Download Exam 1 - B-T Cell development

B Cell development – Immunoglobulin Diversity
Immunoglobulin Diversity:
3 ways to increase diversity:
1. Different combinations in Light Chain V+J and Heavy Chain V+D+J.
2. Introduction of P and N nucleotides at Junctions between gene segments
during recombination. (1 in 3 chance of being a productive
rearrangement)
3. Different combinations of Heavy Chain and Light Chain association.
A 4th way: Mature B-cells that have seen antigen will begin to produce more
immunoglobulin; single-nucleotide point mutations (somatic hypermutation)
can lead to even more Ig diversity. Some of these hypermutated
immunoglobulins will have a stronger affinity for antigen, and B-cells with
these stronger binding immunoglobulins will out-survive the other B-cells –
affinity maturation.
Immunoglobulin DNA includes:
2 light chain types (κ & λ) and 1 heavy chain (μ)
Each chain locus has L (leader) region
Each locus has C (constant) region
Each locus has several versions of the V (variable) region and J (joining) region
Heavy chain locus has 27 D (diversity) regions



κ-light chain (chrom 22) ~30Vs, 5Js
λ-light chain (chrom 2) ~40Vs, 4Js
μ-Heavy Chain (chrom 14) ~65Vs, 6Js, 27Ds
Final hypervariable protein segment is composed of
 1V+1J(joining) in Light Chains
o V+J join in 1 somatic recombination
 VJ = hypervariable region of light chain
 1V+1D(diversity)+1J in Heavy Chain
o D+J join in 1st somatic recombination
o V+DJ join in 2nd somatic recombination
 VDJ = hypervariable region of heavy chain
Unproductive rearrangements in the heavy chain or later in the light chain will
lead to cell apoptosis in the bone marrow.
Recombination enzymes use Recombination Signal Sequences (RSSs) to ensure
the correct order of VDJ. Two of the enzymes in the VDJ recombinase are RAG-1 and
RAG-2 and are only found in early B and T cell stages.
RAG-1 and RAG-2 bind different forms of RSSs and bring them together. Other
DNA enzymes cleave out a section of DNA and join the two ends together while
simultaneously adding in new peptides to increase diversity. (junctional diversity)
Only productive rearrangements will survive.
Order of B-Cell development:
1. Stem Cell
o DNA unchanged
(“germline”)
5. Small pre-B cell
o Light chain V+J rearrangement
o μ-Heavy Chain in ER.
2. Early Pro-B cell
o Heavy Chain D+J
rearrangement
6. Immature B cell
o Heavy chain and κ or λ light
o IgM on surface
3. Late Pro-B Cell
o Heavy Chain V+DJ
rearrangement
7. Mature B cell
o IgD and IgM on surface
o Migrate to periphery
4. Large pre-B cell
o μ-Heavy Chain has formed
o “surrogate” light chain exists
o “Pre-B cell receptor” on
surface
After mature B-cells bind antigen and produce more Ig, the Ig isotype can be changed by
recombination of DNA to join the previously established V region with a different C region.
This switching only happens in the presence of antigen activated T-cells which are secreting
cytokines. Switching to different isotypes is not random, it depends on the help from
Antigen:T-cell and from the body location (e.g. IgA and IgE are favorable in skin, IgG in
interior body).
When B cells develop in the bone marrow, they communicate with the stromal cells:
 CAMs = cell adhesion molecules
 Integrin VLA4 – binds to adhesion molecule VCAM-1
 SCF = stem cell factor on stromal cells
 Kit = SCR receptor on B cell
o Kit activation causes forming B cell to proliferate
 Later Il-7 is required for growth and proliferation
If a developing B-cell encounters self-antigen, it will either apoptose or become anergic
 Multivalent self-antigen – causes strong cross linkage. RAG stays active to try to
make different light chains that will correct this… if not the cell dies: clonal deletion
(apoptosis)
 Soluble self-antigen – a weak signal that results in low IgM heavy chain expression
on surface, but normal IgD on surface which doesn’t activate the cell: anergy.
T-Cell Receptors
T-Cells have antigen receptors on the surface, TcR. They must see antigen presented to
them on human antigen presenting glycoprotins called MHC molecules which get their name
because they are found in a chromosome region called major histocompatibility complex.
TcR are made up of α-chain and β-chain.
 α-chain diversity = V+J
 β-chain diversity = V+D+J
Further diversity via N & P nucleotides added at junction
(note: A 2nd less common type of TcR is made from γ-chain and δ-chain)
Like Ig diversification, these TcR recombination events require RAG-1 & RAG-2

A defective RAG-1 or RAG-2 gene is the cause of severe combined
immunodeficiency disease (SCID). Combined meaning that both B and T cells
are absent. Autosomal recessive.

