11 - B cell Development Flashcards
Transcription factors that initiate differentiation of Pro-B cells
EBF, E2A, Pax5
Phases of B lymphocyte life history
- B cell precursor rearranges its immunoglobulin genes
- Immature B cell bound to self cell surface is removed from repertoire
- Mature B cell bound to foreign antigen is activated
- Activated B cells give rise to plasma cells and memory cells
Stages of lymphocyte maturation
Stem cell –> Pro lymphocyte –> Pre lymphocyte –> Immature lymphocyte –> Mature lymphocyte
Major events during stem cell and pro lymphocyte maturation
- Growth factor mediated commitment
- Proliferation
- Initiation of antigen receptor gene rearrangement
Major events during pre lymphocyte maturation
Selection of cells that express pre antigen receptors
Major events during Immature lymphocyte maturation
Selection of repertoire and acquisition of functional competence
Which antibodies do B cells start off with expressing
IgM
Membrane IgM (and IgD) on the surface of mature B cells
Is associated with the invariant Igβ and Igα molecules, which contain ITAMs in their cytoplasmic tails that mediate signalling functions.
Signal initiation by antigens
Occurs by cross-linking of the BCR and is facilitated by the coreceptors for the BCR, CD21 and C’ proteins
BCR complexes in class switched B cells (including memory B cells)
Contain membrane immunoglobulins that may be of IgG, IgA, or IgE classes
T/F: Each lymphocyte clone produces antigen receptor unique to that clone
TRUE
Functional antigen receptor genes
- Produced in immature B cells in the bone marrow by a process of gene rearrangement
- Generates a large number of variable region encoding exons using a relatively small fraction of the genome
organisation of human Ig loci
- Three separate loci encode all of the Ig heavy chains, the Ig κ light chain, and the Ig λ light chain.
- Each locus is on a different chromosome
- D (diversity) segments are not
found in Ig light chain loci
What is the germline organisation of the Ig genetic loci characterised by
Spatial segregation of many different sequences that encode variable domains, and relatively few sequences that encode constant domains of receptor proteins
Human Ig protein domains
- Proteins formed following the process of recombination of individual gene segments
- The V and C regions of each polypeptide are encoded by different gene segments.
- Contains hypervariable (complementarity-determining) regions
Diversity of antigen receptor genes
From the same germline DNA, it is possible to generate recombined DNA sequences and mRNAs that differ in their V-D-J junctions
V(D)J recombination
- Lymphocyte-specific proteins that mediate V(D)J recombination recognise recombination signal sequences (RSSs), located 3′ of each V gene segment, 5′ of each J segment, and flanking each side of every D segment
-During V(D)J recombination, double-stranded breaks are generated between the heptamer of the RSS and the adjacent V, D, or J coding sequence - The intervening double-stranded DNA, containing signal ends is removed in the form of a circle, and the V and J coding ends are joined
What does the RSS consist of
Highly conserved stretch of 7 nucleotides (heptameter) located adjacent to the coding sequence, followed by a spacer, followed by the nonamer
V genes that do not face each other
- In some V genes, especially in the Ig κ locus, the RSSs are 3’ of a Vκ and 3’ of Jκ, and therefore do not face each other.
- In these cases, the intervening DNA is inverted and the V and J segments are properly aligned
Double-stranded breaks are enzymatically generated at RSS- coding sequence junctions by what
- Recombination-activating gene 1 and recombination-activating gene 2 (RAG1 and RAG2) proteins
- The Rag-1/Rag-2 complex is also known as the V(D)J recombinase
V(D)J recombinase
- Recognises the DNA sequence at the junction between a heptamer and a coding segment and cleaves it
- Makes a nick on one DNA strand
- Forms a covalent hairpin
- Generates a blunt end
- Holds the hairpin ends and blunt ends together before ligation
Terminal deoxynucleotidyl transferase
A lymphoid-specific enzyme that adds bases to broken DNA ends to generate junctional diversity
Junctional diversity
- Addition of nucleotides at the junctions of the V and D, D and J, or V and J segments at the time these segments are joined, increasing diversity
- New nucleotide sequences, not present in the germline
Mechanism of junctional diversity (P nucleotides)
- Coding segments (e.g., V and J gene segments) that are cleaved
by Rag-1 form hairpin loops whose ends are often cleaved
asymmetrically by the enzyme Artemis (one DNA strand is longer than the other) - The shorter strand has to be extended with nucleotides complementary to the longer strand before the ligation of the two segments.
