programmed to act in specialized ways
- Cytotoxic CD8+ effector T cells eliminate host cells
- Helper CD4+ effector T cells secrete cytokine instructions
- Effector cells express different migratory adhesion molecules and chemokine receptors that naive T cells
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2. Effector cells no longer require B7 costimulation to act
Effector T cells can respond to anitgen alone (although other co-stimulatory molecules enhance the response)
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3. Effector cells express different migratory adhesion molecules and chemokine receptors than naive T cells
WHY? Many effector T cells must migrate to where the threats are
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What adhesion molecules does a naive T cell have? What about an effector cell and why?
Naive and Effector T cells use different adhesion molecules to enter different sites
Naive T Cell: L-selectin, LFA-1
Effector T cell: VLA-4, PSGL-1
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How does a naive T cell and an effector compare in terms of the amount of L-selectin on the surface?
- Naive T cell has a moderate amount of L-selectin
- Effector T cell has no L-selectin
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How does a naive T cell and an effector compare in terms of the amount of VLA-4 on the surface?
- Naive T cell has no VLA-4
- Effector T cell has a moderate amount of VLA-4
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How does a naive T cell and an effector compare in terms of the amount of LFA-1on the surface?
- Naive T cell has a moderate amount of LFA-1
- Effector T cell has a high amount of LFA-1
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How does a naive T cell and an effector compare in terms of the amount of CD2 on the surface?
- Naive T cell cell has a moderate amount of CD2
- Effector T cell has a high amount of CD2 (CD2 acts like an LFA)
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How does a naive T cell and an effector compare in terms of the amount of CD4 on the surface?
- Both express a moderate amount
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How does a naive T cell and an effector compare in terms of the amount of CD8 on the surface?
- Both express a moderate amount
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What is the purpose of VLA-4?
To enter inflamed sites (explains why a naive T cell doesn't have any)
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What is the purpose of LFA-1
Enter all sites and form an immune synapseW
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What are the different chemokine receptors that the different effector T cells express?
- Once the T cells become effectors, they shed their CCR7 chemokine receptor they needed to take them to the T cell area
Th1: CCR5
Th2: CCR4
Th17: CCR6
Tfh: CXCR5
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THERES A DIFF BETWEEN CCR5 and CXCR5
CCR5 is expressed by Th1 cells
CXCR5 is expressed by Tfh cells
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What does a Th1 cell use CCR5 for?
To find infected macrophages that are secreting CCL5
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4. All effector T cells secrete cytokines to direct the immune response
By making different cytokines, different classes of effector T cells guide different cell-mediated immune responses
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What cytotoxins and cytokines do CD8 T cells secrete?
Cytotoxins:
- Perforins
- Granzymes
- Granulysin
- Serglycin
Cytokines:
IFN-y
LT
IL-2
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How do cytotoxic T effector cells use granules?
- CD8 T cells encounter and has nonspecific adhesion of target (random nonspecific bind of a target)
- Antigen recognition redistributes cytoskeleton and cytoplasmic components of T cells. Lytic granules are placed at the front of the cell (containing perforins, granzymes, granulysin, and serglycin)
- Once the immunological synapse forms, the lytic granules are aimed at the target
- Granules are released only on target cell. Bystander cells are safe. It causes apoptosis
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There are different types of CD4 cells because?
There are different types of microbes that need a different immune response
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What are the different responses against different microbes (you can also say what T cells are in these areas)
Type-1 response: made for intracellular pathogens that can survive in macrophages (you can have Th1 cells in this response)
Type-2 response: Some pathogens are too large to be phagocytosed, so they are expelled from the body (you can have Th2 cells here) --> weep and sweep response
Type-3 response: Some small pathogens are extracellular and easily opsonized and phagocytosed (Th17 response for improving neutrophil activity)
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Some locations require continual, non-destructive defense (Tregs)
- The type of tissue that a microbe invades presents a different threat
External tissues that are built to withstand some damage and then repair can have Type 1,2, and 3 responses
However, thin mucosal tissues can't tolerate inflammatory destruction because microbiota would flood in and the functions like respiration, nutrient absorption, and reproduction would stop
--- CONSTANT T reg responses help non-inflammatory defenses, liek secretory IgA here
- Type 1,2, and 3 responses are only here when there is a severe threat in these areas
(epidermis of the skin, gut epithelim and respiratory system)
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What guides which which kinds of T cells arise?
