week 6: Major Histocompatibility complex (MHC) and antigen processing/presentation

what is the MHC?

• describe the genes

• expression

• protein product roles

• Tightly linked cluster of genes

• Expressed in all mammals studied

• Protein products 

  • play role in recognition:

    • cell-cell

    • self/nonself

  • present antigens to T cells

    • essential for normal immune response

    • implications for disease susceptibility

what do class I and class II MHC do?

• classical MHCs

• closely related membrane-bound glycoproteins

• form stable complexes with peptides

  • antigen presentation

what is class III

group of unrelated proteins, some may have immune functions

compare the structures of the MHC class I and class II molecules

MHC class I

• has one alpha chain with 3 domains (alpha is variable)

• has one beta chain

MHC class II

• has one alpha chain with 2 domains

• has one beta chain with 2 domains

• alpha domain 1 and beta domain one are variable

*the peptide-binding grooves are where Ag’s will be bound, these grooves differ between MHC1 and MHC2 because their corresponding peptides differ

*alpha gene is encoded in MHC locus and varies from person to person

*the Beta2 microglobulin gene is outside of MHC locus, less variable, more for structure

compare the peptides that bind in the peptide binding groove in MHC class I and MHC class 2

class I:  peptides 8 - 10 amino acids 

• peptides have particular anchor residues (usually hydrophobic) at termini (beginning and end of amino acid chain)

• ex. glycine, proline, leucine, isoleucine, tyrosine, valine

class II:  peptides 13 - 18 amino acids

• because the groove is more open ended so larger peptides can bind

compare which cells express class I vs class 2 MHC and why this is necessary

• Class I molecules

  • glycoproteins on surface of almost all nucleated cells (cells that need to be deleted by Tc)

  • presentation to cytotoxic T cells (T_C)

• Class II molecules

  • glycoproteins expressed mainly on antigen presenting cells (macs, B, dendritic) → cells that help helper T cells

  • present to helper T cells (T_H)

*note: APCs also express class 1

what else is MHC called in humans?

what chromosome is it found on?

• human leukocyte antigen (HLA)

• found on chromosome 6

what else is MHC called in mice?

what chromosome is it found on?

• MHC called H2 complex

• on chromosome 17

haplotypes

• haploid genotype

  • specific combination of linked alleles in a cluster of related genes

  • one haplotype inherited from each parent

• allelic variation (denoted as a superscript)

what are the MHC class 1 genes?

class 2?

• class 1: alpha chains: K, D, L; constant beta chain

• class 2: alpha chains: E, A; constant beta chain (a heterozygote can express any alpha chain with any beta chain - can mix and match mom and dad)

explain how MHC genes are inherited

• how are they expressed?

• mouse strains

• inherited as haloptypes because they are so tightly linked

• MHC is highly polymorphic (vary in population) and very tightly linked (0.5% crossover frequency) → inherit one maternal haplotype and one paternal haplotype (basically never get recombination)

• MHC alleles are codominantly expressed (express the genes from both mom and dad)

• Mouse strains

  • some have been inbred with particular MHC haplotypes

Syngeneic

identical at all genetic loci

Congenic: 

• genetically identical except at a single genetic locus or region

  • any phenotypic differences can thus be attributed to the region that is different

  • strains made by series of specific crosses

MHC congenic: 

• identical at all loci except MHC

  • e.g. strain A.B (genetic background of strain A with MHC of strain B)

  • recombinant strains:  differences at only a few genes within MHC

describe the genes of MHC

• introns/exons?

• alleles?

• Separate exons encode the different regions on the class I and class II molecules

• Polymorphism

  • hundreds of different allelic variants

how many different class 1 and class 2 molecules can each human have?

• Each human individual

  • up to 6 different class I molecules

  • up to 12 different class II molecules

explain MHC diversity

• MHC considered polygenic

  • genes with similar but nonidentical function

• High polymorphism

• Sequence differences mainly in regions encoding antigen-binding domains → which allows for diff Ag binding/presentation

• Implications for disease susceptibility and immune responsiveness

  • the more diverse, the less of a problem 

describe the cellular distribution of MHC class 1

• what cells express them? which express them the most? the least?

