Acquired Immunity

Acquired Immunity

1. B Cells

   • BCR
     • Structure
       • structurally and evolutionarily related to TCRs, both have a highly variable region which binds Ag and a constant region
     • each B cell has a BCR of a single binding specificity
       • GENERATION OF DIVERSITY
         • The body has millions of T and B cells with unique antigen receptors, this number far exceeds the number of genes in the human genome (30,000) so several mechanisms are required to create diversity
         • 1. Random somatic recombination (gene rearrangement) of V, D and J gene segments
           • Hozumi and Tonegawa demonstrated in 1976 that the Ig gene locus was rearranged in mature B cells by illustrating that the C and V regions, which were once in different fragments of germline DNA , are on the same fragment of mature B-cell DNA, thus proving that DNA recombination had occured
           • There are two loci encoding light chains (kappa and lambda) and one for the heavy chain
           • In light chains there is one recombination to give V-J joining, in heavy chains there is DJ joining, then VD joining to give recombined VDJ. this gene is then transcribed, transcript is spliced to remove the region between J and C(constant region). Transcript then translated.
           • Number of H chain combinations = 40(V)x25(D)x6(J)=6000, number of kappa chain combinations = 30(V)x4(J)=120, number of lambda chain combinations = 40(V)x5(J)=200, so total combinations=6000(H)x320(kappa+lambda)=1.2mil
             • Still not enough different receptors
         • 3. Insertion of extra nucleotides at recombination sites
           • recombination is guided by the presence of noncoding sequences flanking the V, D, and J gene segments - recombination signal sequences (RSS)
             • structure: heptamer - spacer - nonamer
               • 5’CACAGTG- (N)12 – ACAAAAACC3’ 5’CACAGTG- (N)23 – ACAAAAACC3’
               • spacer is either 12 or 23 bases long
                 • 12/23 Rule: an RSS containing a 12bp spacer pairs with one with a 23bp spacer
                   • V(lambda) - 23 bp V(kappa) - 12bp V(heavy) - 23bp, D(heavy) - 12bp, J(lambda) - 12bp J(kappa) - 23bp J(heavy) - 23bp
                     • guides which segments can join to each other and in which order
             • MOA
               • Rag1 and Rag2 (recombination activating gene) products are proteins that bind to RSS, then form complexes with another Rag protein to result in DNA hairpins to form 1 coding joint (covalently closed in a hairpin) and 1 signal joint
                 • signal joint contains RSS and forms a circular molecule which is lost from the chromosome
                 • coding joint leads to the productive VDJ recombination, artemis cleaves the hairpin and TdT randomly adds nucleotides to the junction to create JUNCTIONAL DIVERSITY. Finally, DNA ligase ligates the DNA ends which have been added to by TdT(terminal deoxynucleotidyl transferase
                   • CDR3, most variable part of the V region is generated by the VDJ amd VJ junctions
                   • Artemis nuclease cleaves DNA hairpin at a random site near the hairpin to generate a single-stranded DNA end = P nucleotides
                     • TdT randomly adds nucleotides to the ends = N nucleotides (not nucleotide removal can also occur=loss of part of the D region)
                       • Creates junctional diversity
                       • Manny rearranged receptors are non functional due to frame shifts in the J region - D region can often be read in 3 reading frames. non functional receptors will die
                         • If a rearrangement is non functional, then rearrangement of the other allele is attempted
                     • Creates junctional diversity
           • Occurs in Bone Marrow
         • 2. Combinations of two different chains to make Ag-binding site
         • Somatic hypermutation
           • In B cells only
           • extra random mutations occur after B cell activation which are selected for if they increase affinity for the antigen
           • introduces mutations into the V regions of rearranged heavy and light chain genes
           • occurs in the germinal center of secondary lymphoid tissues where B cells are responding to antigen (with the aid of T helper cells)
           • Activation-induced cytidine deaminase (AID) converts cytosine to uracil
           • base changes are distributed along the V region
           • resulting new BCR sequences are tested for binding to Ag
             • outcomes
               • mutations may make BCR non-functional - B cell dies
               • some mutations increase affinity for Ag and are selected for
               • AA changes cluster ing the CDRs - regions of the Ab that directly bind Ag
     • has a secreted form = Ab=Ig/immunoglobulin. the BCR has a hydrophobic C terminal which anchors it in the membrane and Ab lacks this
       • Ab Basic Structure:
         • 2 identical heavy chains (50-77kD) and 2 identical light chains (25kD) linked by disulphide bonds
         • Ag has 2 important domains separated by the hinge region: Fab (fraction antigen binding) and Fc (fraction crystallisable. Fab and Fc can be generated with protease digestion
         • Antibody Classes
           • Ig exists in 5 different classes or isotypes - differ in the heavy chain constant region
           • different classes have different functions
           • During the immune resonse, a particular variable region can be swapped onto a new class of constant region
             • IgM and IgD are the first classes produced and are made by alternative splicing of constant regions in mRNAs
               • class switch recombination generates IgG, IgA and IgE
         • Ig variable region
           • within the Ig variable region are 3 hypervariable regions called HV1, HV2 and HV3 (aka complementarity determining regions - CDR1, CDR2, CDR3)
             • these regions comprise loops which dominate the antigen recognition site in both the heavy and light chain, so 6 projecting loops in all
               • there are similar CDR regions in the TCR responsible for binding Ag peptides bound to MHC

