1
Q
How do you differentiate between B and T lymphocytes?
A
- Terminally differentiated T lymphocytes express CD3
- if they express CD4 antigens they are helper cells
- if they express CD8 antigens they are cytoxic cells
- Terminally differentiated B cells express CD18
- plasma cells express CD38
2
Q
Explain T cell differentiation from its precursor to its terminal cells
- which location does each stage take place in
A
- Orginiates in the bone marrow than mature into CD8+ or CD4+ cells in the thymus
- CD4+ cells can further differentiate into different types of helper cells depending on the types of cytokines they produce
- Th1, Th2, Th17, regulatory T cells
3
Q
What is the difference between the development of B cells and T cells?
A
- B cells develop in the bone marrow whilst T cells migrate from there and mature in the thymus
- B cells are produced throughout the lifespan in the bone marrow, but T cells are produced in the thymus, which involutes at puberty
- decreased T cell production in adulthood
- new T cells can be produced in extrathymic sites, these provide long-lived peripheral T-cell pool ( liver intestine)
- both have diverse repertoires of Ag receptors in gene rearrangement
4
Q
Describe the stages of development for B cells
A
- originates from a common lymphoid progenitor in the bone marrow
- the emergence of pro-B cell when progenitor undergoes genetic rearrangement of diversity (D) and joining (J) segments - DJµ rearrangement
- differentiates to large then small pre-B cells - there is expression of the first receptor on the cell surface
- Immature B cells undergo the process of positive and negative selection
- positively selected cells are released into peripheral blood
- negative selection results in apoptosis
5
Q
Describe the stages of development for T cells (specific)
A
- T cell precursors migrate to the thymus and undergo 4 stages for Double negative (DN) differentiation
- they don’t express either CD4+ or CD8+ receptors
- DN4 cells express pre- T cell receptors then for through an immature single positive phase before becoming Double Positive (DP )for CD4 and CD8
- these DP cells undergo positive and negative selection before being released into peripheral as either CD4 cells or CD8 cells, or if negatively selected, apoptosis
6
Q
What are the common stages of B and T cell development?
(3)
A
- 1st phase: generation of an antigen receptor
- V(D) J gene rearrangement – producing a new antigen receptor
- 2nd phase: refinement of the antigen receptor repertoire
- Antigen receptor is tested for antigen recognition (self-antigens)
- Only those receptors that recognise self-antigen are selected- POSITIVE SELECTION
- Those receptors which bind strongly to self-antigens are deleted - NEGATIVE SELECTION
- 3rd phase: Stimulation by foreign antigen
- Clonal selection of lymphocytes (in the lymph nodes and spleen)
- Generation of effector and memory lymphocytes
7
Q
What are the two classes of B Cell antigens?
A
- Thymus dependent antigens
- dependent on Th cells to induce antibody production
- formed of proteins
- doesn’t have repeating motifs, prevents them from being spontaneously cross-linked not the b cell surface
- Thymus Independent antigens
- not dependent on Th cells to induce antibody production
- formed of polysaccharides, lipids
- have repeating motifs which allows the b cell receptor to be cross-linked on the surface of the b cell
8
Q
Explain the B cell Th cell independent response
A
- T-independent antigens are repetitive antigens so they allow B cell receptors to be cross lined and aggregate on the surface of the B cell
- this provides an activation signal
- still require secondary activation signal provided by TLR engagement
- naive b cells express both IgM and IgD on their surface
- T-independent activation these b cells commonly secret IgM antibodies
- they are not able to class switch as this requires T cell help
- the immune response is short-lived and doesn’t provide immunological memory
9
Q
What type of antigen recognition can B cells and T cells do?
A
- B cells can recognise native antigens
- T cells can only bind processed peptide antigens presented to them in the context of MHC molecules.
10
Q
Explain the B cell Th Dependent response
A
- Antigen binding to BCR provides “Signal 1” to B cell
- Antigen is internalised, processed and antigenic peptides are displayed on MHC-II for T cell recognition
- CD4 Th recognises antigen-MHC complex via the T cell antigen receptor (TCR): provides “Signal 1” to T cell
- CD80/CD86 on B cell binds to CD28 on T cell provides “Signal 2” to T cell
- causes increased T cell activation
- there is also up-regulation of CD40Ligand on these T cells cell-surface
- these bind to CD40 providing “Signal 2” to B cell
- activated T cell also produced Cytokines which further activates B cells…“Signal 3”
- B cell proliferates and undergo somatic hypermutation to improve the affinity of the B cell receptor to the antigen
- ultimately b cells differentiate into antibody-secreting B cells (plasma cell) or becomes a memory B cell
11
Q
What is the structure of T cell and B cell antigen receptors (TCR and BCR)?