• in healhty cells what do they display?

• in infected cells what do they display?

• on almost all nucleated cells, to varying degrees

  • highest on lymphocytes

  • low on fibroblasts, muscle, hepatocytes, neural cells

• displays self-peptide in normal, healthy cells (shows the T cells what is healthy, helpful in T cell differentiation)

• displays viral peptide in infected cells

• expressed on thymic stromal cells for T cell education

  • used in T cell screening by showing the T cell the MHC is self, so don’t respond)

describe the cellular distribution of MHC class 2

• what cells express them?

• describe the level of their expression

• expressed on 

  • antigen presenting cells (APCs)

    • macrophages, dendritic cells, B cells

    • constitutive expression (constantly expressed)

    • level depends on developmental stage

  • thymic epithelial cells and some others if induced

    • helpful in T cell education/screening to show T cell self MHC

explain the hypotheses for explaining how haplotype influences immune response

• Class II gene expression very important (because they present to helper T cells)

• Experiments in mice showed haplotype influences response

  • hypotheses (possible that both are true)

    • determinant-selection model

      • different class II molecules have different abilities in binding antigen

    • holes-in-the-repertoire model

      • T cells that could bind foreign antigens that are similar to self antigens are eliminated

explain MHC and disease susceptibility

• Some diseases occur more (or less) among individuals with particular haplotypes

  • e.g.

    • some autoimmune diseases

    • some viral infections

    • some allergies

    • some neurological disorders

regulation of MHC expression

• what factors are involved

• 5’ promoter sequences regulate expression

  • associated transcription factors

• Cytokines, e.g.

  • interferons (alpha, beta, gamma) 

  • tumour necrosis factor

  • interleukin 4 (IL-4)

  • effect depends on cell type and developmental stage

• Viral infection

  • some viruses can decrease MHC expression (so cell cannot present viral peptides so the virus can go undetected)

  • decreased expression helps virus evade system

explain the concept of self-MHC restriction of T cells

• T cells recognize antigen only when presented by self-MHC 

  • same MHC haplotype as the T cell

Experiment by Rosenthal and Shevach

• describe the set up of the experiment (where the cells came from)

• what they did

• what did the results confirm?

• Proliferation of T_H cells

  • source of T_H cells:  lymph node cells from immunized guinea pigs

    • strain 2, strain 13, (2 x 13)F₁

  • APCs:  macrophages 

    • isolated from nonimmunized guinea pigs

      • from strain 2, strain 13, (2 x 13)F₁

    • “pulsed” with same antigen as used to immunized guinea pigs above

  • coincubation of primed T cells (trained to react to Ag) and pulsed APCs (present the Ag)

• Results confirmed by repeating experiments with congenic and recombinant congenic strains

• need to look at different haplotypes because talking about MHC restriction

Experiment by Rosenthal and Shevach

• results

• + = helper Ts were activated and proliferated

• - = helper Ts were not activated and did not proliferate

• got response (+) when the helper Ts were able to recognize their specific MHC

Experiment by Zinkernagel and Doherty

• describe the set up of the experiment (where the cells came from)

• what they did

• what did the results confirm?

• looked at Cell lysis mediated by T_C cells

  • detected by chromium-release assay (target cells were injected with radioactive chromium, if T cells lyse the cells you see chromium in the media)

  • T_C cells: splenocytes (spleen cells) from mice immunized specific with LCM virus

  • target cells:  LCMV-infected cells of the same or different haplotype as the T cells

  • results confirmed with congenic and recombinant congenic mice

Experiment by Zinkernagel and Doherty

• explain the results

1. no lysis because the T cells only kill infected cells

2. lysis because the infected cells had the correct MHC haplotype to match the T cell

3. no lysis because the infected cells had a different MHC haplotype

Predict whether T_H-cell proliferation or CTL-mediated cytolysis of target cells will occur with the following mixtures of cells.  The CD4⁺ T_H cells are from lysozyme-primed mice and the CD8⁺ CTLs are from influenza-infected mice. 

