4 - Adaptive immune system

Immune gene diversification

• Unique B and T cell receptors and antibodies are achieved through gene rearrangement

• Combinational diversity - random recombination of V, D and J segments, 10^6 combinations

• Mutational diversity - somatic hypermutations, usually deletions, 10^9 combinations, fine tune antibodies to help bind more tightly to antigens

• Antibody functions are controlled by class switch recombination

B and T cell receptors

• BCR and TCR genes are stored in short DNA fragments, cut by enzymes during lymphocyte development

• DNA recombination - random combination of gene segments generates unique coding sequence

• Additional diversity generated at junctions during recombination process

B cell receptors

• B cells can make membrane bound and soluble forms of Ig receptors, encoded by the same genes, only synthesised after stimulation

• Difference lies in the C-terminus of heavy chains, secreted Ig has hydrophilic aa sequence, membrane bound has an extracellular hydrophilic spacer sequence, hydrophobic transmembrane segment and short cytoplasmic tail

• Difference is due to alternative mRNA splicing

• Membrane bound Igs associate with Iga and IgB transduct signals into the cell

Antibody diversity (where BCRs are made)

• Random combination of gene segments from heavy and light chain gene clusters during B cell development (bone marrow)

• kappa, lambda and heavy chain clusters found on various chromosomes

• Kappa and lambda light chains contain variable, joining and constant regions

• Heavy chains contain V,J,C and diversity region

Pre BCR formation

• Heavy chain rearranges first and combines temporarily with a surrogate light chain to form a preBCR

• This complex signals the cell to stop rearranging heavy chains and start L chain recombination

• This continues 1 allele at a time until a functional light chain is created to pair with the heavy chain

• The final IgM BCR prevents further rearrangement to ensure each cell only has one unique BCR

VDJ recombination

• Assembly of a complete variable region assembles from light (V+J) and heavy (V+J+D) chain segments

• Recognise RSSs and ensure only one of each type of segment is included in the recombined heavy and light chains

• Only occurs during B cell development

Recombination signal sequences

• Direct recombination between gene segments, flank each V J and D segment

• Consist of heptamer, spacer (12/23bp) and nonamer

• 12/23 rule - recombination only happens between segments flanked by one 12bp and one 23bp spacer

• Ensures proper joining e.g. a V to a J and not another V

Proteins required for VDJ recombination

• Initated by recombination activating gene products RAG-1 and RAG-2 which recognise RSSs and cleave DNA at junctions (lymphs)

• High mobility group B proteins 1/2 enhance RAG1/2 binding to RSSs (not lymph specific)

• Terminal deoxynucleotidyl transferases (TdT) add non templated (N) nucleotides to the ends of heavy chain V, D and J segments following their cleavage by RAG1/2 (lymphs)

• Non lymphoid specific NHEJ proteins - ku 70/80 stabilises DNA ends, DNA-PKcs activates artemis by phosphorylation, artemic opens hairpin loops, DNA ligation complex and DNA polymerases

Mechanism of VDJ recombination

• RAG1/2 and HMGB1/2 bind to the RSSs and bring V and J segments together through synapsis

• RAG1/2 cuts both segments at RSS borders creating two hairpin ends and two blunt signal ends

• Ku70/80 bind the hairpin ends

• Artemis opens the hairpins and created P nucleotides (palindromic additions)

• TdT adds random N nucleotides

• Exonucleases may trim extra bases

• DNA polymerases fill in any gaps

• Ligase seals the DNA

Sources of diversity so far

• Recombination of V,D,J segments

• Any heavy chain can pair with any light chain

• P and N nucleotide addition

• Exonuclease trimming

T cell receptor

• Two types, both heterodimer (aB or ys), immunoglobulins

• aB bind to ligands made up of antigenic peptides presented on an MHC 1 or 2 molecule

• ys can recognise antigens not presented on MHC

• Variable domain at N terminus for antigen binding, three hypervariable regions within

• Constant domain positions antigen binding site away from cytoplasm and into the extracellular space

• Transmembrane region for interaction with CD3 signal transduction complex

T cell signal transduction complex

• Composed of 3 dimers, delta epsilon, gamma epsilon and zeta zeta or zeta eta

• Each signalling protein contains immunoreceptor tyrosine based activation motif (ITAM) sequences

• When the receptor is activated tyrosine residues in ITAM get phosphorylated, creating docking sites for adaptor proteins that start the signalling cascade inside the cell

TCR co-receptors

• T cell receptors are non covalently associated with a number of accessory molecules

• CD4+ T cells recognise peptide bound MHC2

• CD8+ T cells recognise antigen bound to MHC 1

• CD28 binds to ligands on antigen presenting cells for activation of naive T cells

TCR diversity

• Random selection of a and s (C7) and B and y chains (C14) during development in thymus

• Genes that encode a and y have V, J and C segments

• Genes that encode B and s have V, D, J and C segments

• Variable regions rearrange and are joined to constant regions after transcription by RNA splicing