A
BCR
- light chain and heavy chain - y shaped
- always associated with co-receptors made up of
- Ig-beta and Ig-alpha
- helps transduce signals internally to B cells following BCr engagement with an antigen
TCR
- Alpha chain and Beta chain heterodimer that is the site of recognition
- has co-recoptor
- CD3 - also for signalling
12
Q
What does V(D)J stand for in terms of TCR and VCR formation?
A
- Variable
- Diversity (always in brackets as only the heavy chain contains diversity in BCR)
- Joining
13
Q
Where does V(D)J recombination take place in B cells?
A
- on the Ig heavy chain gene on Chromosome 14
- Diversity segments only occur in the heavy chain
- on the kappa and lambda light chain genes on Chromosome 2 and 22 respectively
14
Q
Where does V(D)J recombination take place in T cells?
A
- on the alpha chain gene - Chromosome 14
- on the beta chain gene found on Chromosome 7
- only undergoes VJ there are no diversity segments
15
Q
How are new gene sequences produced through V(D)J recombination?
A
- Heavy chain rearrangement involves DJ recombination event followed by a VDJ rearrangement
- the DJ recombination happens in the proc T/B cell phase
- B cells in the IgH gene | T cells in the alpha chain gene
- the DJ recombination happens in the proc T/B cell phase
- Light chain rearrangement is a single step VJ recombination
- new pieces of genetic code are transcribed into proteins forming membrane-bound immunoglobulin
- in Immature B cells this is always IgM and IgD
16
Q
How does genetic rearrangement occur?
A
- Rearrangement occurs between specific sites on the DNA called Recombination Signal Sequences (RSSs)
- These sequences contain conserved segments of DNA composed of a heptamer, a spacer (not conserved) and a nonomer
- They are found on the 3’ side of V segments, the 5’ side of J segments and both the 3’ and 5’ side of D segments
- Rearrangement is catalysed by two Recombination Activating Genes: RAG-1 and RAG-2
17
Q
What are RAG-1 and RAG-2?
A
Recombination Activating Genes, used for V(D)J recombination in BCR and TCR
18
Q
What are the steps in V(D)J recombination?
A
- Cleavage
- begins with the binding of RAG1/RAG2 endonucleases to RSSs which flank the coding sequences to be joined
- RAG complex cuts one strand of DNA at the end of the heptamer sequence
- 5’ cut end of this DNA strands reacts with complementary uncut strand - breaking it and forming a double-stranded break at the end of the heptamer sequence
- Repair/Diversity
- through the action of additional proteins such as Ku70:Ku80 that join the complex (indicated in blue), the DNA hairpin is cleaved at a random site to yield a single-stranded DNA end
- this end is then modified by the action of TdT(pink) to create diverse imprecise ends
- Joining
- the two non-modified heptamer sequences are ligated to form a precise signal joint while the coding joint is also ligated by the action of DNA ligase IV
19
Q
Review V(D)J recombination using this image
A
- V(D)J recombination is the primary mechanism for generating diversity in B and T lymphocytes.
- The first recombination event takes place in pro B and pro T cells and involved DJ recombination in the IgH gene and the TCR beta chain gene respectively
- The next step involves the recombination of their new DJ sequences with a new V sequence
- Subsequently, the light chain genes (kappa or lambda) in B cells and the alpha chain gene in T cells undergo VJ recombination
- These new genes are then transcribed into mRNA and translated into proteins, which are assembled and then expressed on the surface of B and T lymphocytes as unique B cell receptors and T cell receptors, respectively.
20
Q
What is Somatic hypermutation?
- when does it occur
- what is the outcome
A
- Somatic hypermutation or SHM is a diversity-generating process
- takes place in the centroblast stage of b cell differentiation
- It adds further diversity to already rearranged (V(D)J recombined) segments through the introduction of point mutations and makes
- produces antibodies with higher affinity for antigens/ lower affinity to antigens/ no change in affinity
- Affinity maturation occurs after SHM selects for B cells that produce the highest affinity antibodies
- The mutation rate is ~1 base per 1000 – this is ~ 1 million times higher mutation rate than is observed during normal cell division
21
Q
What is affinity maturation?
- when does it occur?