1. H-2^k TH cells + lysozyme-pulsed H-2^k macrophages

2. H-2^k TH cells + lysozyme-pulsed H-2^b/k macrophages

3. H-2^k TH cells + lysozyme-pulsed H-2^d macrophages

4. H-2^k CTLs + influenza-infected H-2^k macrophages

5. H-2^k CTLs + influenza-infected H-2^d macrophages

6. H-2^d CTLs + influenza-infected H-2^d/k macrophages

7. H-2^d/k CTLs + influenza-infected H-2^k macrophages

1. yes because helper Ts recognize MHC on macrophage

2. yes

3. no

4. yes

5. no

6. yes

7. yes

roles of APCs

• Internalize antigen proteins (endocytosis)

• Process the proteins into peptides

• Present the peptides on MHC class II molecules

• Provide costimulatory signals (confirmation for T cell to get excited and activated after seeing the Ag)

explain the evidence for antigen processing requirement in APCs to activate T cells

a) APC get fixated (kills APC, so proteins get fixed on surface, blocks Ag pick up), then Ag is given, but the Ag cannot bind because of fixation so no T cell activation

b) APC is given Ag, Ag binds and is processed then but on PM, APC is fixated, T cell gets activated

c) APC is fixated, then given Ag peptides (already cut up/processed Ags), MHC can present the peptides even after fixation because they were already processed, T cells get activated

• this is evidence that Ag needs processing before MHC presentation

• processing was either done by the APC or given processed but either allowed presentation to T cell

describe MHC class 1, MHC class 2

• location

• type of cells they present to

• types of Ags they present

• MHC Class I molecules

  • on most nucleated cells (infected cells)

  • present:

    • to T_C (CD8⁺)

    • “endogenous” (Ags inside the cell, ex. viral, tumor Ags) antigens processed in cytosolic pathway

      • includes antigens of viruses that have infected cells

• MHC Class II molecules

  • on APCs (engulf and process Ags, not infected)

  • present:

    • to T_H (CD4⁺)

    • exogenous (extracellular, anything forgien in serum, need to be taken in but are not infected) antigens processed in endocytic pathway 

if an APC was infected, what MHC class would it present its Ag on?

class 1 (always for infection to present to killer Ts)

describe the endogenous and exogenous processing pathways

endogenous/cytosolic (MHC class 1)

• Ag gets degraded by a proteasome in the cytosol into Ag peptides. Chaperons guide the peptide to assemble it onto MHC in the RER then progresses through the endomembrane system (vesicle, golgi, vesicle, PM), to present on PM

exogenous pathway

• MHC class 2 leaves the RER in a vesicle and travels to golgi

• leaves golgi in a vesicle

• exogenous antigen gets engulfed in an acidic vesicle which degrades the Ag into Ag peptides

• the MHC and Ag peptide vesicles merge so the MHC and peptide assemble and move to PM

describe the evidence for the two different presentation pathways

• experimental design

• Experimental design:  test whether class I-restricted or class II-restricted T_C cells lyse targets after different treatments of targets:

  • infectious virus (positive control to make sure assay works to infect cells with virus and T cells can lyse)

  • noninfectious virus (UV-inactivated)

    • can not get into the cell, can only do endocytic pathway

  • infectious virus + emetine (inhibits viral protein synthesis)

    • prevents viral protein from produced in the cytosol, but virus can get in cell

    • no viral proteins in the cytosol

    • eliminates cytosolic Ag (eliminates cytosolic pathway)

  • infectious virus + chloroquine (blocks endocytic pathway in target cells)

    • exogenous pathway only

    • virus can still infect and get into cytosol

explain the results of the experiment

a) positive control → virus gets into cell by infection, but cell can endocytose virus too, both pathways worked, positive control works

b and c) no viral protein in cytosol, but endocytosis occurs, no class 1 restricted activity but still class 2 restricted activity, therefore T cells in class 2 endocytic pathway still worked, but not cytosolic

d) infectious virus gets into cell and makes viral protein, get cytosolic Ag, endocytic pathway blocked, class 1 restricted activity is there but not class 2, shows cytosolic pathway functions, but not endocytic

thus there must be 2 separate pathways because diff results for each condition

cytosolic pathway

• Processes endogenous antigens (intracellular proteins, including viral proteins if virus has infected cell)

• Features:

  • degradation of proteins in proteasomes (organelles)

    • unique to class 1

  • peptide transport to and through rough endoplasmic reticulum (RER)

  • class I MHC-peptide complex assembly assisted by chaperone proteins

  • shuttled to PM

explain proteolytic degradation

both normal/constitutive proteosomes (all cells have) and immuno-proteosomes (special to immune cells) cut Ag into specific peptides that are ideal for MHC class 1 binding

• ideal aa sequence

• ideal size

overview of peptide transports into RER for MHC class 1

• assisted by?

