BMS150 Phys 3.04 Immunology PDF

Phys 3.04 - Immunology Antigen Presentation Dr. Maria Shapoval BMS 150 Week 2 Overview Functions and structure of the HLA complexes Type I Cell types expressing HLA-2 Antigen processing & HLA-1 expression T-cell activation Type II Cell typ...

Phys 3.04 - Immunology Antigen Presentation Dr. Maria Shapoval BMS 150 Week 2 Overview Functions and structure of the HLA complexes Type I Cell types expressing HLA-2 Antigen processing & HLA-1 expression T-cell activation Type II Cell types expressing HLA-2 Antigen processing & HLA-1 expression T-cell activation Learning Objectives Describe the function of HLA proteins in antigen presentation Recognize the basic structure of HLA-1 and HLA-2 List the cell types able to present antigens via HLA-1 and HLA-2 Describe the endogenous and exogenous pathways of antigen presentation Distinguish the antigen type (intracellular and extracellular) for each HLA type Briefly describe cross-presentation and list the cells types able to preform cross-presentation. Relate the role of antigen presentation in T-cell activation Describe the interactions needed for cytotoxic T cell and T- helper cell activation  T-cells and antigen – necessity of HLA T-cells recognize antigens in a specific fashion: ▪ Antigens must be presented to a T-cell in order for them to recognize the antigen ▪ Antigens are presented by being bound to particular proteins found on other cells In mice and rats, these are called MHC (major histocompatibility) proteins In humans they’re called the HLA (human leukocyte antigen) proteins A wide variety of cells can present antigens using HLA proteins ▪ The HLA protein can help a T-cell distinguish between foreign antigen and self antigen And thus, should know to only mount a response against foreign antigens  T-cells and antigen – necessity of HLA Although HLA proteins are bound to antigens, they are not genetically “shuffled” like lymphocyte receptors More to come on this concept later! ▪ Meaning that the antigen is not bound particularly tightly to these proteins ▪ This also implies that these proteins can present a wide variety of antigens to a wide variety of lymphocytes ▪ There are also a wide variety of genes/proteins called “MHC-like” They have a wide of range of functions beyond simply presenting antigens.  HLA proteins There are two types of HLA proteins: ▪ HLA class I ▪ HLA class II HLA class I proteins interact with cytotoxic T-cells and binds intracellular antigens ▪ Interact with a CD8 co-receptor on the cytotoxic T-cell HLA class II proteins interact with T-helper cells and binds extracellular antigens ▪ Interacts with CD4 co-receptor on the T-helper cell  HLA-I Structure ⍺1, ⍺2, and ⍺3 The antigen subunits form binding site is the glycoprotein found between the ⍺1 and ⍺2 heavy chain subunits FYI - Binds antigens 8- 10 amino acids long The CD8 co- receptor on the β2 microglobulin cytotoxic T cells subunit is not binds to the ⍺3 covalently bound to subunit the heavy chain and is needed for proper folding.  HLA-II Structure Composed of two glycoproteins of similar sizes: β1-β2 ⍺1-⍺2 The antigen binding site is found between the β1 and ⍺1 subunits FYI - Binds antigens 13-18 amino acids long The CD4 co-receptor on the T-helper cells binds to the β2 and ⍺2 subunit HLA molecules - polymorphism Each individual expresses about 6 different class I molecules and 12 different class II molecules ▪ HLA type I molecule subtypes are all indicated by the designation A, B, or C ▪ HLA type II molecule subtypes are all indicated by the designation “D” HLA molecules - polymorphism Clinical Application: ▪ There are hundreds and hundreds of different allelic variants of class I and class II molecules in the human population Differing allelic variants of class I and II HLA proteins is the main reason why we usually reject organs from randomly- matched organ donors ▪ The presence of particular allelic variants have been associated with increased risk of some autoimmune diseases Eg. HLA-B27 predisposes a person to ankylosing spondylitis Eg. HLA-DQ8 predisposes a person to celiac disease HLA-1 expression and function HLA-1 types are expressed on almost all nucleated cells ▪ Level of expression differs – highest expression level found on lymphocytes 50,000 HLA-1 proteins on lymphocyte