- The longer strand serves as a template for the addition of short lengths of nucleotides called P nucleotides, and this process introduces new sequences at the V- D-J junctions
Another mechanism of junctional diversity (N nucleotides)
- Random addition of up to 20 non template-encoded nucleotides called N nucleotides.
- Mediated by terminal deoxynucleotidyl transferase (TdT)
Pre B cell receptor (pre-BCR)
- Complexes of μ heavy chain, surrogate light chains, and signal transducing proteins called Igα and Igβ form the pre-antigen receptor of the B lineage
μ heavy chain and surrogate light chain association
Surrogate light chains are are invariant (i.e., they are identical in all pre-B cells) and are synthesised only in pro-B and pre-B cells
First checkpoint in B cell maturation
Expression of pre-BCR
Allelic exclusion
- An individual B cell can express an Ig heavy chain protein encoded by only one of the two inherited alleles.
- Ensures that every B cell will express a single receptor, thus maintaining clonal specificity
What happens if the first rearrangement of pre BCR is unproductive
- One heavy chain allele is productively rearranged and expressed
- The other is either retained in the germline configuration or non-productively rearranged
Events following the pre-B cell stage
- Each developing B cell initially rearranges a κ light chain gene.
- The assembled IgM molecules are expressed on the cell surface in association with Igα and Igβ, where they function as specific receptors for antigens
- The IgM expressing B cell is called an immature B cells
Light chain recombination
There are no D segments in the light chain loci, therefore recombination involves only the joining of one V segment to one J segment, forming a VJ exon
Immature B cells
Immature B cells do not proliferate and differentiate in response to antigens
Central B cell tolerance
Immature B lymphocytes that recognise self antigens in the bone marrow with high affinity either change their specificity or are deleted.
Receptor editing of self reacting B cells
- If immature B cells recognise self antigens that are present at high concentration in the bone marrow and especially if the antigen is displayed in multivalent form (e.g., on cell surfaces), many antigen receptors on each B cell are cross-linked, thus delivering strong signals to the cells
- The B cells reactivate their RAG1 and RAG2 genes and initiate a new round of VJ recombination in the immunoglobulin (Ig) κ light chain gene locus.
- A new Ig light chain is expressed, thus creating a B cell receptor with a new specificity
Anergy in B cell central tolerance
- If developing B cells recognise self antigens weakly (e.g., if the antigen is soluble and does not cross-link many antigen receptors or if the B cell receptors recognise the antigen with low affinity), the cells become functionally unresponsive (anergic) and exit the bone marrow in this unresponsive state
- Anergy is due to down regulation of antigen receptor expression as well as a bock in antigen receptor signalling
- If the new receptor is no longer self-reactive, the immature B cell migrates to the periphery and matures
Peripheral B cell Tolerance
Mature B lymphocytes that recognise self antigens in peripheral tissues in the absence of specific helper T cells may be rendered functionally unresponsive or die by apoptosis
Anergy and deletion in peripheral B cell tolerance
- B cells that have encountered self antigens have a shortened life span and are eliminated more rapidly in lymphoid follicles than cells that have not recognised self antigens
- The high rate of somatic mutation of Ig genes that occurs in germinal centres has the risk of generating self-reactive B cells.
