Lecture 5 - Antibody Genetics and B cell Development

variable regions

they are encoded by gene segments V, D and J generating a vast repertoire of Ag receptors

somatic DNA recombination

this occurs during lymphocyte development in the bone marrow where VDJ gene segments are rearranged

light chain V

this domain is generated from V and J

heavy chain V

this is generated from V, D and J segments

recombination signal sequences (RSSs)

they control rearrangement, they are conserved heptamer and nonamer sequences flanking V, D and J Ig segments

spacers

they are 12bp or 23bp nucleotides that separate RSSs and get spliced out after rearrangement, joining of gene segments almost always involved these segments (12/23 rule)

RAG1/2 recombinase

precisely cuts DNA for recombination

functional gene segments

they are derived from cloning of DNA from one individual and due to genetic polymorphism these numbers vary in different people, generating antigen diversity of BCR

pseudogenes

excludes all mutated and non-functional versions of the gene

primary antibody repertoire

initially composed of IgM containing variable regions produced by VDJ recombination and constant regions from the u gene segment

somatic hypermutation

results in mutations being introduced into the heavy chain and light chain variable regions, altering the affinity of the antibody for its antigen

how does somatic hypermutation work?

B cells in the germinal centre will undergo this process and cells with mutations that improve affinity for antigen are selected, this can change anywhere from one to a few amino acids in the immunoglobulin producing closely related B cell clones that differ in their affinity for antigen

class switch recombination

this is when the initial u heavy chain C regions are replaced by heavy chain regions of another isotype, modifying the effector function of the antibody but not its antigen specificity, type of nonhomologous DNA recombination that is guided by stretched of repetitive DNA (switch regions), it enables the same Vh exon to be associated with different Ch genes during an immune response

somatic hypermutation experiment

this can track somatic hypermutation by sequencing Ig V regions from hybridomas established at different time points after the experimental immunization of mice, within a few days of immunization, V regions in a particular clone of B cells begin to acquire mutations and overtime more mutations accumulate

B cell somatic hypermutation selection

B cells whose V regions have accumulated deleterious mutations and can no longer bind antigen die while those that improve affinity of BCR for antigen are able to compete more effectively for antigen and receive signals that drive their proliferation and expansion, the antibodies they produce also have this improved affinity

what happens after B cell selection?

the process of mutation and selection can continue in the lymph node germinal center through multiple cycles in response to secondary and tertiary immune responses elicited by further immunization with the same antigen and therefore the antigen-binding efficiency of the antibody response is improved overtime

activation induced cytidine deaminase (AID)

this enzyme initiates somatic hypermutation in V genes, they are expressed by B cells in the extrafollicular foci and germinal centres, it is the initiator of mutations in somatic hypermutation, gene conversion and class switching

how does AID work?

it is only expressed in B cells and requires access to the cytidine side chain of a single-stranded DNA molecule which is normally prevented by the hydrogen bonding in double stranded DNA, it initiates a nucleophilic attack on the exposed cytosine ring and this is resolved by the deamination of the cytidine to form uridine, the UG mismatch is repaired in several ways and results in a single mutation at the originally altered base or more mutations in the surrounding DNA sequence

CDRs

act as hotspots for somatic hypermutation

affinity

Ig can bind with specific antigen with more strength so the fit becomes better

avidity

more antigen binding sites become available, so instance IgM has high avidity because it has 10 binding sites due to its pentameric form

germinal centres

sites of intense proliferation and selection of antigen-specific lymphocytes, a small proportion of lymphocytes in this area are antigen-specific T cells and this area grows in size as an immune response proceeds and disappears as an infection resolved over 3-4 weeks

B cells location

found in the dark zone, light zone and mantle zone of the germinal centre

proliferating B cells (location)

they are visible in the dark zone

follicular dendritic cells

they are found mainly in the light zone

CD4 T cells (location)

they are seen in large masses in T cell zones which separate the follicles but a significant amount are also in the light zone of the germinal centre

sources of antibody diversity

multiple V, D and J elements, combinatorial diversity, assortment of heavy and light chains, recombinational inaccuracy and somatic hypermutation

IgM and IgD co-expression

this is regulated by RNA processing, some transcripts are spliced to produce mRNA while others are spliced to produce IgD mRNA, the same VDJ is used for both so IgM and IgD from the same cell have the same specificity

constant region genes organization

Cu is always next to the Jh locus, C(delta) is next to Cu if its present and the locus evolves by gene duplication

Ig isotypes

they are encoded by a cluster of Ig heavy chain C region genes arranged in a cluster in both mouse and humans

IgM and IgE

lack a hinge region but each contains an extra heavy chain domain

Ig mice isotype classes

IgM, IgD, IgG1, IgG2a, IgG2b, IgG3, IgA and IgE

when does class switching occur?