cell membranes Much, much less on fibroblasts, muscle cells, hepatocytes, and most neurons Other cells have intermediate levels of expression ▪ Cells without nuclei do not express HLA-1 What is an example of a cell without a nuclei? HLA-1 expression and function For the most part, HLA-1 proteins bind intracellular antigens via the endogenous pathway ▪ Most of the time these are self-antigens ▪ In the case of infection or malignancy, the peptide can be foreign (ie not a self antigen) During viral infection, some HLA-1 molecules on a cell will express viral peptides, while some will express host peptides HLA-1 expression: endogenous pathway Antigen processing: endogenous pathway (the basics) ▪ Source of the antigenic peptide is from the cytosol The peptide is derived from proteasomal degradation of foreign/altered proteins (or normal self-antigens) ▪ Review – how to proteins destined for degradation by proteosomes get tagged? Retrieved from: https://upload.wikimedia.org/wikipedia/commons/8/8b/MHC_En dogenous_and_Exogenous_Pathways.png HLA-1 expression: endogenous pathway Antigen processing: endogenous pathway (The basics) ▪ source of the antigenic peptide is from the cytosol The peptide is derived from proteasomal degradation of foreign/altered proteins (or normal self-antigens) ▪ The peptide is then transported into the RER and loaded onto the HLA-1 protein Retrieved from: https://upload.wikimedia.org/wikipedia/commons/8/8b/MHC_En dogenous_and_Exogenous_Pathways.png HLA-1 expression: endogenous pathway Antigen processing: endogenous pathway (The basics) ▪ Source of the antigenic peptide is from the cytosol The peptide is derived from proteasomal degradation of foreign/altered proteins (or normal self-antigens) ▪ The peptide is then transported into the RER and loaded onto the HLA-1 protein ▪ The loaded HLA-1 is then expressed on the cell surface Retrieved from: https://upload.wikimedia.org/wikipedia/commons/8/8b/MHC_En dogenous_and_Exogenous_Pathways.png HLA-1 expression: endogenous pathway Antigen processing: endogenous pathway (the details) ▪ Cells that are very good at presenting HLA-1 bound peptides to T-cells have specialized proteosomes, called immunoproteosomes. ▪ Cell types: antigen presenting cells & some other cell types Immunoproteosomes have slightly different subunits that are substituted into the “regular” proteosome ▪ This can be induced by cytokines - IFN-gamma and TNF-alpha The peptides that are produced by immunoproteosomes bind with better Details of image FYI – for affinity to HLA-1 visualization only HLA-1 expression: endogenous pathway Antigen processing: endogenous pathway (the details) ▪ The protein that translocates the peptide fragment into the RER for loading onto the HLA-1 is called TAP FYI – some viruses have the ability to block TAP, preventing their viral peptides from FYI – note the being requirement expressed on of ATP and HLA-1 hydrolysis Eg. HSV during translocation HLA-1 expression: endogenous pathway Antigen processing: endogenous pathway (the details) ▪ There are a variety of other proteins that help with loading onto HLA-1 once in the RER All other proteins except TAP are FYI HLA-1 expression: endogenous pathway Antigen processing: endogenous pathway (the details) Once the peptide is loaded onto the HLA-1, what happens? HLA-1 expression continued Presence of intracellular invaders (eg. viruses) will trigger an increase in transcription of HLA-1 proteins ▪ This occurs via binding to NOD-like Receptors (NLRs) ▪ Review - NLR are PRRs found within the cytosol complex PAMP and DAMP receptors Binding of NLR will upregulate the expression of HLA-1 ▪ NODs activate NF𝜅B Details of image FYI – for visualization only Remember the NF𝜅B pathway? Note - in this case it is the intracellular NLR that triggers the NF-𝜅B pathway rather than a cell surface receptor Points to remember: ▪ Upon activation of the pathway, an inhibitory protein (I𝜅B) is phosphorylated and degraded Review – how is I𝜅B degraded? ▪ NF𝜅B is free to travel to the nucleus and promote the transcription of a NF𝜅B target gene In this case, promoting the transcription of Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 15-62. Pg 874 HLA-1 HLA-1 expression continued Cytokines can also increase the expression of HLA-1 molecules ▪ Both type 1 and type 