2. T Cells

   • Activation: 2 signals required
     • Generation of CTL against Viral antigen:
     • Generation of T helper 1 (Th1) cells
     • Generation of T helper 2 (Th2) cells
     • (1) TCR-peptide-MHC interaction and (2) costimulatory signal that is only given by professional APCs
       • expression of costimulatory molecules (e.g. B7) is induced by PAMPs - restricts the activation of T cells to infectious situations
   • TCR
     • Structure
       • has a variable region to bind Ag and a constant region that anchors into cell membrane
       • TCR alpha locus, like light Ig chain contains V and J segments, TCR beta locus, like Ig heavy chain, contains V, D and J gene segments
     • Generation of diversity
       • Arranged in a pattern similar to Ig gene segments and are rearranged by the same mechanisms and enzymes
         • no. of receptors= (V)52x70x(D)2x(J)13x61=~6mil due to random somatic recombination
         • TCR vs BCR diversity
     • Each T cell has a TCR of a single binding specificity
   • Self-reactive T cells are anergised when they first leave the thymus if they see self Ag without costimulation

3. MHC

   • required for antigen presentation to TCR (but not BCR - BCR can recognise intact protein)
     • Ag must be degraded to an Ag peptide fragments (antigen processing) to be displayed on MHC (MAJOR HISTOCOMPATABILITY COMPLEX) molecules on the surface of APC (antigen presenting cells)
   • MHC class I
     • display peptides from intracellular proteins on the cell surface (antigen presentation
     • recognised by CD8 T cell to CTL
     • Expressed on all nucleated cells
     • Structure:
       • • 1 alpha chain and 1 beta2 microglobulin
       • ends of peptide binding cleft are more enclosed, so smaller peptides bind (8-10 AA)
       • binding is to N and C termini and 2 other anchor residues, either
         • aromatic residue + lysine
         • or tyrosine + hydrophobic/basic residues near the C terminus
         • anchor residue side chains fir into pockets in binding groove
     • One MHC can bind many different peptides
     • Antigen processing for MHC1 pathway
       • MHC1 translocated into the ER during synthesis, as are peptides to be expressed on MHC1
         • MHC1 does not fold properly and is not transported to the cell surface unless a peptide is bound
       • getting peptide fragments into the ER
         • 1. proteasome unfolds and cleaves proteins into peptide fragments
           • Proteasome = hollow tube with 28 subunits. Its inner surface has catalytic sites of proteases. alternative catalytic subunits are induced by interferon gamma to cause a change in cleavage pattern, leading to increased hydrophobic and basic residue cleavage. cleaves N terminal with aminopeptidases. selective proteasomal processing increases the efficiency of the formation of some peptide fragments. it is also responsible for digesting 25% newly synthesised proteins within 10 mins of production (significant energy wastage shows how important this process is for immune surveillance
           • 2. TAP transporter (TAP1+2 heterodimer) draws peptide fragments (>8AA to confer specificity) into the ER, TAP = ATP dependent and selective peptide pump
             • 3. Tapasin (bound to TAP) and calreticulin = chaperones bound to MHC1 to prevent MHC folding until Ag binds -> leads to proper folding
               • 4. once Ag binds, chaperones released from MHC, then, vesicle with MHC1 is exocytosed to cell surface
       • Pathogen resistance
         • herpes simplex: protein binds TAP to decrease protein transport and MHC on cell surface
         • Adenovirus: protein retains MHC in ER
         • cytomegalovirus: MHC degradation
         • host defence against this is NK cells which target ells with lower than healthy MHC levels on cell surface
     • cross presentation of exogenous Ag on MHC1
       • explains CTL responses to viruses which don't infect DCs or tumours, which aren't professional APCs
         • only occurs in a subset of DCs, these DCs endocytose proteins and display them on surface MHC1
           • is advantageous for viral Ag which handicap MHC1 presentation in infected cells, because DC pathways differ for MHC expression, so it is less likely to be repressed
   • MHC class II
     • display peptides from proteins taken up into vesicles and from bacteria resident in phagosomes
     • recognised by CD4 T cell to T helper cell
     • expressed only on professional APCs (B cells, macrophages, DCs)
     • Structure:
       • • 1 alpha and 1 beta chain
       • ends of peptide binding cleft are more open (so larger peptides bind, >13AA)
         • bind with sequence anchoring residues: 1. hydrophobic 2. -ve charge 3. +ve charge 4. hydrophobic
     • Processing Ag for MHC2 Pathway:
       • 1. pathogen engulfed
         • 2. vesicle acidification and acid proteases e.g. cathepsins to make petides. MHC2 buds off from ER in vesicle
           • 3. MHC2 vesicle fuses with peptide vesicle. Cathepsins cleave off, CLIP fragment of invariant chain remains bound to MHC
             • 4. HLA-DM associated with MHC2 catalysis CLIP dissociation and binding of peptides. Catalytic activity continues until a peptide with high affinity binds
               • 5. with a high affinity ligand having bound, MHC2 is sent to cell surface -> can be displaces for several days
         • invariant chain blocks MHC2, therefore peptides meant for MHC1 do not bind while in the ER
   • MHC in mouse = HLA in human
   • MHC gene clusters
     • MHC genes cluster together on one chromosome together with a few other immune-related genes
       • therefore MHC genes are usually inherited as a block of genes, on paternal and one maternal
     • polygenic: several MHC1+2 genes
     • Polymorphic: many alleles for each gene, most people are heterozygous as it gives a disease resistance advantage
       • different alleles cause increased variation in peptide binding cleft
       • haplotype: combination of different alleles on a single chromosome possessed by an individual
       • Highly variant in the population - infections endemic to different areas have driven selection of MHC variants. Differences between alleles are mainly in the peptide binding cleft
   • TCR-Peptide-MHC interaction
     • in Ab and TCR the antigen binding site is made by the combination of CDR1, CDR2, and CDR3 (3 CDRs from each of 2 chains
     • most variability in the TCR is in CDR3 (junctional diversity)
     • CDR3 forms the major contact with displayed peptide
     • CDR1+2 have more contact with the MHC