A
- the selection process for antigen binding that occurs in follicular dendritic cells the light zone of the germinal centre
- it selects for B cells that produce the highest-affinity antibodies
- B cells that have undergone SHM but bind antigens with lower affinity are outcompeted and undergo apoptosis
22
Q
Where does Somatic hypermutation take place in genes?
A
- occur at very restricted loci - hypervariable regions
- These regions correspond to the complementarity determining regions CDRs - the sites involved in antigen recognition on the antibody.
- The most common type of mutation involves nucleotide substitution rather than addition or deletion
23
Q
What are the two important enzymes in the SHM process?
- how do they work to generate mutation
A
- Activation-induced cytidine deaminase (AID)
- triggers SHM, it deaminates cytidine into uracil
- this base mismatch is incorrectly repaired to generate mutations via mismatch repair and base excision repair
- Uracil N-glycosylase,
- removes uracil bases generated by AID-mediated deamination - base excision
24
Q
What is Class Switch Recombination?
A
- The process of CSR allows for the substitution of one isotype for another, thus providing the antibody with new functions and a different distribution throughout the body
- the same receptor with a different constant region, allowing the cell to perform different effector functions
- this is how different Immunoglobulins are formed
- It allows long term B cell memory to occur as class-switched B cells become long-lived plasma cells or memory b cells
25
What gene loci are associated with Class switch recombination (CSR)?
- how is IgD formation different?
* CSR induced by different cytokines caused by cellular interactions of the B cell with different T cells
* interactions direct B cells towards a specific isotype switch
* this occurs exclusively at the heavy-chain antibody locus
* CSR DNA sequences lie upstream of each set of constant regions - **Switch regions (S-regions)**
* each S-region is associated with an upstream promoter and exon, which allows for the production of sterile (noncoding) germline transcripts through the S-region
* IgD lacks a defined S-region. it uses an alternative splicing mechanism with the mu constant region
26
How is Class switch recombination achieved (CSR)? (mechanism)
* the first target region is always the Su switch region which lies downstream of the VDJ sequences of the heavy chain
* the other partner switch regions is determined by the cytokines released by the T helper cell involved in the B cell activation process
* the cytokines they secret determine which transcription factors are activated and which switch region is targeted by AID
* _Su region_ and the _Sgamma1 region_ are targeted by _AID._
* AID changes cytidine bases for uracils which are then excised by uracil N glycosylase creating DNA strand breaks.
* These double-stranded DNA breaks at both switch regions are brought together by the DNA damage repair machinery and are spliced together
* this removes all of the intervening DNA
* leading to a new active transcript downstream of the VDJ sequence resulting in
* the transcription of a new constant region
27
What is the significance of alternative splicing in B cell formation
* IgD is generated through an alternative splicing mechanism rather than CSW as there is no defined S-region for IgD
28
# Th2, Th1, Treg
What cytokines are associated with the formation of Ig classes during CSR
- Th2
- Th1
- Treg
29
What are the different responses/ functions of the various Immunoglobulins?
- IgM
- IgD
- IgG
- IgA
- IgE
30
Why do T cells need antigen presentation?
* they can't recognise native cells they need to be processed and presented to them
31
What are the main professional antigen-presenting cells?
- what is their niche
* **Dendritic cells (DCs)**
* the broadest range of antigen presentation
* _potent TH cell activator_ as they express co-stimulatory molecules _CD80 + CD86_
* **Macrophages**
* **B cells**
* internalises the antigens that bind to its BCR and presents incorporated to MHC II molecules
* best at presenting antigens specific to its own BCR
32
What is the significance of the MHC classes
* MHC class I - found on all nucleated cells and they present intracellular antigens such as viruses
* MHC class II - present extracellular antigens such as bacteria
T cells need to be able to distinguish between external antigens (taken up by APCs) and internal antigens (infected cell)
33
Explain Antigen Processing
* Enzymatic process of degrading proteins through proteases into antigenic peptides - this requires energy and movement of endocytic vesicles
34
Give an overview of Antigen Processing in MHC Class I vs MHC Class II presentation
_Class 1_
* endogenous antigens inside the cell are presented to CD8+ T cells
_Class 2_
* exogenous antigens in endosome are presented to CD4+ T cells
35
What are the two antigen processing pathways?
* Endogenous antigens in cytosol presented on class I MHC molecules to CD8+ T cells
* Exogenous antigens in endosomes presented on Class II MHC molecules to CD4+ T cells
36
Which proteasomes generate peptides for MHC class I presentation?