2 interferons (IFN) ▪ Tumour necrosis factor alpha (TNF⍺) The source of these cytokines is typically a cell that has either been activated or infected (or both) in the nearby neighbourhood ▪ The first to produce them are local antigen-presenting cells TNF-alpha and type-1 interferons ▪ Later activated T-helper cells (Th) cells will contribute to the cytokine production Source of IFN-gamma HLA-1 – What happens next Once a peptide-bound HLA-1 is expressed on the surface of a cell, what happens next? ▪ It can bind a CD8+ T-cells Cytotoxic T cell and activate it. ▪ Review - What is the basic function of a cytotoxic T cell? Once activated, a cytotoxic T- cells can kill infected cells by inducing apoptosis ▪ More to come next class HLA types summary HLA-1 HLA-2 Cell types expressing HLA class Antigen type (intracellular or extracellular) Endogenous or exogenous pathway of presentation? - Describe the basic steps Activates which type of T-cell? (name the co-receptor) HLA-2 expression and function HLA-2 types are expressed exclusively on Antigen presenting Cells ▪ Antigen presenting cells (APCs) “Professional” ▪ Dendritic cells ▪ Macrophages ▪ B-cells “Non-professional” ▪ Fibroblasts (skin) ▪ Glial cells ▪ Pancreatic beta cells ▪ Thymic epithelial cells ▪ Intraepithelial lymphocytes ▪ Vascular endothelial cells HLA-2 expression and function HLA-2 types are expressed exclusively on antigen presenting Cells ▪ However, some of these APCs won’t even express HLA-2 unless they’ve been activated Dendritic cells constitutively (aka always) express high levels of HLA-2 and are very good at co-stimulating helper-T-cells Macrophages need to be activated before they express HLA-2, but they are also good at co-stimulating helper T-cells B-cells constitutively express HLA-2 at low levels and are good at co- stimulating helper-T-cells Non-professional APCs will only express HLA-2 under particular conditions ▪ Eg. Sustained inflammation HLA-2 expression HLA-2 proteins bind extracellular antigens via the exogenous pathway ▪ For some APCs, first we need to up-regulate the expression of HLA-2: HLA-2 expression is up-regulated by particular cytokines ▪ Interferon-gamma is particularly good at this Note - In B-cells, IFN-gamma actually down-regulates HLA-2 ▪ IL-4 is good at upregulating HLA-2 on B-cells HLA-2 expression: exogenous pathway HLA-2 proteins bind extracellular antigens via the exogenous pathway (the basics) ▪ Phagocytosis needs to be upregulated in concurrence with HLA-2 expression Phagocytosis is the source of the peptides that are loaded onto the HLA-2 ▪ Phagocytosis can occur through the “regular” pathway ▪ Or, phagocytosis can also be antibody mediated in the case of B-cells Retrieved from: https://upload.wikimedia.org/wikipedia/commons/8/8b/MHC_Endogenous_and_Exogenous_Pathways.png HLA-2 expression: exogenous pathway HLA-2 proteins bind extracellular antigens via the exogenous pathway (the basics) ▪ Phagocytosis needs to be upregulated in concurrence with HLA-2 expression Phagocytosis is the source of the peptides that are “loaded onto the HLA-2 ▪ Phagocytosis can occur through the “regular” pathway The phagocytosed antigen is processed in an phagosome and then merges with a vesicle containing the HLA-2 Retrieved from: https://upload.wikimedia.org/wikipedia/commons/8/8b/MHC_Endogenous_and_Exogenous_Pathways.png HLA-2 expression: exogenous pathway HLA-2 proteins bind extracellular antigens via the exogenous pathway (the basics) ▪ Phagocytosis needs to be upregulated in concurrence with HLA-2 expression Phagocytosis is the source of the peptides that are “loaded onto the HLA-2 ▪ Phagocytosis can occur through the “regular” pathway The phagocytosed antigen is processed in and phagosome and then merges with a vesicle containing the HLA-2 The antigen is loaded onto the HLA-2 and expressed on the surface of the cell Retrieved from: https://upload.wikimedia.org/wikipedia/commons/8/8b/MHC_Endogenous_and_Exo genous_Pathways.png HLA-2 expression: exogenous pathway HLA-2 proteins bind extracellular antigens via the exogenous pathway (the details) ▪ Phagocytosis by B-cells can be antibody-mediated Antigen binds to specific antibody on the surface of the B-cell (aka B-cell receptor) Antigen and the antibody are phagocytosed together (process is called receptor-mediated