Lecture 13 (Matuschewski) Flashcards
Gene rearrangements: Antigen receptors
Adaptive Immunity Hallmarks
Adaptive Immunity Hallmarks
- Recognize & Remember antigen exposure (recall response)
- T-Cells & Antibodies, produced from B-cells
- Specificity for distinct antigens
- A diverse repertoire to recognize a wide variety of antigens
- occurs in in Vertebrates
- Acquired, slow, 10*20 possible antibodies, memory, 575 genes
Innate Immunity
Innate Immunity
- First line of defense ⟶ no exact recognition
- Inherited
- Fast
- low diversity
- 100 receptors
- no memory
- 850 genes
Antibody variations
Antibody variations
- lgM: Primary Response ⟶ high avidity (sum of affinity), in circulation (made before antigen exposure)
- lgG: Secondary response, binds specific targets internally, passes into the extravascular spaces
- IgA: abundant on mucosal surfaces and on secretions in the respiratory tract and the intestine, binds pathogens before they cross barriers
- IgE: associated with the allergic response and with defense against parasites. It is secreted on mucosal surfaces of the respiratory tract
- IgD: Plays a role in the initiation of B-cell activation. It is found on immature B cells (made before antigen exposure)
IgG Antibody structure
IgG Antibody structure
- Fc Region: Effector molecules bind here via the Fc receptor & selectively distributes different antibody classes across the body
- Fab region: Ends of the light and heavy chains make up the antigen binding site ⟶ contains variable domains to target different antigens
- Hinge Region: connects Fc with Fab region and improves the efficiency with which the antibody can cross-link antigens (because of its flexibility)
Molecular Structure of IgG
Molecular Structure of IgG
- Contain four peptide chains
- 2γ heavy chains, that consist of one variable and three or four constant domains
- 2 identical light chains, that consist of one variable (VL) and one constant (CL) domain
- 2 genes expressed for 1 Antibody
- Disulfide bonds link the chains to each other
Complementarity-determining regions (CDRs)
Complementarity-determining regions (CDRs)
- 3 hypervariable domains (part of Fab) which form loops of ß-strands at the far end of the variable domain when folded
- Variability leads to specificity
- Grab the Antigen ⟶ Transmit signal when there is a fit
- Shape complements that of an antigen
Clonal Selection Theory
Clonal Selection Theory
- explains how the immune system responds to pathogens
- The entire immunological repertoire develops spontaneously in the host (one gene per cell)
- Each antibody pattern is the specific product of a cell (presented on the cell surface) and specific to a single antigen
- Antigen reacts with any cells carrying its specific receptor to signal cell proliferation and differentiation (Antibody formation)
- Some daughter cells differentiate to form Plasma antibody producing B-cells, whereas others survive as clones of memory cells
B-cell receptor variations
B-cell receptor variations
- Every individual possesses about 10^9 different BCR sequences (encode B-cell receptor)
- The clonal overlap of naive B cell repertoires across individuals is low (<1%) ⟶ repertoires are highly diverse
- Everyone has different antibody producing cells
Overview V(D)J Recombination
Overview V(D)J Recombination
- Process by which B lymphocytes create diverse antigen receptors by rearranging specific DNA segments
- V: Variable segment, D: Diversity segment (present only in heavy chains), J: Joining segment
- In B cells, separate V, D, and J gene segments undergo somatic rearrangement to form a single V(D)J exon
- This recombination occurs in germline immunoglobulin (Ig) genes, in somatic lymphocyte precursors during their development, these are initially organized as separate DNA segments in all cells.