only after B cells have been stimulated by antigen (from IgM to another class)

rearranged Ig heavy chain locus organization (before class switch)

each domain is encoded by a separate exon, the hinge is encoded by an exon, RNA splicing removes introns and switch sequences upstream of all C regions except for C (delta)

transcription

initiation guides class switching, this occurs due to RNA polymerase through regions from promoters located upstream each S sequence, due to the repetitive sequences, RNA pol. can stall within the S regions allowing these regions to serve as substrates for AID

AID (class switching)

along with other enzymes, it introduced high density of single stranded nicks into the non-template DNA strand and the template strand, staggered nicks are converted to double strand breaks and these breaks are then recognized by the cell’s double strand break repair proteins

what happens after AID acts in class switching?

the two switch regions are brought together by the repair proteins, the switch is completed by excision of the intervening region of DNA (including constant regions) and ligation of the S regions

isotype switching steps

switch sequences upstream of all C regions except C (delta), alignment of switch sequences depending on particular isotype class switching that is occurring and lastly deletion of sequences between Su and the switch sequence for the downstream isotype induced by AID

Ig

it cna be expressed on the cell surface or secreted, the same VDJ is used for both membrane and secreted forms of IgM meaning they are derived from the same heavy chain sequence by alternative RNA processing, follows a prediction of the clonal selection hypothesis

single B cell clone

progeny may produce different Ig isotypes, switching occurs after a given clone has expanded, different daughter cells can express different isotypes, after isotype switching only one isotype at a time is expressed in a single cell, the same V exon is used in each case so antigen specificity is maintained, all of the class switching can be influenced by cytokines in the microenvironment

lymphoid progenitors

they are heterogeneous and each one is biased to give rise to different subsets of lymphoid cells after successive stages of differentiation in either the bone marrow or thymus, there is considerable plasticity in their pathways so in certain conditions progenitor cells may switch their commitment

EILPs

they generally produce innate lymphoid cells, including NK cells

CLPs

biased toward producing B cells

ETPs

migrate to the thymus and preferentially produce T cells

B cell lineage commitment

precedes rearrangement of Ig genes, induced by bone marrow microenvironment, involved induction of specific combinations of transcription factors, some of them are lineage restricted while others are not, transcription factors regulate expression of lineage specific genes

stages of B cell development

the B cell precursor rearranges its immunoglobulin genes so BCRs are generated in the bone marrow, the immature B cell bound to self cell surface antigen is removed from the repertoire by receptor editing or apoptosis so negaitve selection, the mature B cell bound to the foreign antigen is activated so migration of B cells through the circulatory system to lymphoid organs and B cell activation then activated B cells give rise to plasma cells and memory cells in bone marrow and lymphoid tissue

B cell precursor

B cells develop in the bone marrow and migrate to peripheral lymphoid organs where they can be activated by antigens, in the first phase of development, progenitor B cells in the bone marrow rearrange their Ig genes, this is independent of antigen but is dependent on interactions with bone marrow stromal cells, they are much smaller and rounder, they cluster around stromal cells (tight association)

Immature B cell

they carry an antigen receptor in the form of a cell surface IgM, leading to the second phase of development where the immature B cell can now interact with antigens in its environment, still within the bone marrow, those that are strongly stimulated by antigen at this stage are removed from the repertoire by receptor editing or by apoptosis thus removing many self reactive B cells from the repertoire and if this fails it causes autoimmune disease

mature B cell

in the 3rd phase of development, the surviving immature B cells emerge into the periphery and mature to express IgD as well as IgM, these mature B cells can now be activated by encounter with their specific foreign antigen in a peripheral lymphoid organ

activated B cells

they proliferate and differentiate into antibody-secreting plasma cells and long lived memory cells, the plasma cells have massive ER and enhanced golgi apparatus cause producing proteins at such a high level

progression of B cell development

it starts with hematopoietic stem cell, then B cell lineage commitment where Ig gene rearrangements can start to occur, then there is pro-B cell, pre-B and immature B cell till we get the mature naive B cell form that express mIg (BCR, both IgM and IgD),

bone marrow stromal cells

they are required for B cell commitment and development after birth, they are non hematopoietic cells, part of the connective tissue foundation of the bone marrow, they support B cell development by secreting required chemokines and cytokines which drives development and making required cell-cell contacts with developing B cells, this induces developing B cells to express transcription factors that initiate critical gene expression required at each developmental stage

lymphoid myeloid primed multipotent progenitors (LMPPs)

they can produce both lymphoid and myeloid cells but are no longer self renewing stem cells, they express transcription factors and receptors required for development of multiple hematopoietic lineages

what is required for development to the immature B cell stage?

