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Topic 3 – antigen presentation to T lymphocytes by MHCClass 1: CD8+ - All cells express MHC1 so all cells can demonstrate presence of viral pathogen within it Class 2: CD4+ - Only some cells use MHC2 (specialised thymus or gut cells) EXAM Q: WHAT ARE 3 TYPE S OF ANTIGEN RECEPTOR A: T -CELLS, B -CELL...

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Topic 3 – antigen presentation to T lymphocytes by MHC Class 1: CD8+ • All cells express MHC1 so all cells can demonstrate presence of viral pathogen within it Class 2: CD4+ • Only some cells use MHC2 (specialised thymus or gut cells) EXAM Q: WHAT ARE 3 TYPES OF ANTIGEN RECEPTOR A: T-CELLS, B-CELLS AND MHC MOLECULES HOW CELLS ARE INFECTED 2 main intracellular compartments in cell separated by membranes 1) Cytosol • Caused by Ag from the inside/intracellular environment • Usually a virus that binds to cell, becomes internalised and hijacks cell to generate more viral protein in cytosol • Ag degraded and loaded onto MHC1 • Ag trapped and transmitted to outside via secretory system and Golgi • Displayed on cell membrane 2) Vesicular system • Ag is extracellular where bacteria/pathogen is in surrounding tissue fluid • Cell takes in Ag via phagocytosis using endosomes and lysosomes/ membrane bound vesicles • Ag degraded • Fragments displayed on cell membrane by MHC2 DELIVERING PEPTIDES TO CELL SURFACE USING MHC MOLECULES Cytosolic compartment: • Cytosolic pathogens like virus or self proteins • These are degraded to small proteins within cytosol • Bind to MHC1 • Presented on CD8+ T-cells (cytotoxic T-cells) • Results in cell death Vesicular compartment: • Pathogens taken in in membrane bound vesicle or endocytic vesicles • Endocytic vesicles acidic and low pH • Causes degradation of pathogens

Peptides bind to MHC2 Ag presented to CD4+ (effector or helper t-cells) Either get signals activating macrophage to kill vesicular pathogens OR signals to activate B-cell to become plasma cell and secrete immunoglobulins • Ig will go on to eliminate other extracellular pathogens of the same type Significance of using two different compartments to degrade Ag: Separates two arms of the adaptive immune system Viral proteins stimulate cytotoxic t-cell response Extracellular pathogens stimulate t-helper cells and eventually the B-cell response • • •

TAP COMPLEX (and cytosolic pathway - MHC1 molecules) peptides that bind to MHC1 actively transported from cytosol to ER via TAP complex Infected cell has both degraded viral protein AND normal host cell protens that were misfolded, old or not correctly functional So Peptides can be from pathogens (viruses/intracellular bacteria) or degraded self proteins.

These are transported to ER via Tap1 and Tap2 which together make the TAP complex TAP complex: • active transporter that binds ATP • It forms a pore • Hetrodimeric protein • Polymorphic protein: can exist in a variety of forms and each form can transport a different petide. Therefore there is some selectivity of peptides that can go via the TAP complex Go to ER as molecules in ER able to feed degraded peptides from cytosol to region in ER where MHC1 is made

Peptides are generated in cytosol but proteolytic digestion occurs in proteasome (organelle): • 3D structure of 4 rings on top of each other, forming a pore • Proteasome activator domains and catalytic core in centre

1) 2) 3) 4)

Proteins labelled/flagged with ubiquitin to flag it for degredation in proteasome Fed through catalytic chamber of proteasome Proteolytically digested and degraded into small peptides Peptides fed through TAP complex in ER

In times of infection the proteasome alters its beta units to become an immunoproteosome • Produces peptides with hydrophobic C terminals • Allows better recognition of the viral and self proteins • Allows better binding in the groove of MHC1 Newly synthesised MHC1 are retained in ER until they bind a peptide:

The RER and cytosolic ribosomes are making new self proteins If they are good and functional these stay in cytosol or are transported through ER to go on and perfrom their particular function Some non-functional or misfolded proteins (called DRiP’s – defective ribosomal proteins) are designated for ubiquitination and destruction • • •

Both defective and viral proteins fed through proteosomal complex Degraded to peptide fragments Transported to TAP complex and into ER

MHC molecules are inserted into membrane Its heavy chain is an alpha chain with 3 alpha domains