Partially defective RAG-1 or RAG-2 is Omenn Syndrome which leads to lack of
B cells, but some malformed T cells present. T-cells infiltrate the skin, gut, liver,
spleen
o Diffuse erythroderma
o Protracted diarrhea
o Failure to thrive
o Hepatosplenomegaly
o Rapid death
To leave the ER, the TcR associates with 4 CD3 molecules (2ε, δ, γ) and 2ζ(zeta)
molecules.
MHC Structure & Function
MHC Class I
 α-heavy chain (α1,α2,α3) and β2-Microglobulin.
o α1,α2 fold to create peptide binding site
MHC Class II
 α-heavy chain (α1,α2) and β-heavy chain (β1, β2)
The lower portions of the MHC molecules are immunoglobulin-like. They are important
in binding to CD4 or CD8 co-receptors.
The binding site/groove is formed by α-helix (walls) and β-pleated sheets (floor).
MHC Class I Groove
 Groove limited by pocket-like ends
 Holds peptides 8-10aa long.
 Best for hydrophobic or basic residues
 Uses carboxy or amino terminus for binding
MHC Class II Groove
 Groove ends are opened to allow binding of larger peptides
 Binds peptides 13-25aa or more
 Does not rely on carboxy or amino terminus for binding
For peptide molecules to bind MHC Class I & II, they don’t need a specific sequence of
amino acid residues, but just a few “anchor residues” that are either always the same
residue or always similar (i.e. always hydrophobic, etc.). All other non-anchor residues
can be “random.”
MHC Class I molecules are expressed on almost every nucleated cell in the human body
and thus CD8 cells can find infection almost anywhere. MHC Class II cells are less
prevalent, mostly on professional APCs. Interferons IFNγ and IFNα can upregulate
expression of MHC-I & II.
MHC Class I Antigen Presentation:
 A virus resides in cell cytosol and uses host cell to produce virus protein
 Some virus protein is degraded by cell’s normal proteasome activity
 Peptide fragments 8aa or longer are transported into ER by TAP transporters
 Also in the ER, the MHC Class I molecule is being folded.
o Calnexin chaperone assists in folding of the α-heavy chain (Ca2+
dependant)
o When β2-microglobulin binds, Calnexin leaves and is replaced by
calreticulin and tapasin chaperones.
o Tapasin holds the MHC molecule in close proximity to the the TAP
transporter.
 Tapsin and calreticulin leave upon peptide binding to MHC.
 MHC with bound peptide leaves ER bound to vesicle membrane.
MHC Class II Antigen Presentation:
 Antigen agent is internalized by endocytosis or phagocytosis
 Lysosomes fuse and begin to degrade the antigen into peptides
 Developing MHC Class II molecule leaves ER in vesicle with an invariant chain
attached to the peptide groove.
 All but a small part of the invariant chain is degraded. The CLIP portion that
remains binds to the peptide groove.
 This vesicle fuses with the vesicle containing the antigen peptides.
 HLA-DA molecule removes CLIP and allows peptide to bind MHC Class II
MHC Genetics:
 Important alleles for HLA Class I
o A, B, C
 Important alleles for HLA Class II
o DP, DQ, DR
 Each chromosome copy has a set of alleles (haplotype) for a total of 6
alleles in the genotype.
 HLA alleles are co-dominant, meaning both haplotypes are expressed at
the same time.
 HLA alleles are organ important for transplant success.
 HLA typing can be determined by several techniques
o Microcytotoxicity test (serology)
 Ab for specific HLA type will kill sampled cells with that
specific type thus indicating the presence of that HLA
o DNA PCR polymerase chain reaction with probe for specific HLA
 If a match for the specific HLA probe isn’t present in the
DNA, no DNA will be amplified by PCR.
 View these pieces in electrophoresis gel for visualization.
(using one lane for each batch of DNA with a specific
probe)
Humoral Response
B Cell IgM immunoglobulins bind Ag with multiple binding sites which causes a
clustering of IgM on the B cell. Surface receptors (Ig-α and Ig-β) associated with the IgM
interact with tyrosine kinases on the cytosol side.
 Blk, Fyn, Lyn (Src family)
o Phosphorylate ITAMs on Igα and Igβ to start signaling pathway
 Syk – binds to phosphorylated Igβ; activates PI3 Kinase.
 Btk – (Bruton’s tyrosine kinase) – member of Tec family of tyrosine kinases.
o Acts downstream of Src kinases and Kyk.
o Recruited to cell membrane by activated PI3 kinase
o Phosphoylated by Src family kinases
After all the signaling pathways, the final result is activation of transcription factors
(NFκB, NFAT, AP-1) in the nucleus. Transcribe genes needed for proliferation and
differentiation.
B cell activation is amplified with antigen has complement proteins bound:
C3d on bacteria binds CR2(CD21) on B cell which interacts with CD19 signalling
molecule for ITAM
Thymus Independent Ag – An antigen that has surface characteristics that can costimulate B-cells for B-cell activation without the use of T-Helper cells.
TI-1
 LPS (lipopolysaccharide) of Gram-negative bacteria
o Binds LBP (LPS-Binding Protein) which binds CD14 on B cell.
 LPS can also be an antigen itself and bind to IgM
TI-2
 Composed of repetitive carbohydrate or protein epitopes present at high density
on microorganism surface.
 Only B-1 cells usually respond (which don’t really function before age 5)
 Overrides need for T signals by causing very strong cross-linking of BcR and coreceptors.
B cells require T cell help for:
 Isotype switching
 Affinity maturation
 Memory cell generation
B cell has CD40 receptor and T cell has CD40Ligand.
B cell also has receptors for cytokines produced by the T cells
Hyper-IgM Syndrome – due to a lack of CD40L on T cells which prevents loss of
isotype switching., affinity maturation, and memory cell generation.