antigen presentation

week 3 immunology

antigen processing is required to activate T cells

• antigens must be processed and presented to immune cells

• T cells recognise linear peptides derived from protein antigens

• presentation is mediated by specialised protein molecules found on surface of APCs

  • MHC class I → CTLs (killer T cells)

  • MHC class II → Th cells

antigen peptides originate from

endogenous (intracellular) pathway

exogenous (extracellular) pathway

antigen processing and presentation

endogenous antigen processing: the endogenous antigen (a)

• endogenous antigens can be cytosolic proteins but also virus particles

• have to be processed to the appropriate size

• 8-10 amino acids optimum peptide length for MHC class I presentation

endogenous antigen processing (immuno)proteasome cleavage (b)

• the proteasome cleaves polyubiquitinated proteins

• peptides generated via proteasomal degradation

• cytokine IFN γ increases 3 specialised catalytic proteosomal subunits:

  • β₁i

  • β₂i

  • β₅i

• these can replace homologous catalytic subunits in the housekeeping proteasome (immunoproteasome)

• modifies cleavage specificity to tailor peptide production for class I binding (optimum peptide length)

endogenous antigens processing: peptide transport into ER and class I/peptide loading complex formation ( c )

• peptides are transported into the endoplasmic reticulum (ER) by transporters

• transporters associated with Antigen Processing (TAP1a and TAP2)- ATP dependent

• TAP1/2 with calreticulin, tapasin and ERp57 form the peptide loading complex (PLC)

• PLC loads the peptides into the class I MHC molecule

• β-2 microglobulin (β2m): essential component of MHC class I molecule, required for expression of all MHC class I on the cell surface

endogenous antigens processing: release of class I/peptide from peptide loading complex (d)

• following peptide loading, stable peptide MHC β2m complex (trimer) is released from PLC

endogenous antigens processing: transport through Golgi (e)

• peptide- MHC β2m complex traverses the Golgi system

endogenous antigens processing: class I/peptide complex (f)

• peptide- MHC β2m complex appears on surface ready for presentation to the T cell receptor (TCR)

exogenous antigen processing: exogenous protein uptake

• exogenous protein is taken up by endocytosis

• intracellular vesicles of dendritic cells, macrophages and B cells

• in endosome, GILT (interferon γ - induced lysosomal thiol reductase) breaks any disulphide bonds in the engulfed proteins

• progressive acidification in early endosomes’ proteolytic enzymes to cleave proteins into peptides

• late endosomes contain lysosomal associated membrane proteins (LAMPS) which are implicated in enzyme targeting (autophagy)

exogenous antigen processing: MHC class II and li

• MHC class II molecules assemble from α and β chains in the ER with the transmembrane invariant chain (li)

• trimer recruits 3 more MHC class II molecules

li functions

• ensures correct folding of nascent class II molecule

• occupying MHC groove to inhibit spontaneous binding of peptides in the ER

• combination of li with the αβ class II heterodimer inactivates a retention signal and allows transport to the Golgi

• targeting motifs in the N terminal cytoplasmic region of li ensure delivery of the class II containing vesicle to the endocytic pathway

exogenous antigen processing: fusion

• late endosomes fuse with the vacuole containing the class II-li complex forming the MHC class II enriches compartments (MIICs)

• serine proteases cathepsin S and L and asparagine endopeptidase (AEP) degrade li, leaving only the part bound to MHC II groove

• this part of li bound to MHC II is called CLass II-associated Invariant chain Peptide (CLIP)

exogenous antigen processing: removal of CLIP, antigen presentation

• an MHC related dimeric molecule, DM, catalyses the removal of CLIP and keeps the groove open so peptides generated in the endosome can be inserted

• initial peptide binding is determined by peptide conc and its on-rate but DM assists in removal of lower affinity peptides to allow their replacement by high affinity peptides

• acidic pH required for exchange of peptides

• complexes are transported to the cell surface for presentation to Th

MHC class II-like molecules HLA DM and HLA DO regulate the exchange of CLIP for other peptides

• MHC II- CLIP can’t be released to cell surface unless another peptide replaces it

• HLA-DM removes unstably bound peptides (peptide editing) to ensure stable peptide: MHC class II complexes that can survive long enough to stimulate CD4 T cells

• HLA DO binds to HLA DM in same manner as MHC class II molecules (negative regulator)

MHC class II like molecules like HLA DM and HLA DO regulate exchange of CLIP for other peptides

• in acidified endocytic compartment, HLA DO dissociates slowly from HLA DM

• HLA DMA can then catalyse peptide editing for MHC class II molecules

• IFN-γ ↑ HLA M expression but not of the HLA-DOβ chain

• hence, IFN-γ produced by T cells and NK cells can ↑ expression of HLA DM and overcome inhibitory effects of HLA DO

advantages of peptide editing by DM

• provides important safeguards

• peptide: MHC complex must be stable at the cell surface

• if peptides were to dissociate too readily, an infected cell could escape detection