When first made MHC alpha chains are unstable so they use chaperone proteins to help overcome instability For MHC1 chaperone used for the alpha chains in Calnexin As MHC1 heacy chain made it is co-translationally insterted into the ER membrane and will be associated with its chaperone • Isnt in correct conformation and is a partially folded chain • The beta macroglobulin (non-membrane part of MHC1) binds here but MHC1 still unstable • Chaperone changes to calreticulin • MHC1 associates with Tapasin molecule allowing the partially folded macroglobulin chain to bind close to the TAP complex. After the peptide fragments go through the TAP complex they are further processed by ERAAP • = ER aminopeptidase associated with antigen presentation • It is an aminopeptidase • Therefore has function of trimming peptidase to correct size (8-10aa) before they go past the MHC binding groove • If they have correct peptide motifs they bind • MHC is now able to adopt its final conformation • MHC loses chaperone • MHC becomes its recognisable shape and is released from the ER, through Golgi and then cell surface Therefore, MHC molecules do not go to cell surface unless they bind to the correct peptide as they do not have their final conformation until they bind to this peptide. Therefore MHC molecule rarely found on cell surface without a peptide bound to it Many viruses produce immunoevasins that interefere with Ag presentation by MCH1 and evade this process Viruses subvert the immune system operational mechanisms e.g adenovirus produces E19 protein • Inhibits binding of MHC to TAP by coming iin between MHC and Tapasin • This inhibits peptide engaging with MHC • Without MHC bound to peptide the cell wont be recognised by T-cells as it cannot present Ag to them • Cell evades cytotoxic T cell response Subverting immune response limits the virus’s detection by the immune system

PEPTIDE PRESENTATION TO MHC2 – PEPTIDES GENERATED IN ACIDIFIED ENDOCYIC VESICLES

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MHC2 takes exogenous Ag Ag brought into cells by vesicles (Ag never comes into contact with cytosol) In early stages endosome has neutral pH so proteases are inactive Overtime vesicle becomes more acidic Causes activation of proteases and acidic environment also has denaturing effect on the Ag Ag proteins broken up onto peptide fragments Peptides come into contact with MHC2 which is also in a vesicle. Vesicles fuse Ag peptides and MHC2 bind Ag fragments presented on outside of cells

MHC2 is made in the ER but not loaded with proteins intended for MHC1 Why doesn’t MHC2 bind proteins in the ER like MHC1 if it is also made in the ER? • •

Invariant chain sits as a trimmer in the ER Binds to 3 MHC2 molecules

Roles of invariant chain: 1) Prevents peptides binding to binding groove MHC2 whilst it is in the ER • So even though peptides have passed through TAP complex they cannot bind to MHC2 and bind to MHC1 instead 2) Trafficking • Part of the chain goes through surface of ER • As MHC transported around cell this region of the chain helps target the MHC2 molecules into the endosomal pathway • There is a signalling portion of this region • Initiates signal for MHC2 complex to leave ER into vesicles and come into contact with endosomal pathway with degraded Ag before breaching cell surface

Directing the MHC2 molecules to the endosomal pathway:

At this stage the groove is blocked with invariant chain but we need peptide to enter the groove. The Ii region of the invariant chain is in the binding groove (this is the region involved in targeting the delivery of MHC2 to endosomal pathway) Ii region progressively cleaved Clip (class II-associated invariant chain-peptide) region remains (blue in panel 3) This prevents incorrect molecules binding as you start to release MHC2 from the imvarient region in the ER MHC2 molecules will be loaded with peptide in the MHC class 2 compartment (MIIC) • Specialised, intracellular compartment •

immunogold is an antibody (Ab) bound to gold fragment

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Ab labelled with gold fragments of diff sizes

• Small gold particles recognise MHC2 and large particles attached to Ab that recognise the invariant chain • Immunolocalization: large particles found I golgi and small within the MIIC which shows it is in these compartments that Ii is cleaved and peptide loading occurs •

SO we start with lots of large particles in the Golgi as the Ab is labelling the CLIP and then this changes to small particles being apparent in the MIIC which means that the CLIP must have been removed and we can assume been replaced by AG peptide.

HLA-DM FACILLITATES LOADING OF AG ONTO MHC2

Still need to remove clip region from MHC2 binding groove HLA-DM acts as peptide exchanger by altering conformation MHC2 • Causes CLIP to fall away • Groove now accessible to peptide (cognate Ag) MHC2 doesn’t need peptide editing like MHC1 as MHC2 peptides are longer than those in MHC1 and can be anywhere in the peptide chain despite having anchor residues As long as peptide has correct anchor residues it will bind into the groove So 1000’s of different peptides can bind into the groove • Peptide binding into the groove stabilises MHC2 structure after HLA-DM has acted • Ag peptides then put onto cell surface Last panel: MHC2 on surface of cell with peptide bound into Ag groove If MHC2 does not bind peptide after dissociation from the invariant chain, specifically the Ii group, they are unstable in the acidic environment and are rapidly degraded. CROSS PRESENTATION cross presentation of Ags where cellular Ags can be presented on MHC2 in APCs and exogenous material can be expressed on MHC1 in other situations. 1) HOW IMMUNE RESPONSE ENSURES A CD8 RESPONSE TO ALL VIRUSES EVEN IF THEY FAIL TO INFECT APC’S Normally for cd8+ t-cells to become active they need to eb stimulated by correct APC’s bearing correct stimulating AG via MHC1

