Cellular and Genetic Basis of Antibody Specificity Flashcards
Side-Chain Hypothesis of antibody generation
- theory that a single B cell has potential to generate many different antibodies and had many surface R
- suffered from its inability to explain how a given B cell would secrete only the antibody that was needed and none against the self…
4 components of the clonal selection hypothesis of lymphocyte activation
- Monospecificity: all antigen receptors on a given lymphocyte possess the identical specificity
- Receptor engagement: physical binding of antigen-specific receptors on lymphocytes by antigen is necessary for lymphocyte activation
- Receptor=product: for B cells, the specificity of the Antigen R on a given cell is identical to the specificity of the antibodies produced upon activation of that cell
- Clonal integrity: specificity of a given lymphocyte and its daughter cells does not change during the course of an immune response (no change in which Ab is made)
_______ selects naive lymphocytes bearing receptors that bind to an epitope with sufficient affinity.
-Antigen
A responding lymphocyte can give rise to _________ daughter cells
-5,000 to 50,000
Only __________ can give rise to antibody-secreting plasma cells.
-Ag-activated B cells
For B and T cells, the primary response to antigen results in _______________.(general answer)
- generation of increased (10-50 fold) numbers of cells specific for the original antigen
- therefore, increased numbers of Ag-specific cells are available to respond against the antigen upon secondary exposure
3 general actions upon Ag exposure
- Ag-bind B-cells that have R with sufficient affinity
- Ag-activated B-cells divide (clonal selection)
- Release antibodies
The secondary response can also be called the _______ response.
-memory
What is one way to think about why secondary responses are more protective than primary responses?
-there are a greater number of responding clones that were generated at clonal selection from the primary response
Antigen-driven antibody responses are _________.
heterogeneous and NOT mono-clonal
-thousands of different clones (each with unique VL-VH pair) will respond to any given antigen
Antigen-activated cells and their clonally-related daughter cells differentiate into effector cells. Name who these effector cells are for B and T cells
- B cells differentiate into antibody producing cells (plasma cells….and memory cells)
- T-cells differentiate into cytokine producing cells that regulate other immune cells (helper T cells) and killer cells (cytotoxic T cells) that lyse infected host cells
Although antibody responses are very polyclonal, monoclonal antigen-specific antibodies can be obtained by…..
- fusing B cells with myeloma cells to create hybridomas
- these hybridomas can be propagates in culture indefinitely and secrete large amounts of monoclonal antibodies
Concept and utility of inbred mouse lines
- mice are genetically identical to one another and distinct from other inbred mice
- inbred mice are homogeneous for all genes
Generating Ig molecules in B cells requires at least ____ genes.
- 2
- heavy chain gene and a light chain gene
T/F: The processes required for generating antigen R on B cells could not be influenced by antigen
True; since these receptors are needed to recognize the antigen to begin with
A germ-line view of antibody and antigen receptor diversity
-a B cell precursor has genes for every unique VH- VL pair and would turn on only one Ig heavy chain gene and only one Ig light chain gene
What does the germ-line view diversity fail to account for?
- how so many antibodies with unique specificities could use identical constant regions
- how antibodies with identical specificity might switch constant regions (from IgM to IgG
Somatic model for diversity of antibodies and receptors
- all cells have only 1 Ig heavy, kappa, and lamda gene
- these are mutated intentionally in pre-B cells, spreading unique mutation patterns across Ig genes in each pre-B cell
Where do the point mutations occur according to the somatic model?
-only in the B-cell precursors
3 contrasts between somatic model and germline model
- somatic mutations would not be inherited. In this case, they could occur only in cells of the B cell lineage
- entire antibody repertoire would be generated from a very small number of genes in somatic model
- somatic model requires the intentional mutation of specific genes
Steps in generating a light chain gene
- for each light chain locus, one of 100s of V segments is combined with 1 of a small number of J segments
- afterwards, the resulting V region coding unit is combined with a constant region exon via RNA processing
- DNA recombination event (V-J), RNA transcription, RNA splicing
A variable region domain of an antibody molecule is the result of the recombination and juxtaposition of ____ (for light chains) or 3 (for heavy chains) genetic elements or gene segments. This gene rearrangement process is called ____.
- 2 for LC or 3 for HC
- V(D)J recombination
Steps in generating IgG heavy chain locus
- D-J segment rearrangement
- V-DJ segment rearrangement
- After assembly of VDJ coding exon, RNA is generated and the region between the VDJ and first constant region exon (C-mu, for IgM) is spliced away by RNA splicing
Final mRNA for IgH chain
-VDJCmu
Does light chain or heavy chain recombination occur first?