* **26S proteasome** (or standard proteasome): which is expressed by most cells;
* **the immunoproteasome:** which is expressed by many immune cells
* **the thymic-specific proteasome:** expressed by thymic epithelial cells
37
How are peptide levels in cells regulated?
* the rate of production of a protein and the rate of degradation
* in eukaryotes, ubiquitin marks target proteins for proteolysis which occurs in the proteasomes
38
How are endogenous antigens proteins transported?
* TAP proteins (**_T_**ransporters associated with **_A_**ntigen **_P_**rocessing)
* TAP 1 and TAP 2 form heterodimer in the membrane of ER to facilitate _selective transport of peptides from cytoplasm into lumen of ER_
* TAP pump preferentially transport peptides with a length of 8–15 amino acids
39
Give an overview of the peptide loading onto MHC Class 1 molecule
* MHC-I alpha chains are held intially held in the ER with the chaperone protein **calnexin**
* MHC-I alpha chains bind with **Beta-2 microglobulin** to form a complete MHC-1 molecule
* it is then released from calnexin and joins a peptide loading complex consisting of
* tapasin (Tps), ER-protein-p57, and calreticulin (CRT)
* **Tapasin** associates with **TAP transporters** causing
* proteins degraded in the cytosol by proteasomes to give polypeptides which are transported to the ER
* the protein binds to the MHC-I molecule which can now be released an exported from the ER to the plasma membrane
40
What are CTLs and what do they do
* they are effector CD8+ T cells
* eradicate infected cells,
* can be activated against cancer cells
* they target neo-antigens
41
How does Immune evasion occur in MHC I?
* Viruses can interfere with Class I MHC expression to escape killing by CTLs
* Herpes Simplex Virus (HSV) protein ICP47 can selectively bind to TAP and inhibit the transfer of peptides into ER so they are not presented as MHC-1 complexes the plasma membrane
* Some parasites invade de-nucleated red blood cells, which do not express MHC-I
42
How are antigen peptides generated in the exogenous pathway?
- overview
* Acidification of endocytic vesicles activates proteases that degrade proteins into fragments
* the digestions occur in endolysosomes/ phagolysosomes
* vesicles containing peptides fuse with vesicles containing MHC II molecules
* These peptide fragments are loaded onto MHC class II molecules
43
Explain the trafficking of MHC class II molecules
* MHC class II a and b chains associate in the ER
* In the trans golgi network, MHC class II is sorted into vesicles
* These vesicles deliver MHC class II to specialised compartments where peptide loading occurs
44
What prevents MHC class II from binding “self” peptides in the ER?
The Invariant Chain
* new MHC II molecules in the trans-golgi network are associated with an **invariant chain (Ii)** peptide
* this prevents inappropriate binding of antigens and enables the MHC II molecule to exit the ER
* the complex of Ii and MHC II is transported through the golgi to an acidic **endosome compartment**
* due to the acidic pH the **cathepsin proteases** are activated and they digest the invariant chain leaving a
* **residual Class II-associated Invariant chain peptide (CLIP)**
* in the compartment, they will ultimately encounter an antigen that has been internalised and partly degraded
* the two endosomes form **multivesicular bodies (MIIC)** where the Ii protein is replaced by the antigenic peptide this process requires the chaperone
* **HLA-DM** or in B cells **HLA-DO**
* tubular vesicles bud from the MIIC and carry the new MHC II peptide complex to present on the cell surface
45
How are exogenous antigens recognised?
- what happens after they are recognised
* they have to be taken up by APCs to get noticed by CD4+ T cells
* this leads to the activation of macrophages and the production of secreted antibodies by plasma cells
46
How does Immune evasion occur in Class II MHC
_Viral Inhibition of Class II MHC_
* Adenovirus interferes with Class II upregulation in APCs
* The HSV viral envelope protein, glycoprotein B, reduces MHC Class II processing and inhibits the production of invariant chain peptide
* HIV interferes with Class II processing
_Pathogens that evade lysosomes_
* Leishmania & mycobacteria (tuberculosis) prevent phagosome-lysosome fusion
204: Theme 1 - Immunology
flashcards
Decks in class (13)
# Cards
-
Innate Immunity44
-
Adaptive Immunity46
-
Immunological Tolerance23
-
Vaccines and Vaccine Development40
-
Regulating Immune Response: Cytokines17
-
Immunological Diseases18
-
Mucosal Immunology36
-
Autoimmune Diseases43
-
Pharmacological aspects of Immunology53
-
Psychoneuroimmunology16
-
Systemic Inflammatory Disease Symposium27
-
Immunodeficiency Diseases22
-
Allergy22