endocytosis) The antibody is the recycled back to the surface of the cell and the antigen is processed in an endosome. HLA-2 expression: exogenous pathway HLA-2 proteins bind extracellular antigens via the exogenous pathway (the details) ▪ How do we ensure an cytosolic antigen isn’t loaded onto an HLA-2 (rather than an HLA-1) in the RER? In the RER, the HLA-2 protein associates with the invariant chain (FYI - aka CD74) ▪ This prevents a cytosolic antigen from being loaded onto the HLA-2 while in the RER HLA-2 expression: exogenous pathway HLA-2 proteins bind extracellular antigens via the exogenous pathway (the details) ▪ How do we ensure an cytosolic antigen isn’t loaded onto an HLA-2 (rather than an HLA-1) in the RER? As the HLA-2 containing vesicle merges with the phagosome/ endosome containing the antigen, the invariant chain is chopped up ▪ The “chopped version” is called CLIP, and it will remain bound to the HLA-2 peptide binding region until displaced HLA-2 expression: exogenous pathway HLA-2 proteins bind extracellular antigens via the exogenous pathway (the details) ▪ How do we ensure an cytosolic antigen isn’t loaded onto an HLA-2 (rather than an HLA-1) in the RER? When a peptide binds with sufficient affinity to HLA-2, CLIP is displaced HLA-2 expression: exogenous pathway HLA-2 proteins bind extracellular antigens via the exogenous pathway (the details) ▪ How do we ensure an cytosolic antigen isn’t loaded onto an HLA-2 (rather than an HLA-1) in the RER? When a peptide binds with sufficient affinity to HLA-2, CLIP is displaced HLA-2 with bound extracellular antigen is then expressed on the surface of the cell HLA-2 expression: exogenous pathway HLA-2 proteins bind extracellular antigens via the exogenous pathway (the details) ▪ How do we ensure an cytosolic antigen isn’t loaded onto an HLA-2 (rather than an HLA-1) in the RER? FYI - HLA-DM and HLA-DO are two HLA-like molecules that associated with the HLA-2 and either help or inhibit the loading of the antigen onto the HLA-2 HLA-DM helps with loading onto an HLA-2 How do you keep antigens separate? In summary: ▪ The mode of antigen entry into cells and the site of antigen processing determine whether antigenic peptides associate with: HLA class I molecules in the rough endoplasmic reticulum HLA class II molecules in endocytic compartments Under certain circumstances however, exogenous antigens may be assembled with MHC class I molecules via cross- presentation and vice versa. ▪ Let’s take a look at this briefly Antigen-processing exceptions Exogenous antigens can be presented by HLA-1, and endogenous antigens can be presented by HLA-2 in some circumstances: ▪ 1. An infected cell dies and is phagocytosed Viral particles in the cytosol of the infected cell will be presented on HLA-2 of the phagocyte (eg. Macrophage) ▪ Thus an “intracellular” antigens becomes extracellular antigens Autophagy can also results in peptides from the cytosol being presented on HLA-2  Antigen-processing exceptions Exogenous antigens can be presented by HLA-1, and endogenous antigens can be presented by HLA-2: ▪ 2. Cross-presentation – not fully understood Likely a blend of the exogenous and endogenous pathways ▪ Dendritic cells are best at cross presentation, but all antigen presenting cells can do it. ▪ Antigens can leak out of the endosome after phagocytosis into the cytosol, then the antigen can be presented to HLA-1 Retrieved from: https://upload.wikimedia.org/wikipedia/comm ons/1/1a/APC_Cross-Presentation.png Antigen-processing exceptions Exogenous antigens can be presented by HLA-1, and endogenous antigens can be presented by HLA-2 ▪ 2. Cross-presentation continued Allows antigen presenting cells to either sequentially or simultaneously activate both T- helper and cytotoxic T cells HLA-2 – What happens next Once a peptide-bound HLA-2 is expressed on the surface of a cell, what happens next? ▪ It can bind a CD4+ T-cells, ie. a helper T-cell, and activate it. Let’s take a closer look! T-cell activation An antigen presenting cell is “presenting” an antigen via HLA-2 on its surface ▪ Helper T-cells have randomly-reorganized T-cell receptors that are highly specific for a particular peptide However, remember, they will only recognize the antigen in the context of an HLA-2 molecule They also need a