4. Lipid Presentation:

   • recognised by TCRs are glycolipids and lipoproteins displayed on CD1
     • human CD1 molecules bind lipid molecules - particularly from mycobacteria
       • CD1 = structurally analogous to MHC 1
         • has a hydrophobic binding groove

5. Professional Antigen Presenting Cells (APCs)

   • Macrophages
     • resting = low MHC2 + B7 costimulator
       • activated by PAMPs
     • widely distributed in tissues and LNs
     • function as APC at site of infection
   • Dendritic Cells
     • Specialised for Ag uptake and presentation
     • the most potent cells for naive T cell activation
     • probably the only cell type activating naive T cells in vivo
     • A diverse cell type
     • immature DCs reside in tissues and the blood
     • = sentinels in the tissues for detecting infection
     • very active in the uptake of soluble antigens and whole pathogens
     • =vehicles for transporting Ag to LN for display to T cells
       • Upon pathogen encounter they are activated to mature and migrate to LN
         • There they activate T cells which recognise peptide on MHC, contact is maintained for ~20hrs
     • Also important for maintaining tolerance to self proteins by display of self peptides on non activated DCs
     • Immature DC Ag uptake mechanisms
       • 1. phagocytosis 2. macropinocytosis 3. Receptor-mediated endocytosis for isolated molecules
     • Migration and Maturation
       • Immature DCs have:
         • 1) high Ag uptake 2) low surface MHC2 3) low surface costimulatory molecules
       • Maturation is mediated by:
         • 1) PAMPs 2) DAMPs 3) inflammatory cytokines (IL-1beta, TNF-alpha)
       • Effects
         • 1. formation of peptide-MHC complexes
         • increased capacity to activate T cells
         • 2. subsequent decreased Ag and pathogen uptake
         • 3. increased costim. molecules (B7)
         • 4. increased surface area, so increased interaction and display surface available
         • 5. altered surface expression of integrins and chemokine receptors for migration
         • 6. Increased expression of CCL18 chemokine to attract naive T cells
   • B cells
     • Ag uptake is via BCR - highly specific
     • B cell presentation via MHC2 functions during B cell activation - they present to previously activated T helper cells, the T helper cells then aid B cell differentiation to plasma cells

6. Tasmanian Devil Facial tumour disease

   • an infectious cancer - tumour cells passed on by biting
     • no viral involvement indicated
       • tumour should be rejected due to foreign MHC, this may not occur due to:
         • 1. low NHC diversity in the population 2. tumour evades immune surveillance
           • mechanism not yet fully understood