interaction of B cell progenitors with bone marrow stromal cells and signal molecules and/or receptors are required for differentiation to the common lymphoid progenitor

FLT3

this is a cell surface receptor tyrosine kinase that LMPPs express, this binds to the membrane bound FLT3 ligand on stromal cells, signaling through it requires differentiation into CLP

CXCL12

this is a chemokine that acts to retain stem cells and lymphoid progenitors at appropriate stromal cells in the bone marrow

cytokines

they induce proliferation of B cell progenitors when they interact with their receptors, they are what drive all B cell developmental processes dictating what the cell can do and what it ends up doing (ex; SCF, stem cell factor, a cell surface cytokine)

IL-7

this is the interleukin-7 receptor that is present at the common lymphoid progenitor stage and those produced by stromal cells are required for the development of B-lineage cells, soluble cytokine produced by stromal cells, late pro-B and pre-B cells need it to grow

CAMs

these are cell-adhesion molecules, it allows progenitor cells to associate with stromal cells, these adhesive interactions induce the proliferation of B cell progenitors, they include integrins (VLA-4), selectins and other adhesion molecules

cultured stromal cell structure

they have extended processes

B cell organization in the bone marrow

the more mature B cells are located toward the centre while the less mature are found in the periphery closer to the bone so as cells mature they can exit into the circulation

Ig heavy chain gene rearrangement in B cell development

the stem cell is found in the germline, then in the early pro-B cell D-J rearrangement occurs, in the late pro-B cell stage V-DJ rearrangement occurs, the lastly VDJ is rearranged at the large pre-B cell stage

Ig light chain gene rearrangement in B cell development

germline DNA persists from the stem cell stage up to the large pre B cell stage, then V-J rearranging occurs at the small pre-B cell stage, then its completed in the immature B cell cause the light chain has no diversity segment

surface Ig expression

it is absent from the stem cell to late pro-B cell stage, then in the large pre-B cell the u chain transiently at surface as part of the pre-B-cell receptor, mainly intracellular, the small pre-B cell has an intracellular u chain and IgM is expressed on the cell surface of the immature B cell, in the mature form IgD and IgM are made from alternatively spliced H-chain transcripts

what are the first Ig gene rearrangements detected?

they are the D-J heavy chain rearrangements in early pro-B cells, no functional u protein is expressed after DJh rearrangement although transcription occurs and D-Jh often occurs on both chromosomes

what happens after DJh rearrangement?

Vh rearranges in the large pre-B cell, this induces transcription from the Vh promotor and there is still no light chain rearrangement, recombinational inaccuracy so rearrangement can be productive or non productive and the u protein will be made only if rearrangement is productive

functional u heavy chain production

this causes the expression of pre-B cell receptor (pre-BCR), pro-B cells express a surrogate light chain and the functional u heavy chains associate with it to form the pre-BCR, an Iga/Igb signalling heterodimer is also expressed and this defines the pre-B cell stage

surrogate light chain (SLC)

it is made of a VpreB( (V-like protein) and a λ5 (C-like protein)

pre-B receptor

expressed on the cell surface in association with IgA and IgB, most pre-BCR is held in ER, some gets to the cell surface and its expression regulates the next stages of B cell development, this receptor initiates signaling through spontaneous dimerization induced by VpreB and λ5 which substitutes for a light chain and binds to a heavy chain, thus allowing its surface expression, VpreB substitutes for the light-chain V region in this surrogate interaction while λ5 takes part of the light chain constant region

how does pre-BCR expression result in cross-linking signals?

VpreB and λ5 contain unique amino terminal regions not present in other Ig like domains, dimerization generates signaling from the pre-B cell receptor that is dependent on the presence of the ITAM containing signaling chains Iga/Igb, signals inhibit RAG1 and RAg2 expression and the proliferation of the large pre-B cell

what would happen if a B cell makes two productive VDJh rearrangements?

there would be problems for self non-self specificity and for clonal selection there would be a problem cause you can’t have one particular plasma cell producing multiple antibodies/isotypes, only one

allelic exclusion

signaling through the pre-BCR complex down regulates RAG expression and inhibits further VDJh rearrangement so a single B cell will only express the product of a single functionally rearranged heavy chain

consequences of a failure to express pre-BCR

if the first VDJh rearrangement is non-productive then the second allele will do it but if the second is non productive too then the cell will die (almost ½ the pro-B cell population will die at this stage of development

Igh (a/b)

allelic exclusion means that in this Igh gene, the individual contains both a allotype expressing and b allotype expressing cells meaning that some B cells express the a allotype while some express the b allotype but not both/no co-expression

what occurs as result of pre-BCR signaling?