• if peptides could too easily be acquired from other cells, healthy cells might be mistakenly targeted for destruction

stages of endogenous and exogenous antigen processing

cross-presentation of antigens

• 25% of MHC class I present antigen of exogenous origin

• up to 20% of MHC class II molecules present peptides from endogenous origin

non lysosomal antigen processing

• most CTL recognise antigens generated via a non-lysosomal pathway

• protein synthesis is required for non-lysosomal antigen processing

inactive viruses vs infectious viruses

antigens generated by endogenous and exogenous antigen processing activate different effector functions

viruses have evolved mechanisms to hide from host immune system

• MHC class I antigen presentation pathway is targeted by viral immune evasion proteins

  • inhibition of proteasome function

  • TAP-mediated peptide transport

  • chaperone facilitated peptide loading

  • transit of MHC class I from the ER

MHC class molecules

• all cells present antigen on the surface via MHC

• humans: HLA (human leukocyte antigen)

• all nucleated cells display MHC class I molecules on their surface

• professional APCs display MHC class I and class II

  • macrophages

  • dendritic cells

  • B cells

MHC class molecule structure

• MHC class I: long heavy chain and β2 macroglobulin

• MHC class II: α and β chain

molecular structures of class I and class II MHC peptide complexes

• the basic structures of MHC-I and MHC-II molecules are very similar but the way the peptide is bound and presented in binding cleft different between class I and class II

HLA locus

• gene coding for MHC I and MHC II are located on chromosome 6

• class I genes: A, B, C

  • each codes for a 3 domain peptide, associated with invariant β2microglobulin

• class II genes: DP, DQ, DR

  • each codes for individual α and β chains that interact

• HLA genes are highly variable (polymorphic) in sequence between individuals

antigen on MHC class I and T cell recognition

• 8-10 AA peptides noncovalently interact with the domains on the class I molecule and complementarity-determining regions (CDRs) on the T cell receptor (TCR) stabilised by the CD8 molecule

antigen on the MHC class II and T cell recognition

• 13-25 AA peptides interact with domains on the class II molecule within the peptide binding groove, allowing presentation to CD4+ Th cells

  • interactions with the TCR are stabilised by CD4 recognition of conserved regions on the class II molecule

T cell receptor (TCR)

• transmembrane heterodimer

• each T cell carries a TCR of only a single specificity

• 95% express α and β chains on their surface

• 5% express γ and δ on their surface

• individual T cell can express either an αβ or γδ heterodimer, never both

• TCR is always expressed with the CD3 complex, which is required for signal transduction

TCR diversity: α- and β-chain genes are composed of discrete segments that are joined by somatic recombination during T cell development

α chain (top part)

β chain (lower part)

α chain

• Vα gene segment rearranges to a Jα gene segment to create a functional V-region exon

• transcription and splicing of the VJα exon to Cα generates the mRNA that is translated to yield the TCR α-chain protein

β chain

• variable domain is encoded in 3 gene segments

  • Vβ

  • Dβ

  • Jβ

• rearrangement of these gene segments generates a functional VDJβ region exon that is transcribed and spliced to join Cβ

• resulting mRNA is translated to yield the TCR β chain

TCR diversity is high

• somatic rearrangement mechanism shared by immunoglobulin and TCR

• overview of the number of human TCR gene segments and the sources of TCR diversity compared with those of immunoglobulins

• TCR diversity is higher

TCR conc diversity in the 3rd hypervariable region (CDR3)

• TCR antigen recognition site looks similar to that of the antigen-recognition site of an AB molecule

• most variable parts of the T cell receptor interact with the peptide of a peptide: MHC complex

T cell activation

• antigen induced activation of naive T cells initiates changes in:

  • morphology

  • metabolic activity (from predominantly oxidative phosphorylation to aerobic glycolysis)

  • progression into cell cycle

T cell activation: sequential waves of signalling protein activation, gene expression and cell cycle progression

• transcription factors (NFAT, AP-1, NFκB) rapidly undergo changes in expression and/or post-translational modifications by signalling kinases upon activation by TCR and CD28 signalling

• these factors then bind regulatory elements in many genes to activate their expression, including the genes encoding IL-2, CD69, and IL-2Rα

• activation of the Akt–mTOR signalling cascade induces metabolic changes in the activated T cell to prepare it for transition from G0 to G1 as it enters the cell cycle

• overall, leads to rapid cell division and clonal expansion

functional TCR complex

• composed of antigen binding TCR α:β heterodimer

• two ε

  one δ

  one γ

  a homodimer of ζ (zeta)