These B cells may be actively eliminated by the interaction of FasL on helper T cells with Fas on the activated B cells
Signalling by inhibitory receptors
Inhibitory receptors set a threshold for B cell activation, which allows responses to foreign antigens with T cell help but does not allow responses to self antigens (eg. CD22 inhibitory receptor)
Fas
Death receptor
B cell maturation
Immature B cells that are not strongly self reactive leave the bone marrow and complete their maturation in the spleen before migrating to other peripheral lymphoid organs
What Ig do mature B cells express
IgM and IgD
B-1 lineage
- Most B cells that develop from foetal liver derived stem cells differentiate into B-1 lineage
- Found in peritoneal and pleural cavity fluid
B-2 lineage
- B lymphocytes that arise from bone marrow precursors after birth give rise to the B-2 lineage.
- Two major subsets are derived from B-2 B cell precursors
B-2 linage subsets
- Follicular B cells are recirculating lymphocytes (populate follicles in spleen and lymph nodes)
- Marginal zone B cells are found in spleen and lymph nodes and enrich MZ of spleen
Outcomes of follicular B-2 cells
- T dependent, isotope switched, high affinity antibodies
- Long lived plasma cells
Outcomes of marginal B-2 cells
- T independent, mainly IgM
- Short lived plasma cells
Outcomes of B-1 cells
- T dependent, mainly IgM
- Short lived plasma cells
Antigen presented to B cells
Generally in its intact, native conformation and is not processed by APCs
Where are antigens from tissue sites transported to
Lymph nodes by afferent lymphatic vessels that drain into the sub capsular sinus of the nodes
Subcapsular sinus macrophages
Capture large microbes and antigen-antibody complexes and deliver these to follicles, which lie under the sinus
Interaction of B and T cells in lymphoid organs
- Naive CD4+ T cells are activated in the T cell zones by antigen processed and presented by dendritic cells, and differentiate into helper T cells
- Naive B cells are activated in the follicles by the same antigen
- The helper T cells and activated B cells migrate toward one another and interact at the edges of the follicles, where the initial antibody response develops
- Some of the cells migrate back into follicles to form germinal centres, where the more specialised antibody responses are induced.
Extra follicular plasma cells
Short lived (~ 3 days)
Follicular/germinal centre plasma cells
- Long lived, migrate to bone marrow, MALT
- Memory cells
Where does somatic hypermutation and class switching occur
Germinal centres of secondary lymphoid organs
Germinal centre reaction
The characteristic events of helper T cell–dependent antibody responses occur primarily in organised structures called germinal centres that are created within lymphoid follicles during T-dependent immune responses.
Events of helper T cell dependent antibody responses
Affinity maturation, isotype switching, and generation of long-lived plasma cells and memory B cells
Ig heavy chain isotope switching
In T dependent responses, some of the progeny of activated IgM- and IgD-expressing B cells undergo heavy chain isotype (class) switching and produce antibodies with heavy chains of different classes, such as γ, α, and ε
How does Ig heavy chain isotope switching happen
The Ig heavy chain DNA in B cells is cut and recombined such that a previously formed VDJ exon that encodes the V domain is placed adjacent to a downstream C region, and the intervening DNA is delete
Activation-induced cytidine deaminase (AID)
Tnitiator of mutations in somatic hypermutation and class switching
Affinity maturation
- Leads to increased affinity of antibodies for a particular antigen as a T-dependent humoral response progresses
- Result of somatic hypermutation of Ig genes followed by selective survival of the B cells producing the antibodies with the highest affinities
Somatic hypermutation
Phenomenon in which a high frequency of point mutations are generated within a 1–2-kb segment in the variable region of expressed immunoglobulin genes in response to the presence of an antigen.
Molecular events in antigen- independent phase of B cell development (bone marrow, foetal liver)
V(D)J rearrangement
Cellular events in antigen- independent phase of B cell development (bone marrow, foetal liver)
proB > preB > mature B cell
Molecular events in antigen- dependent phase (spleen, lymph node)
Class switch, somatic hypermutation
Cellular events in antigen- dependent phase (spleen, lymph node)
B cell activation, memory and plasma B cell differentiation