• ideal peptides

• Assisted by TAP for presentation on MHC class 1

  • transporter associated with antigen processing

  • heterodimer encoded within MHC class II

  • spans RER membrane

  • ATP-dependent

• Ideal peptides for MHC:  

  • 8-10 amino acids

  • hydrophobic or basic carboxy termini

describe TAP structure

• ARP dependent

• 2 chain transporter

• selects by size based on what it allows into RER

• spans RER membrane

explain TAP activity

• proteosome degrades Ag into aa (to be recycled) and into peptides

• some of the peptides go through TAP (ATP dependent manner) into RER lumen

• in RER lumen the peptide is ready to be loaded onto MHC class 1 molecule (with the help of chaperones to prevent peptide degradation in RER)

name and describe the chaperones involved in Peptide-MHC 1 complex assembly in RER

• Assisted by chaperone proteins

  • calnexin:  membrane RER protein

    • promotes folding of free class I α chain

    • released when β2 microglobulin binds α chain

  • calreticulin

    • remains bound to class I while it has no peptide

  • tapasin (TAP-associated protein):  

    • membrane-bound

    • brings TAP close to class I molecule, promotes peptide transfer

    • releases MHC after peptide bound

  • ERp57 (protein with enzymatic activity)

    • disulfide bond with tapasin and association with calreticulin

  • ERAP1 (exopeptidase in the ER)

    • trims peptides before they bind to MHC

describe the endocytic/exogenous pathways

• what is processed

• time?

• features

• MHC class 2 (APCs presenting to helper Ts)

• Processing of exogenous antigens (they must be endocytosed)

• Takes 1-3 hours

• Features

  • association of invariant chain with class II molecules (in peptide binding groove to stabilize it and block other things, acts as a place holder) when MHC class leaves RER, until it meets the peptide

  • invariant chain gradually gets degraded through endomembrane system and becomes CLIP structure

  • HLA-DM and HLA-DO (positive and negative regulators of the exchange of CLIP and peptide in the peptide binding groove)

  • generation of peptides in endocytic vesicles

describe Invariant chain (Ii) and CLIP

• Trimeric protein

• Associates with 3 pairs of class II α and β chains in the RER

• Blocks peptide-binding cleft of class II molecules

• Guides transport of class II molecules out of ER and to endocytic vesicles (where peptides are)

• Gets gradually degraded

  • small fragment remains bound to cleft:

    • CLIP (class II-associated invariant chain peptide)

describe HLA-DM

• Encoded by class II MHC

• Catalyzes exchange of CLIP for peptide

•  α and β chains, but nonclassical MHC, not polymorphic

• Reside in endosomes

• Inhibited by HLA-DO

describe HLA-DO

• Hinders HLA-DM

• In B cells

  • downregulated in germinal centre B cells

• Recently found in DCs

  • downregulated during DC activation

• In thymic medullary epithelial cells

what is cross-presentation of exogenous Ag?

• Exogenous antigens internalized and presented via the endogenous presentation pathway (or vice versa)

which would cells do cross-presentation?

why is it important?

• dendritic cells

• killer T cells need support by APCs to be able to recognize MHC1

• need to get exogenous Ag to MHC1 (not MHC2) so that killer T cells can recognize

• need to ensure killer Ts does not kill the dendritic cell

• allows dendritic cell to not only activate helper Ts but also killer Ts

describe dendritic cell licencing and cross presentation

a) dendritic cell presents Ag to helper T to activate it. helper T interacts with DC to license it to allow it to cross present

b) DC can now also present on MHC class 1 to activate killer T cells now (naive). helper T still is around and supports (releases cytokines)