But if virus does not affect APC then partially degraded Ag in endosomal pathway can be loaded into MHC1 binding groove by translocation in cytosol = CROSS PRESENTATION 2) LIKEWISE MHC2 CAN PRESENT CYTOSOLIC PROTEINS MHC2 loaded with cytosolic proteins via autophagy •

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Autophagy is the natural process by which some damaged organelles and cytosolic proteins are delivered into the endosomes by autophagosomes for proteolytic breakdown. Peptides can be expressed in context of MHC2

MHC COMPLEX GENETICS AND FUCNTIONS • • • •

Cluster of genes All have related function Clusters usually derived from gene duplication Results in series of genes in a row on a chromosome which happened for MHC molecules HLA: • region encoding MHC molecules • Human leukocyte antigen • Closely related genes that evolved from one another by gene duplication followed by divergence • Can be 3 different MHC1 (red) alpha chains as they are encoded by the three genes: HLA-A, HLA-B and HLA-C

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MHC2 (yellow) genes also genetically linked. Alpha and beta chain encoded by separate genes in closely related region adjacent to MHC1 3 major groups DP, DQ and DR and sometimes DM Overview of MHC1 and MHC2

PROTEIN PRODUCTS OF MHC1 AND 2 ARE POLYMORPHIC AND DIVERSE

1) Gene products of MHC1 • • • •

3 MHC loci regions on the chromosome encoding the three alpha chains The numbers show the number of allelic variations that are known Most variation on alpha chain B The large numbers show the large variations of alleles at loci A, B and C

2) MHC 2 gene products • Also shows large variation in populations Chromosome 6 encodes the genes for MHC1 and MHC2 but REMEMBER you have TWO chromosomes (one from each parent): MHC1:

• both chromosomes from each parent expressed as a haplotype – CODOMINANCE • 6 diff types MHC1 genes can be expressed

MHC2: • 6-8 different types MHC2 possible in an individual so it is polygenic • Polymorphic as on a population level there are many different alleles.

Overall: Variation is due to many number of genes present on chromosomes in an individual and it is polygenic and polymorphic due to population diversity Function of the selection for polymorphism is the ability to have variation in Ag binding grooves which gives variation in the peptides that can be held More types of MHC molecule you have the more diverse genome is so there is a greater ability to change the amino acids contributing to the binding site which maximises the ability to present any type of peptide MHC RESTRICTION

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Ensures T-cell recognition only occurs for MHC molecules and that it is a selective process Tcr only able to recognise self MHC and cognate peptide and not just MHC in general

SUPERANTIGENS • • • • •

They bind strongly to Ag receptors and TCR Not limited by TCR recognition Can only bind to T-cells Cause large immune response even if super Ag not in the binding groove Lead to toxic shock syndrome – sometimes fatal

They don’t comply with selectivity of T cells and can bind to any T-cell and MHC receptor causing a large T-cell response.

Topic 4 – T-cell development and cell-mediated immunity OVERVIEW T-CELL DEVELOPMENT IN THYMUS AND ACTIVATION BY FOREIGN ANTIGENS Many stages involve notch signalling

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B and T cells originate from stem cells in bone marrow - Derived from precursors involving the Notch pathway: a conserved signalling pathway - Pathway influences if the stem cell becomes the B or T cell - Involves membrane bound receptors and cell contact If cells are signalled to become a T-cell they migrate to the thymus (panel 1) - Thymic education occurs here – where T-cells learn what self MHC is - Occurs as a result of +ve and -ve selection (panel 2) - Now TCR are chosen that don’t interact too strongly or too weakly with MHC and receive a survival signal - Example of self-preservation. - Those not chosen are driven to apoptosis Tells migrate to peripheral secondary lymphoid organs and come into contact with Ag (panel 3) - If they interact with cognate Ag via APC’s they are activated - APC’s gives specific effector cells Activated T-cells migrate to the mucosal surfaces of the gut and then migrate back to the site of infection