7. T Cell Classes

   • Conventional
     • CD8 cytotoxic T cells
       • kill virus-infected cells
       • targets viruses and some intracellular bacteria
     • CD4Th1 cells
       • activate infected macroophages by producing interferon gamma
       • help B cells for Ab production
       • targets microbes that exist in macrophage vesicles and extracellular bacteria
     • CD4Th2 Cells
       • release IL-4 to help stimulate B cells to produce Ab and Ig switching to IgE
       • target helminth parasites
     • CD4Th17 cells
       • enhance neutrophil influx by producing IL-17
       • promote barrier (skin + intestine) integrity
       • targets Klebsiella pneumoniae
     • Tfh Cells
       • =follicular helper cells
       • are usually CD4 Tfh cells
       • help isotype switching and Ab production in LNs and spleen
       • located near B cells to assist for their function
       • targets all pathogens
     • CD4 regulatory T cells
       • suppress T cell responses
       • bring immune response back to baseline levels after the pathogen is killed/reduced
       • Some bacteria and cancers induce these cells to reduce the immune response against them
   • Other subsets
     • Natural Killer T (NKT) cells
       • rapidly produce cytokines (INFgamma, IL-4, IL-13, IL-17) that exist in pre-activated state and are located in tissues for immediate use
       • bind glycolipids and lipids bound to CD1d
       • cannot be boosted
       • different subtypes exist
       • have semi -invariant TCR with highly conserved alpha chain
         • in humans: TRAV10/TRAJ18 (TCRVa24/Ja18)
         • In mice: TRAC12/TRAJ18 (TCRVa14/Ja18)
       • a component of innate immunity bridging to adaptive immunity
       • involved in tumour immunity, T1D and response to infecition
     • MAIT (Mucosal associated invariant T cells) Cells
       • TCR alpha chain is highly conserved
         • TRAV1-2/TRAJ33 (mice=TCRVa19/Ja33, humans=TCRVa7.2/JJa33)
       • recognise microbial vitamin B metabolites complexed to MR1, a self protein that displays foreign molecules
       • prominent population in mucosal compartments, liver and blood
       • Participate in immunity to bacteria and yeast (not viruses because they do not produce their own vitamin B)
       • produce INFgamma, TNFalpha, IL-17
     • Epithelial gamma:delta cells
       • expression of TCRgammadelta rather than the coonventional TCRalphabeta
       • recognises a wide array of peptide and non-peptide ligands including stress signals on cells (lipids, glycolipids, phosphoantigens)
         • does not need to be displayed on MHC to be seen, these cells can observe directly from the cell surface
       • different types exist in different tissues
       • predominantly CD4 + CD8 negative
       • early producers of cyto+chemokines (IL-17) to increase the inflammatory response
       • B cell help and IgE production by releasing IL-13
       • prime alphabeta T cells via antigen presentation
       • role in DC maturation
       • increase wound healing
       • cause lysis of infected or stressed cells
   • Activation of T Cells
     • Spleen acts as a priming site for DC carrying pathogens found in the blood
     • Ag carried by DCs to T cells in LNs via lymphatics
       • 1. Naive T cells enter LN cortex from the blood via high endothelial venules
         • 1: cell circulates
           • 2: rolling adhesion: L-selectin on T cell to GlyCAM-1 of HEV
             • 3: Activation: chemokines binding onto ECM activate LFA-1 or Mac-1
               • 4: Firm adhesion: LFA-1 (on T cell) binds to ICAM-1 (on HEV)
                 • 5: Diapedesis and migration: lymphocyte migrates into LN
               • CCL21(SLC on HEV) binds to CCR7 (a key marker of lympoid homing T cells) on T cells
             • L-selectin=CD62L
         • 2b. T cells not activated by Ag on DCs exit the LN via cortical sinuses
         • 2a. T cells activated by Ag presented by DCs start to proliferate and lose the ability to exit the LN
           • 3. Activated T cells differentiate to effector cells and exit the LN
     • Third signal - cytokines - influences the type of effector T cell that eventuates
       • • CD4 also characterised by FoxP3 txn factor upregulation
     • T cells inhibited by:
       • CTLA-1 after engagement with B7
       • PD-1 (on T cell) binding to PD-L1/2 on APC
       • many others
     • TCR signalling
       • 1. TCR associated with CD3 chain due to opposite electrostatic charges -> tyrosines (of ITAMs) are phosphorylated -> started by the Lck associated with CD4
         • 2. Lck recruits ZAP-70, this recruits other 2nd messengers
           • 3. PLC is activated
             • 4. increased cellular calcium ions, PKC and MAP kinase pathways
               • 5. induce IL-2 production due to txn factors NFkappaB, NFAT and AP-1

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