Flashcards in B Cell Development and Primary Antibody (Ab) Repertoire Deck (37):
Describe the three gene families and how they can be divided.
Each of the 3 gene families, the kappa light chain family, the lambda light chain family and the heavy chain family can be divided into V-region genes and C-region genes.
The kappa, lambda and H chains are located on separate chromosomes. Each set of genes, kappa, lambda and heavy chain, has a similar basic organization.
The Ig heavy and light chain loci are composed of multiple genes that give rise to the V and C regions of the proteins, separated by stretches of non-coding DNA.
Where are V region exons found?
At the 5'end of each Ig locus are the V region exons each about 300 base-pairs (bp) long, separated from one another by non-coding DNA of varying lengths
Where are joining (J) and diversity (D) segments found?
What do they code for?
Downstream of the V genes are additional coding sequences, 30 to 50 bp long, which make up the joining (J) segments and, in the H chain locus only, the diversity (D) segments
The J and D gene segments code for the carboxy terminal ends of the V regions, including the third hypervariable (complementarily-determining) regions of antibody molecules
What encodes the variable and constant regions in light chain and heavy chain proteins?
Thus, in an Ig light chain protein (kappa or lambda), the variable region is encoded by the V and J exons and the constant region by a C exon. In the heavy chain protein, the variable region is encoded by the V, D, and J exons. The constant region of the protein is derived from the multiple C exons and, for membrane-associated heavy chains, the exons encoding the transmembrane and cytoplasmic domains.
Describe the C region genes.
Where are they? How are they arranged?
What does eaach heavy chain C region gene consist of? (How many exons?)
At varying distances 3' of the V genes are the C region genes. In both mouse and man, the kappa light chain locus and a single C-kappa gene and the genes for heavy chain C regions (CH) of different isotypes are arranged in a tandem array.
Each heavy chain C region gene actually consists of three to four exons (each similar in size to a V region exon) that make up up the complete C region, and smaller exons that code for the carboxy terminal transmembrane (TM) and cytoplasmic domains of the heavy chains.
Describe how Ig genes are expressed in B-lineage cells and non-B-lineage cells.
All cells except B-lineage, including plasma cells contain Ig genes in the germline configuration. The Ig genes are expressed only in B-lineage cells.
What is the first step in production of antibodies?
Rearrangements of Ig genes are the essential first steps in the production of antibodies.
Describe how DNA rearrangements occur in regards to antigen stimulation.
DNA rearrangements occur in a precise order and occur independent of antigen stimulation.
What is the first Ig gene rearrangement in the heavy chain?
(First step to make immature B lymphocyte)
Heavy chain - DJ. The first Ig gene rearrangement involves the heavy chain locus and leads to joining of one D and one J gene segment with deletion of the intervening DNA.
What follows the DJ rearrangement in the heavy chain?
What is important to note about this rearrangement?
(2nd step to make immature B lymphocyte)
Following the DJ rearrangement, one of the many V genes is joined to the DJ complex, giving rise to a rearranged VDJ gene. At this stage, all D segments 5' of the rearranged D are also deleted.
This VDJ recombination occurs only in cells committed to become B lymphocytes and is a critical control point in Ig expression because only the rearranged V gene is subsequently transcribed. The C region genes remain separated from this VDJ complex by an intron.
Describe the rearrangement following VDJ.
(3rd step to make immature B lymphocyte)
Light chain - VJ. The next somatic DNA recombination involves a light chain locus. One V segment is joined to one J segment, forming a VJ complex, which remains separated from the C region by an intron, and this gives rise to the primary RNA transcript. Splicing of the intron from the primary transcript joins the C gene to the VJ complex, forming an mRNA that is translated to produce the κ protein. The light chain assembles with the previously synthesized μ to form the complete membrane IgM molecule, which is expressed on the cell surface, and the cell is now the immature B lymphocyte.
What does a single B cell make (major rule of antibody specificity).
What does a single plasma cell make?