Cytokines. Dendritic cells are a major source of fate-specifying cytokines
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Cytokines impacting T cell fate
- In a lymph node during activation of a naive T cell it gets signal 3
- Different combinations of cytokines are present
- The predominant cytokines dictate which path the activating CD4 cells follow
- These are called fate-specifying cytokines
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What combination of cytokines create Th1 cells? What is the transcription factor that exist? What is its characteristic cytokine
Combination:
- IFN-y
- IL-12
TF:
Tbet
Characteristic Cytokine:
IFN-y
IL-2
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What combination of cytokines create Th2 cells? What is the transcription factor that exist? What is its characteristic cytokine
Combination:
- IL-4
TF:
GATA3
Characteristic Cytokine:
IL-4, IL-5, IL-13
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What combination of cytokines create Th17 cells? What is the transcription factor that exist? What is its characteristic cytokine
Combination:
- IL-6
- IL-23
- TBG-B
*23-6 is 17
TF:
- RORyt
Characteristic Cytokine
- IL-17
- IL-twenty two
*it's strange but they don't come from IL-17
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What combination of cytokines create Tfh cells? What is the transcription factor that exist? What is its characteristic cytokine
Combination:
- IL-6
TF:
Bcl-6
Characteristic Cytokine:
IL-21
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How do these fate-specifying cytokines get in the environment?
- Cytokine concentrations vary in depending on the threat that innate cells sense. These innate cells in the lymph node can sense this because remember there is antigen flowing in this area
- cDCs are a source of fate-specifying cytokines. The cDC1 subset provides IL-12. The cDC2 subset provides IL-23, IL-6, and TGF-B
- cDCs supply Il-12, Il-6, IL-23, and TGF-B
- Basophils, ILC2, eosinophils supply IL4
- NK cells/ILC1 supply IFNy
- Macrophages supply IL-12, IL-6, TGF-B
- Epithelial cells supply TGFB, IL-23, IL-6
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Activating T cells reinforce their subset
Activating TH1 - IL 2, IFNy
Activation TH2 - IL4
Activating Treg- TGF-B
While they are activating, they make the cytokine that makes them in a positive feedback loop, it is not necessarily their characteristic cytokine
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Is signal 3 for T cell activation just IL-2?
No, it is also the fate specifying cytokines it happens to have in that environment
They make their fate specifying cytokine and also IL-2
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How are Tregs differentiated by cytokines?
- Mucosal tissues that are healthy make lots of TGFB
- TGF-B floods the lymph nodes in the yellow areas (the peyer's patches) and induces all CD4 T cels that activate here to automatically differentiate into Tregs
- Tregs will suppress inflammation when they bind antigen
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What are the functions of TGF-B?
- One role is a fate-specifying cytokine for making induced Tregs
- Another function is to suppress pro-inflammatory activities of TH1 and TH2 effectors. It's automatically produced in HIGH amounts at mucosal barriers
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Typically, epithelial cells express TGF-B to create Tregs. But when they recognize a MAMP, what do they also release (IN ADDITION TO TGF-B)
They also express IL-6 and IL-23 to create a TH17 response. This will only occur when a threat is detected
They want a type 3 response because they want a quick and effective response that hopefully isn't super destructive
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TH1 Effectors activate destructive M1 macrophages
Monocytes or macrophages become M1 macrophages with 2 singals
1. IFN-gamma (IFNy) is 1 activation signal for this infected macrophage
2. CD40L co-stimulatory molecule is a 2nd activation signal
THESE SIGNALS ARE GIVEN BY THE TH1 CELL
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Effector Functions of M1 macrophages
1. Increased phagocytosis and phagolysosome activity
2. Make DESTRUCTIVE reactive radicals to kill microbes
3. More antigen presentation
express co-stimulatory molecules like B7 and CD40
increased antigen processing and MHC expression to activate effector T cells at infected site
4. Release pro-inflammatory cytokines (like TNF-alpha, lymphotixin, IL-12, chemokines)
to recruit and activate immune cells - this is destructive
to change local blood vessels - this is destructive
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Can Th1 cells kill macrophages?
- Thq cells activate destructive M1 macrophages
- They kill macrophages that are overwhelmed using FasL and LT-beta in case an infected macrophage is overwhelmed
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What are all Th1 effector function ?