  • all collectively called CD3

• each CD3 chain has one immunoreceptor tyrosine based activation motif (ITAM) segment

• each ζ chain has 3 ITAMs

• transmembrane regions of each chain have unusual acidic or basic residues (indicated by plus and minus)

  • positively charged lysine of the α chain interacts with the 2 negatively charged aspartic acid of the CD3δ:ε dimer while positive arginine interacts with the negative charges of aspartic acid and glutamic acid in the CD3δ:ε dimer

• co stimulatory receptors in T and B lymphocytes

• naive lymphocytes require co-stimulatory receptors for activation

• CD28 family of proteins (naive T cells)

• TNF receptor superfamily/CD40 naive B cells

• while naive T cells primarily utilise CD28 as the co-stimulatory receptor, naive B cells use the TNF receptor family

• enhance the antigen receptor signals that induce transcription

  • factor activation and PI 3-kinase activation, thereby ensuring activation of the T or B cell

TCR signalling is initiated by tyrosine phosphorylation within ITAMs

• CD3γ, δ, and ε r each contain one ITAM and each ζ contains 3, giving the T cell receptor a total of 10 ITAMs

• each ITAM contains 2 tyrosine residues that become phosphorylated by specific protein tyrosine kinases upon ligand binding

• when both tyrosines of the ITAM are phosphorylated, tandem SH2 domain containing proteins such as Syk or ZAP 70 are recruited

T cell activation- protein interactions

• peptide: MHC complex must bind directly to the TCR

• engagement of co-receptors (CD4 or CD8) with the TCR enhances ITAM phosphorylation

• co-receptor-associated kinase Lck leads to phosphorylation (pink circles) of ITAMs in CD3γ, δ, and ε, and in the ζ chains

• tyrosine kinase ZAP-70 binds to phosphorylated ITAMs through its SH2 domains enabling ZAP-70 to be phosphorylated and activated by Lck

• ZAP-7P then phosphorylates other intracellular signalling molecules

roles of co-receptors CD4 and CD8

• signal transduction

  • binding of the CD4 and CD8 molecules serves to transmit stimulatory signals to the T cells, signal transduction properties of both CD4 and CD8 are mediated through their cytoplasmic domains

• stabilisation of TCR peptide: MHC interaction

  • additional binding of a co-receptor to the MHC molecule is thought to stabilise the interaction by increasing its duration

  • thereby providing time for an intracellular signal to be generated

T cell co stimulation enhances antigen receptor signalling pathways

• activated Akt enhances cell survival and upregulates cell metabolism

• recruitment of the kinase ltk to the membrane is critical for the full activation of PLC-γ

NFAT, AP-1 and NFκB stimulate expression cytokine IL-2, which is essential for promoting T-cell proliferation and differentiation into effector cells

• MAPK pathway activates AP-1

• calcium activates NFAT

• protein kinase C activates NFκB

• all three pathways are required to stimulate IL-2 transcription

• gene activation requires binding of NFAT and AP-1 to a specific promoter element and additional AP-1 binding to another site

• Oct1 is required for IL-2 transcription

• Oct1 is constitutively bound to the promoter, hence not regulated by TCR or CD28 signalling

T cell activation leads to different cell subsets

• naive CD8 T cells differentiate into cytotoxic T cells (often called cytotoxic T lymphocytes or CTLs) which are specialised for killing target cells bearing their cognate antigen

• naive CD4 T cells differentiate into several types of Th or regulatory T effector cells

CD4 T cell subsets

• TH1 cells

• TH2 cells

• TH17 cells

• TFH cells

• regulatory T cells

TH1 cells

• produce cytokines, activating macrophages, (IFN-γ), enabling them to destroy intracellular microorganisms more efficiently

TH2 cells

• produce cytokines that recruit and activate eosinophils (IL-5) as well as mast cells and basophils (IL-4) and promote enhanced barrier immunity at mucosal surfaces (IL-13) to eradicate helminths

T17 cells

• secrete IL-17 family cytokines that induce local epithelial and stromal cells to produce chemokines that recruit neutrophils to sites of infection

• produce IL-22 which activates epithelial cells at barrier sites to enhance barrier integrity and repair and produce antimicrobial peptides that kill bacteria

TFH cells

• form cognate interactions with naive B cells through linked recognition of antigen and traffic to B cell follicles (where they produce germinal center response)

• produce cytokines characteristic of other subsets that participate in type 1, 2 and 3 immune responses to influence isotype class switching

• primarily produce IL-21, important for optimal production of high affinity, class switched ABs

regulatory T cells

• suppress naive T cell responses and produces immune regulatory cytokines such as IL-10 and TGF-β which regulate response of effector T cells directly or via repression of pro-inflammatory cytokines