A single B cell makes only one kind of antibody specificity (one VH and one VL), i.e., allelic exclusion occurs
Also, a single plasma cell makes only one kind of antibody; i.e., 1 kind of H chain & 1 kind of L chain; B cells may violate this rule & synthesize two or more heavy chain isotypes simultaneously for the cell surface, eg. IgM and IgD
What are ProB cells.?
These cells are precursors of PreB cells. They have IgH DJ gene rearrangements and no light chain gene rearrangements.
From what do all B lymphocytes arise?
Describe what the earliest cell type that synthesizes a detectable Ig gene product contains.
Where are pre-B lymphocytes found?
What is the pre-B receptor composed of?
All B lymphocytes arise in the bone marrow from a stem cell that does not produce Ig. The earliest cell type that synthesizes a detectable Ig gene product contains cytoplasmic mu-heavy chains composed of variable (V) and constant (C) regions. This cell is called the pre-B lymphocyte and is found only in hematopoietic tissues, such as the bone marrow and fetal liver. The pre-B receptor is comprised of surrogate light chain, mu-chain, Ig-alpha and Ig-beta
Describe immature B cells.
What happens at the next identifiable stage in B cell maturation?
What functions as specific receptors for antigens?
What are immature B lymphocytes? Why are they called this?
At the next identifiable stage in B cell maturation, kappa or lambda light chains are also produced.
These associate with mu heavy chains and then the assembled IgM molecules are expressed on the cell surface, where they function as specific receptors for antigens.
IgM-bearing B cells that are recently derived from bone marrow precursors are called immature B lymphocytes because they do not proliferate and differentiate in response to antigens. Once a B cell expresses a complete heavy or light chain, it cannot produce another heavy or light chain containing a different V region.
Describe mature B cells. Where are they most often found?
What do mature B cells express? (chain wise)
What membrane proteins do they protein? (Ig)
Having acquired a complete Ig and, therefore, an antigen specificity, B cells migrate out of the bone marrow and can be found in the peripheral circulation and lymphoid tissues. They continue to mature, even in the absence of antigenic simulation.
Mature B cells co-express mu and ?(see h/o) heavy chains in association with the original kappa or lambda light chain and, therefore, produce both membrane IgM and IgD. Both classes of membrane Ig have the same V region and hence the same antigen specificity. Such cells are responsive to antigens.
What is allelic exclusion?
Why is it necessary for each B cell to express only one set of Ig heavy and light chain V genes through its life? Why does this occur?
Only one IgH and one IgL allele are productively rearranged.
Each B cell clone and its progeny are specific for only one antigenic determinant. It is, therefore, necessary for each B cell to express only one set of Ig heavy and light chain V genes throughout its life. This occurs because only one functional heavy chain VDJ and one functional VJ gene rearrangement occur in each cell. The expression of only one allele in a cell is termed allelic exclusion.
What is RSS? Where are they located? What does it do?
DNA Recognition sequences
The recombination of V, D and J gene segments is mediated by specific DNA recognition sequences (RSS) located in the intervening DNA 3' of each V exon and 5' of each J segment and flanking both sides of each D segment. The RSS are highly conserved stretches of seven or nine nucleotides separated by non-conserved 12 or 23 nucleotide spacers. In a light chain gene, each heptamer or nonamer adjacent to a V exon recognizes a complementary stretch adjacent to a J exon
What do RAG1 and RAG2 do?
The enzymes RAG1 and RAG2 recognize and align two RSSs and cleave the DNA between the exon and the RSS; the two exons are ligated together by a DNA ligase. Not all rearrangements are functional.
Describe the excision circles.
The other product of V, D and J gene segments is the excised DNA which circularizes via signal joints.
PCR of these excision circles, designated B cell recombination excision circle (BREC) can be used to demonstrate that B cells are developing in the bone marrow, e.g., after bone marrow transplantation.
Describe how secreted and membrane forms of mu-chain result from alternative RNA splicing.
See h/o p 8
primary transcript RNA
(cleavage at second poly(A) addition site and splicing)
(translation, protein processing)
There is co-expression of membrane IgM and membrane IgD on B cells. Given that one cell makes only one antibody, how can mu and delta heavy chains be produced simulataneously by the same B Cell?