NOTCH SIGNALLING DRIVES THYMOCYTES (T-CELLS) DOWN SPECIFIC PATHWAYS (NB: thymocyte is a young T-cell that has just left bone marrow and entered thymus) Two lineages: CD4+ and CD8+ If cell expresses both, they are double positive and are called CD4+CD8+ cells T-cells enter thymus as thymocytes • are expressed as double negatives • don’t express CD4 or CD8 or TCR As T-cells migrate through thymus they differentiate • characterized by different gene expression patterns first division: become either • primitive gamma/delta (blue) • or normal t-cell (yellow) with alpha/beta type TCR which change their TCR genes to go from double negative to double positive second division: • further genetic rearrangement • selection process using NOTCH signaling and thymus cell interactions to educate the thymocytes • help them decide to become CD4+ or CD8+ (single +ve thymocytes) Third division: • mature CD4+ or CD8+ produced • released from thymus as naïve lymphocytes to interact with Ag in lymph nodes Although T-cells proliferate extensively in the thymus, only a few percent of precursors survive and most cells die by phagocytosis in thymic cortex. This is due to the education and selection of self acting T-cells.

ARCHITECTURE OF THYMUS made up of lobules which have distinct areas outer cortical region and inner medullary region and different cells operate indifferent parts of the thymus. Cortex – • Immature thymocytes closely associate with branched cortical epithelia cells. • The cell to cell interaction influences the destination of immature thymocytes that are self acting •

Macrophages get rid of apoptotic thymocytes(the ones that are self acting).

Medulla – • Mature thymocytes associate with local dendritic cells – undergoing positive and negative selection Thymus important as all cells must go through it. •

T-cells go through thymus to become functional.

= Thymic education – mature CD8+ or CD4+.

The amount of T-cells that leave thymus may be much less than the number going in due to the education and selection processes. • Only TCR that recognise self MHC and non-self Ag are given survival signals and move onto the peripheral tissue. • The rest die. THYMOCYTES UNDERGO REARRANGEMNT OF TCR AND CHANGES IN EXPRESSION OF CELL SURFACE MOLECULES IN THYMUS (Be able to map stages of TCR and what is expressed on cell surface during the developmental stages.) Top of figure shows different phases Double negative (neither CD8 or CD4 expressed) has 4 stages:

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Beta chain rearrangement occurs first in DN2-4 for D-J and DN 3-4 for V-DJ Alpha chain begins to rearrange as the cell leaves double negative form and become double positive Also other chains expressed here o Pre Tau alpha (pTa): surrogate light chain § produced early on DN1 through to Late DP until the alpha chain is fully expressed § Surrogate now no longer needed. § pT alpha is a very important chain because without the expression of the surrogate alpha chain you can’t get expression of the beta chain. § role of pTa is to enable the beta chain to be expressed at the cell surface membrane o CD3: a series of molecules that are expressed § Help signalling process when TCR engages with MHC and Ag § Produced during early stages of development from DN3 through the double positive and single positive stages o CD4 and CD8: § start in the DPs § get refined to become EITHER CD4 OR CD8 in the final stage of development to give you the SP cell.

So to get effective signaling from a TCR you need to have both the alpha and beta chain plus this collective set of molecules CD3 Importantly these phases of development here, the DN1 – 4, the DP and SP their differentiation occurs in different parts of the thymus. DIFFERENT STAGES OF THYMOCYTE DIFFERENTIATION ARE FOUND IN DISTINCT PARTS OF THE THYMUS – thymic education need to know what happens to gene arrangement for tcr and what molecules are expressed at the surfaces in reference to CD8 and CD4 Thymocytes come into thymus via venule walls • they are in DN1 stage they move from medullary regions to the cortical region and go through the stages of double negativity progression from DN2-DN4 to immature DP occurs in outer cortex • when you reach DP stage you have functional alpha and beta chain • therefore have a functional TCR • pTa no longer expressed

• CD3 molecules and CD4 and CD8 expression • Here thymocytes are interacting with cortical epithelial cells (form of dendritic cell) These dendritic/cortical epithelial cells in the cortex express MHC1 and 2 • Help immature T cells decide if they will become CD4/CD8 • From this interaction, the TCR’s that recognise MHC either too weakly or too strongly undergo negative selection • They are given a signal to undergo apoptosis • Those that interact and recognise MHC correctly undergo positive selection • Given a survival signal • If a thymocytes TCR is not engaged at all then they are programmed to die in 3-4 days In medulla they are single positive • Interact with medullary epithelial cells • Interact with macrophages and dendritic cells which display self Ag • Here single positive T cells are positively selected for if they do not bind to this self Ag • If they do bind to self Ag they are destroyed. This is this process of thymic education so it’s during this process that you get rearrangement of the TCR genes here, production of the mature CD4 or CD8+ cells and the cells recognise self MHC and the cells become restricted to only interact to AG presented on self MHC. TCR ALPHA/BETA REARRANGEMENT IN T-CELL DE...