1. Th1 cells produce IFN-y and CD40L to activate M1 macrophages
2. FasL and LT-Beta produced by Th1 cells induce apoptosis of bacteria-laden macrophages
3. IL-2 produced by Th1 cells act on naive CD4 and CD8 T cells which alters the balance of Th1 versus Tfh differentiation to favor Th1; influences differentiation of CD8 CTLs and memory CD8 T cells
4. IL-3 and GM-CSF stimulate production of monocytes by bone marrow
5. Th1 cells produce TNF-alpha and Lt-alpha which act on local blood vessels which allow monocytes to exit the endothelium into the site of infection
6. CCL2 produced by Th1 cells is a chemoattractant for monocytes
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How do Th2 cells recognize pathogen and respond?
Th2 effector T cells recognize pathogen antigens presented by MHC II on macrophages and (not rlly) eosinophils at an infested site
The Th2 effector T cell responds by secreting IL-3, IL-4, IL-5, IL-9, IL-13 to activate eosinophils and mast cells at the site
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What do eosinophils activated by Th2 cells do?
Release many toxic mediators that weaken or kill parasites
- Eosinophil peroxidase
- Eosinophil collagenase
- Major basic protein
- Eosinophil cationic protein
- Eosinophil-derived neurotoxin
MAJOR BASIC PROTEIN
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What do mast cells activated by Th2 cells do?
Activated mast cells degranulate and secrete inflammatory mediators... like histamine
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Histamine induces
- Smooth muscles cells to contract
- Blood vessels to dilate
- Mucosal cells to secrete mucus
SBM
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Th2 cells effectors activate non-destructive M2 macrophages
M2 macrophages
1. Enhanced MHC II to present antigen
2. Use arginase to crease amino acid building blocks for tissue repair
3. Release ornithine to stimulate smooth muscle contraction
4. Block pro-inflammatory IL-1alpha with antagonists and decoy receptors
5. Suppress inflammation by secreting TGF-Beta and IL-10
EURBS m2!
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What do TGF-B and IL-10 always do?
Suppress
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What is the type 2 response referred to as?
Weep and sweep which means use a lot of fluids to flush away large pathogens
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Th2 effector cell functions
- Th2 cells produce Il-13 while increases epithelial cell turnover and mucus
- IL-13 produced by Th2 increases smooth muscle contraction that enhances worm expulsion
- IL-3 and IL-4 cause recruitment of M2 macrophages
- IL-5 recruits and activates eosinophils
- Th2 cells drive mast cell recruitment via IL-3, IL-9. Specific IgE arms mast cells against helminths
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How do Th17 (type 3) cells recognize pathogen and respond?
Type 3 response against small pathogens at mucosal barriers
- TH17 effectors recognize a pathogen antigens presented by macrophages and DCs at a barrier
- Then TH17 effectors improve barrier defenses. by secreting more antimicrobial defensins to lyse bacteria and replacing compromised epithelial cells
TH17 cells communicate with the mucosal epithelial cells, not even the neutrophils at this point
- They also CAL phagocytic neutrophils to eliminate any extracellular microbes at the barrier but this is a last resort because there is a lot of damage associated with this response. The type 3 response is typically brief and tries to get it done in one wave, if not A TH1 RESPONSE FOLLOWS
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How do Th17 cells impact neutrophils?
- IL-17 produced by Th17 cells activates stromal cells and myeloid cells to produce G-CSF which stimulates neutrophil production in bone marrow to increase the amount circulating
- IL-17 produced by Th17 cells activates stromal cells and epithelial cells to produce chemokines that recruit neutrophils
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What are the two ways CD4 T cells can become regulatory?
- During selection in the thymus (natural T regs)
- During antigen activation in secondary lymphoid tissue (pTregs aka iTregs)
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pTregs
arise from naive T cells recognizing foreing antigen in the presence of mainly TGF-Beta -- only a minor threat, like commensals on the surface of a barrier
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nTregs
arise from self-reactive T cells in the thymus that were programmed to use Foxp3 --- there really is not a threat, it is self-antigen that nTregs respond to
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So do nTregs respond to antigen?
Not really, they are responding to self. pTregs are responding to antigen which are just commensal microbes
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What do both pTregs and nTregs do?
Both recognize peptide:MHC on an APC and then suppress that APC and neighboring T cells
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Actions of regulatory T cells are examples of
PERIPHERAL TOLERANCE
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Purpose of regulatory T cells at epithelial barriers?
- To tolerate commensal microbiota outside of barrier
- To tolerate food antigens
- To tolerate air-borne allergens
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What are the two examples of peripheral tolerance?
- Eliminating B cells that bind antigen in the bloodstream of secondary lymphoid tissue
- Using Tregs to suppress the activity of T cells on an APC presenting self-antigen