the surrogate light chain expression is down regulated, the pre-B cells proliferate, cell surface pre-BCR is diluted out, pre-BCR signaling ends (self-limiting) and there is re-induction of RAG1 and RAG2, this reduction in pre-BCR signaling triggers gene rearrangement to begin in the light chain loci

gene rearrangement in light chain genes

pre-B cells usually rearrange k before λ and this rearrangement can be productive or non-productive, the expression of the light chain leads to intact mIgM expression so the surface IgM associated with Iga and Igb inhibits further light chain rearrangement, repeated ones can occur at a single light chain locus due to replacement of non productive VJ junction by rearrangement of upstream V to downstream J meaning there is a high probability of generating a functional light chain

what are different ways of generating a functional light chain?

there are 4 chances because there are 2 light chain isotypes (k and λ), this means that it can rearrange k gene on the first chromosome or the second one and same for λ but if it fails al 4 times the cell dies by apoptosis, if it is successful it can lead to mIgM expression blocking further light chain rearrangement

central tolerance

screening of immature B cells while they are still immature and before migration to the periphery, where they might need their antigen, it will eliminate anti-self lymphocyte specificities prior to maturity

immature lymphocytes

universal or open repertoire, all specificities exist, may potentially recognize foreign antigen and self antigens, they are found in primary lymphoid tissues

mature lymphocytes

central tolerance mechanisms will select out and eliminate any anti-self lymphocytes prior to maturity, once mature lymphocytes are a functional repertoire (self tolerant) and they recognize foreign antigens, they are found in the periphery

immature B cell selection

they are selected before migration to the periphery, multivalent self molecules undergo clonal deletion or receptor editing so that specificity is eliminated, soluble self molecules migrate to the periphery where they are anergised (not functional), lower-affinity non-crosslinking self molecules and no self-reaction molecules are selected to migrate to the periphery and mature

how does receptor editing work?

this is induced by the strong ligation of IgM by self antigen, replacement of light chains by receptor editing can rescue some self reactive B cells by changing their antigen specificity, when a developing B cell expresses antigen receptors that are strongly cross linked by multivalent self antigens (ex; MHC molecules on cell surfaces), its development is arrested, surface expression of IgM is decreased but RAG genes are not turned off, continued synthesis of RAG proteins allows the cell to continue light chain gene rearrangement, this combies with the previous heavy chain majing a new receptor

receptor editing (new receptor specificity)

if the new receptor is not self reactive, the cell is rescued and continues normal development, if the cell remains self-reactive it may be rescued by another cycle of rearrangement, however if it continues to react strongly with self antigen, it will undergo apoptosis resulting in clonal deletion of the self reactive cell from the repertoire of B cells

what are the consequences of binding to self-antigen with intermediate strength?

if there is low avidity between mIg and self Ag and some receptor cross linking this causes the induction of anergy so anergic B cells will migrate to the periphery and cannot respond to antigen, have a reduced lifespan, if the soluble self Ag has no avidity and there is no receptor cross linking this causes the release to periphery so potentially self reactive B cells migrate to the periphery and may be controlled by peripheral tolerance

conventional B cell lifespan

this is determined by recirculation, in the bone marrow there is an open repertoire of mature B cells and tolerance induction, these cells go to the blood and secondary lymphoid tissues where additional tolerance induction, self tolerant immature B cells and anergized B cells are located, then if there is no positive selection, B cells fail to enter lymphoid tissues so B cells have a ½ life of 3 days but if positive selection does occur B cells successfully enter the lymphoid follicles where they are long lived mature recirculating naive B cells with a ½ life of about 3-8 weeks, they get stimulated by antigen so longer lived mature recirculating memory B cells, expressing high affinity IgG, IgA or IgE

conventional B cells/B-2 cells

produced throughout life at a roughly constant rate

B-1 cells

produced in the fetus, have a restricted diversity and few N nucleotides in VDJ junctions, they are self renewing whereas conventional B cells are continually produced from mIgM- precursors and they are also frequently found in body cavities rather than secondary lymphoid organs, their limited repertoire is biased towards spontaneous production of IgM antibodies with specificity for carbs with little hypermutations and most autoimmune antibodies are derived from them

burkitt’s lymphoma (chromosomal translocation)

translocation places the MYC gene under the regulation of the Ig promoter/enhancer, this is caused by RAG1/RAG2 mediated recombination recognizing RSS like sequences close to c-myc, this type of lymphoma is caused by a mature memory B cell

hodgkin’s lymphoma

caused by mutation in the germinal center B cell

waldenstrom’s macroglobulinemia

caused by a mutation in the IgM secreting B cell

multiple myeloma

caused by mutation in the plasma cell, various isotypes