- The recombined V(D)J region forms the variable region of B cell receptors (BCRs) and antibodies (Abs), while the constant region (e.g., IgM, IgG) determines the antibody class and function
Overview Ig heavy & light chain gene recombination and expression
Overview Ig heavy & light chain gene recombination and expression
Sequential Events in V(D)J Recombination
Recognition signals
Sequential Events in V(D)J Recombination
Recognition signals
- Lymphocyte-specific proteins that mediate V(D)J recombination recognize DNA sequences called recombination signal sequences (RSSs)
- Each RSS consists of a heptamer (7-bp) separated by an either 12- or 23-bp spacer from a nonamer (9-bp)
- The rule that governs the joining reaction is that an RSS with one type of spacer can be joined only to an RSS with the other type of spacer
- They are located 3′ of each V gene segment, 5′ of each J segment, and flanking each side of every D segment
- Enhancer in proximity to promotor allows transcription
Sequential Events in V(D)J Recombination
Nonhomologous recombination
Sequential Events in V(D)J Recombination
Nonhomologous recombination
- Lymphocyte-specific proteins RAG1 and RAG2 (Tetramer) recognize the homology of heptamer RSS Sequences flanking V, D, and J segments
- then introduce double-strand breaks at specific sites, creating blunt ends and hairpins on the DNA formed at the coding ends
- The enzyme Artemis, a exonuclease activated by the DNA-PK complex, opens the hairpins, creating overhangs or blunt ends.
- TdT (Terminal deoxynucleotidyl transferase) adds random N and P nucleotides
- DNA ligase joins the coding ends, forming the recombined gene and resulting in one glued together exon
- DNA recombination brings the enhancer in proximity to promoter
How is Diversity in B and T cells created
The diversity of the B cell repertoires is created by
- Random combinations of germline gene segments being joined together (VDJ Recombination)
- The addition or deletion of sequences at the junctions between these segments (Junctional Diversity)
- 1 in 3 chance of having an open reading frame (ORF).
- Most cells fail to produce a functional heavy chain.
- Non-functional cells must be degraded.
How is junctional diversity of antigen receptors achieved?
How is junctional diversity of antigen receptors achieved?
- Because of junctional diversity, antibody molecules show the greatest variability at the junctions of V and C regions, which form the third hypervariable region, or CDR3
achieved through mechanisms during V(D)J recombination:
- Exonuclease Activity: Exonucleases trim nucleotides at the ends of the gene segments
- Random Addition of Nucleotides by DNA Polymerase adding P nucleotides and TdT (Terminal deoxynucleotidyl transferase) adding N nucleotides at the junctions of V, D, and J segments
- Imprecise Joining: DNA ligase joins the segments with slight variations in the alignment
Pre B-cell receptors
Pre B-cell receptors
- composed of 2 Ig heavy chains and 2 temporary light chains, which associate with the signaling molecules Igalpha and Igbeta (help send signal to cell)
- The pre-BCR acts as a “checkpoint” to test whether the heavy chain works properly before moving to the next stage of development.
- The pre-BCR mediates signals resulting in heavy chain allelic exclusion (to only produce 1 heavy chain), proliferation of pre b-cells, light-chain recombination and shut off temporary light chain transcription
Checkpoints in lymphocyte maturation
Checkpoints in lymphocyte maturation
- negative selection, because self-antigens could otherwise be recognized and attacked
How can human antibodies be produced in mice?
How can human antibodies be produced in mice?
- Humanized Mouse Genome: Mice are genetically modified to replace their native immunoglobulin (Ig) genes with human V gene segment. This modification includes around 6 Mb of human DNA encoding the antibody genes (Heavy & Light Chain)
- Immune Response: When exposed to an antigen, these “humanized” mice generate immune responses using the human Ig genes, leading to the production of human-like antibodies.
- Antibody Isolation: The antibodies produced are harvested from the mice and can be used for research or therapeutic applications, such as monoclonal antibody therapies
- for example monoclonal antibodies like REGN-COV2, developed for infectious diseases ⟶ decreases viral load & prevents mutational escape
What will happen if you target RAG 1 Gene in Mice?
What will happen if you target RAG 1 Gene in Mice?
- no VDJ Recombination possible ⟶ no functioning B and T cells