-Heavy chain recombination is first
Which segment is found in heavy chain loci but not in light chain loci?
-D segments
In contrast to a single heavy chain locus, there are _______ light chain loci
-2; kappa and lamda
2 additional sources of diversity, in addition to V(D)J recombination
- Junctional diversity: recombination joining events are not precise (acentric cleavage of hairpin), causing overhangs that must be filled in with the appropriate nucleotide
- Enzyme TdT (Terminal deoxyribonucleotide transferase) adds additional nucleotides called N regions
N and P region of VDJ recombination
- P region: due to imperfect recomination
- N region: due to TdT activity
Transcriptional expression of Ig genes
- VDJ recombination brings promoter sequences close to the enhancer
- the enhancer promotes transcription of the rearranged V gene
4 sources of diversity for generating unique antibodies
- V(D)J recombination
- junctional diversity aka P nucleotide additions
- N-sequence addition by TdT
- combination of heavy and light chains
What enzymes are unique to lymphocytes and initiated V(D)J recomination? How do these enzymes know where to cut?
- RAG 1 and RAG2 “recombinase activating genes”
- recomination signal sequences (RSS) tell RAG1/2 where to cut
What are Recombination Signal Sequences (RSS) and what do they do?
- conserved heptamer and nonamer sequences separated by 12- or 23- bp spacers that are located next to V and J exons for light chain loci or to V,D, and J exons in the heavy chain loci
- rearrangement always uses what is called the 12/23 rule
- these signal sequences are recognized by RAG1 and RAG2 and tell them where to cut
V(D)J recombination is an ordered process: what genes rearrange first?
-heavy chain genes rearrange before light chain genes
Successful rearrangement of an Ig heavy chain allele is required for ______ to generate ________. Ig light chain rearrangements occur only in _____________ cells. Therefore, what would a mutation in RAG1 or RAG2 cause?
- pro-B cells to generate pre-B cells
- pre-B cells
- prevent formations of pre-B cells and immunodeficiency
In pro-B cells, heavy chain protein forms a receptor that signals differentiation. What is this receptor? What comprises this receptor? What does this receptor do? What happens if this process fails?
- proB cell receptor is called a pre-B cell receptor (pre-BCR) that consists of heacy chain and a common surrogate light chain
- pre-BCR signals proB cells to proliferate and differentiate into pre-B cells, allowing light chain recombination events to initiate.
- any mutation that results in suboptimal pre-BCR expression or function causes an immunodeficiency due to loss of B cell development
Describe the levels of RAG1/2 during B-cell development in the face of allelic exclusion
- On during pro-B cell to create heavy chain locus in order to make Pre-BCR. If this is productive and produces a signal, RAG1/2 is downregulated, so no recombination can occur
- After early pre-B cells proliferate, RAG1/2 expression is reactivated so that light chain recombination can occur. If productive, the resulting new BCR signals to terminate RAG1/2 expression
Describe the process of eliminating self-reactive B cells soon after they are generated
- Immature B cells (IgM, but no IgD) should not be able to bind and signal antigens, if they do, they are destroyed; clonal deletion
- another safety net is that IgD is not functionally added to a B cell until a short time after they exit the bone marrow. This ensures it will not be activated in the presence of self-antigen and if it is, it will become tolerant
- *While in bone marrow, they should not signal at all. If they do, they die. Shortly after they leave bone marrow, new B cells remain tolerance sensitive so if they bind an antigen, they will signal tolerance.
Mutations in any required mediator of V(D)J recombination will result in what deficiency?
- combined (B and T) immunodeficiency
- example is loss of RAG genes
Normally every B cell have a unique configuration of Ig heavy and light chain genes, reflecting the polyclonal nature of the B cell antigen receptor repertoire. But, because cancers are almost always monoclonal, the detection of a dominant Ig rearrangement is diagnostic for what?
- lymphoma or leukemia
- ex if all B cells express kappa instead of lamda (these 2 should be fairly even)
Lymphoid malignancies often harbor translocation in the Ig or TCR loci. Explain why this is plausible.
-VDJ recombination and class switching produce dsDNA breaks that contribute to translocations which, in turn, lead to overexpression of a cellular growth or survival promoting gene
Acute Lymphoblastic Leukemia (ALL) and a common diagnostic indicator.
- leukemia derived from preB or preT cells
- expression of TdT is a common diagnostic indicator for ALL