IgM and IgD on a given B cell have the same (VH + VL)
The answer is, mu and delta chains with the same VH domain result from alternative splicing of primary transcripts (nuclear RNA).
Describe the generation of antibody diversity..
The germline contains multiple germline VH and VL genes that have different sequences and produce Ig molecules with different specificities. D and J gene segments also contribute to diversity.
The somatic recombination of Ig DNA participates in the generation of antibody diversity in several ways. The combinatorial associations of different V, D, and J gene segments lead to a large potential for generating different antibody specificities. The maximum possible number of combinations is the product of the number of V, D (if present), and J gene segments at each locus. Every clone of B cells and its progeny express a unique combination of V, D, and J genes.
In addition to these mechanisms operative at the level of Ig genes, the combination of different H and L chain proteins also contributes to diversity because the V region of each chain participates in antigen recognition.
What can be added to the junctions of rearranged VDJ genes during rearrangement?
What mediates this process?
Nucleotides, called N sequences, which are not present in the germline, can be added to the junctions of rearranged VDJ genes during rearrangement.
This addition of new nucleotides is a random process mediated by an enzyme called terminal deoxyribonucleotide transferase (TdT).
What happens to B lineage cells in the bone marrow that have non-functional V(D)J gene rearrangements or that express self-reactive antibody?
B cells with non-functional V(D)J genes are deleted. B cells with anti-self reactivity can become anergic, can be deleted, or they can be rescued by receptor editing.
SEE PAGE 10
What are the most common T cell antigen receptors (TCR)?
Most T cells have alpha/beta TCR
approx. 5 percent of T cells have gamma/delta TCR
Describe the structure of the alpha/beta TCR
alpha/beta TCR is a disulfide linked heterodimer of alpha and beta chains
Each chain has 2 Ig - like domains; The N-terminal domains are variable
(V) regions; The C-terminal domains are polypeptide constant regions
- The overall structure of gamma delta TCR is similar to alpha/beta except that the
polypeptide chains are designated gamma and delta.
Describe the overall organization of TCR genes.
What encodes the V regions of alpha and gamma chains?
How are V,D,J genes similar to Ig genes? What does this mean for TCR genes and Ig genes?
How many TCR's might a T cell express? Why?
Is similar to that of Ig genes
- The V regions of alpha and gamma chains are encoded by V and J gene segments.
- The V, D and J gene segments are associated with the same conserved heptamer and monomer nucleotide sequences found in Ig genes. Thus, TCR genes rearrange by the same mechanism as Ig genes.
- Most importantly - as in B cells, only one VDJ and one VJ gene rearrangement occur in each T cell. Therefore, each T cell expresses a single TCR, specific for one particular antigenic determinant.
Immunoglobulins are encoded by genes located on one chromosome.
Immunoglobulins are encoded by 3 gene families: for heavy chain, kappa and lambda light chains. The 3 gene families each reside on a separate chromosome.
Within one immunoglobulin molecule there may be two types of light chain.
The light chains of a single antibody are identical and the heavy chains are identical also. Therefore, one immunoglobulin may be either lambda or kappa.
The immunoglobulin combining site (for antigen) is contributed by the variable regions of the heavy and light chains.
IgG and IgM molecules are distinguished by differences in their heavy chain constant region sequences.
Myeloma proteins are the result of polyclonal B cell activation.
Myeloma proteins are homogenous and result from 1 B cell becoming concerous.
V gene segments are not joined with J gene segments in cells other than lymphoid cells.
Allelic exclusion refers to the phenomenon where only the heavy or light chain is produced by the cell but not both.
Allelic exclusion means that only one allele of each gene (H, and kappa or lambda) is expressed at the protein level - i.e., either the maternal or paternal heavy chain allele is expressed - as well as that for either kappa or lambda. The result of allelic exclusion is the expression by the B cell of only one immunoglobulin and one immunoglobulin specificity (clonal expression).
In pre-B cells, both heavy and light chain genes are rearranged.
Light chain genes are in germline configuration.