co-receptor to bind to the HLA-2 molecule ▪ This co-receptor is known as CD4 It is the major CD that distinguishes a Th from a cytotoxic (Tc) T-cell What was the co-receptor for a cytotoxic T cell? Finally, they also need co-stimulatory activation ▪ A key co-stimulatory interaction is CD28 (on T-cell) with CD80/86 on the APC. T-cell activation 1a. T-cell receptor interacting with HLA-2 TCR HLA-2 CD4 CD28 CD80 2. Co-stimulatory interaction: CD28 1b. CD4 co-receptor interacting with CD80 interacting with HLA-2 (or 86) More on co-stimulators Although most T cells express CD28, most cells in the body do not express its ligands (CD80 or 86) ▪ Only professional APCs have the capacity to express CD80/86. Mature dendritic cells, the best activator of naïve T cells, appear to constitutively express CD80/86 Macrophages and B cells have the capacity to up-regulate CD80/86 after they are activated by an encounter with pathogen More on co-stimulators How does CD28 signaling add to the effects of TCR signaling described previously? ▪ 1. Enhances the strength of signal between the T-cell and the Antigen presenting cell ▪ Initiates a cell-signaling cascade to promote T cell survival and proliferation CD28 will recruit PI3 Kinase T-cell activation: A closer look The interactions between the T-cell and the antigen presenting cell can be divided into two categories: ▪ cSMAC (central Supramolecular Activation Complex) TCR & HLA-2 (with co-activator CD4) Co-stimulator interaction (CD28 & CD80 or 86) ▪ pSMAC (peripheral Supramolecular Activation Complex) Includes other receptor-ligand interactions that help to strength the connection between the T-cell and APC to sustain the signal ▪ FYI – eg. LFA-1 & ICAM-1 T cell activation - What happens next Once the T-cell Receptor (TCR) interacts with the antigen bound to HLA-2 (With the help of it’s CD4 co- receptor & costimulatory interactions) ▪ Initiates cell signaling cascades to promote T-cell function, survival, and proliferation Note: activation of PLC cascade & Ras cascade Details of image FYI – for visualization only T cell activation - What happens next In addition to TCR and HLA-2 (with CD4+ co-receptor) interactions and co-stimulatory interactions, there will also be paracrine signalling between the antigen presenting cell and the T-cell ▪ These signals will help polarize Naïve Th cell the helper T-cells Polarization depends on: HLA-2 ▪ A) the type of APC interacting with the T- helper cell ▪ B) the cytokines present during the time of activation Stay tuned – this is covered next class! HLA types summary HLA-1 HLA-2 Cell types expressing HLA class Antigen type (intracellular or extracellular) Endogenous or exogenous pathway of presentation? - Describe the basic steps Activates which type of T-cell (name the co-receptor) References Punt et al. Kuby Immunology. WH Freeman and Company Parham P. The Immune System. Garland Science. Images ▪ https://upload.wikimedia.org/wikipedia/commons/1/1a/APC_Cross- Presentation.png ▪ https://upload.wikimedia.org/wikipedia/commons/8/8b/MHC_Endogenous_an d_Exogenous_Pathways.png ▪ Alberts et al. Molecular Biology of the Cell. Garland science Study Guiding Questions Build a table comparing the endogenous antigen presentation pathway to exogenous antigen presentation pathway. ▪ In your table, include: HLA types Antigen type (intracellular or extracellular) Description of the steps of the pathway ▪ When is the antigen loaded onto the HLA protein? Describe how T-cells are activated: ▪ List all the interactions needed to activate a T-cell ▪ How are cytotoxic T cells activated? Which type of HLA is used to present the antigen? What types of cells might present the HLA bound antigen? ▪ How are helper-T cells activated? Which type of HLA is used to present the antigen? What types of cells might present the HLA bound antigen? Extra Review slides – Won’t be tested PI3 Kinase pathway AKT Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-64. Pg 1017 Extra Review slides – Won’t be tested Ras-MAP Kinase pathway Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 15-49. Pg 856 Extra Review slides – Won’t be tested PLC pathway Can activate or inhibit transcription factors Can activate or inhibit co-regulators Can activate enzymes travel to the nucleus and that activate transcription factors