Cytokine Families: Complement Pathways: Classical Pathway -Initiated by IgG: can also be IgM, but mainly IgG. -Two IgG antibodies interact at a particular distance on the surface of a pathogen by their heads, while their tails interact with C1 complex, activating it -The activated C1 complex cleaves C4 into C4b and C4a. -Sticky C4b attaches to the pathogenic membrane and cleaves C2 into C2a (sticky) and C2b (small, not sticky). Then, sticky C4b binds sticky C2a forming C4b2a called (C3 convertase) -C3 convertase cleaves C3 into C3b and C3a (pro-inflammatory). -C3b sticks to the pathogen as an opsonin to signal for it to be phagocytosed. -Some C3b will stick to C4b2a making C4b2a3b named (C5 convertase) -C5 convertase cleaves C5 into C5b and C5a (pro-inflammatory). -C5b does not act like opsonin. It instead gets stuck to the plasma membrane and recruits other complements. Lectin Pathway: -Mannose-binding lectin (MBL) is used instead of C1 complex and IgG’s. -Mannose binding lectin binds to carbohydrates on the surface of the pathogen (mannose). -In order to activate the complement molecules, Mannose-binding lectin also binds serine proteases called MASPs (MBL-associated serine proteases). -Then, you get cleavage of C4, C2 (by MASPS) followed by the rest of the pathway (cleavage of C3). Alternative (Tickover) Pathway: -C3 spontaneously interacts with water (the thioester bond is unstable in water) -C3(H2O) are bound -Recruit Factor B (which is a complement) making C3(H2O)B which recruits Factor D. -Factor B is cleaved by Factor D to Bb and Ba, Bb binds to C3(H2O) to make C3(H2O)Bb which is fluid phase C3 convertase -Fluid phase C3 convertase cleaves C3. -The C3 that is cleaved doesn’t really do anything unless there are pathogens around. It can stick to water and just get flushed out. -If there are pathogens around, then C3b binds to their membrane as an opsonin. -C3b can bind to factor B that is cleaved by Factor D into Bb and Ba à C3bBb which is a membrane bound C3 convertase. -The C3bBb mentioned above in the alternative pathway is a convertase that is unstable without properdin. -Properdin stabilization of this convertase leads to lots of C3 cleavage. -It can cleave and bind C3b, making C3bBbC3b (C5 Convertase) -This process is most likely to occur around pathogen surfaces because the microenvironment around a pathogen is different from that of a mammalian cell. MAC Formation: -As you can see, all three pathways lead to C5 convertase which cleaves C5 into C5b and C5a -C5b (stuck to membrane of pathogen) recruits C6 à C7 à C8 à C9 à C9 Polymerizes to form a membrane attack complex à punches holes in pathogen membrane. Leukocyte Receptors: B Cell Receptors: -B-cell receptors recognize tertiary and quaternary protein shapes. -Receptors are Immunoglobulins -They have 2 identical light chains and 2 identical heavy chains. -The hinge region can be seen between the tips and the tail. This is the location at which these receptors can be separated. We use this property to study different parts of these receptors in a lab. -They have 2 binding sites that bind identical epitopes. -There are about 1012 different antigen binding sites. -While the tips of the antigens bind epitopes, the tail region is the effector because it is the part that actually passes down signals. The effector region only consists of the heavy chain. -There are only 5 variant tails/effectors. Due to 5 different heavy chains. -Below are the 5 different isotypes that you can see for immunoglobulins. They have different heavy chains. -IgG (g), IgD (d) and IgE (e) are monomers. -IgA (a) is a dimer (rarely found as monomer). -IgM (µ) (which is most abundant) is a pentamer. -Depending on the isotype, you will have a different heavy chain which means you have a different effector region and you will therefore trigger a different effect. -Alternative splicing can determine whether these immunoglobulin receptors are going to be in their secreted-form (free antibodies) or in their membrane-bound form. T Cell Receptors: -T-Cell receptors recognize 8-10 amino acid antigen segments presented by MHC receptors. - There are no soluble T-cell receptors like free antibodies of B-cells. All T-cells are membrane bound. -There are 2 types of T-cell receptors: αβ and γδ -These receptors also have V and C regions. Co-Receptors: -Co-receptors actually bind (contact) the cell receptors -They help T-cells recognize MHC I and II receptors. -Co-receptor 4 (CD4) help T-helper cells recognize MHC II receptors. -Co-receptor 8 (CD8) help T-killer cells recognize MHC I receptors. -CD21 interacts with B-cell receptors, it has a complement receptor: recall that complements bind antigens and now these can be recognized by co-receptors such as CD21. -Now you have the co-receptor recognizing the antigen bound complements and the actual Bcell receptor recognizing epitopes. This makes the b-cells more likely to bind an antigen. MHC: InterLeukin-1 (IL-1) - Promoters of inflammation - Part of the innate immune system - IL1 binds to target cell surface receptorà activation of MyD88 adaptor protein in cytosolic side à activation of IRAK (IL-1 Receptor Activated Kinases) à activation of TAK1 (TGFβ-associated Kinase 1)à Activation of AP-1 and NF-κBà transcription of cytokine. SEE TLR cascades in week 3 not -IL-1 initiate cascades that lead to the release of more cytokines & a resultant inflammation. Hematopoietin (Class I): -Cytokines in this category share common structural motifs (four helix bundle). -They’re organised into subfamilies based on the type of receptor they bind to. The receptors include: -γ chain bearing receptor -β chain bearing receptor -GP130 receptor -Signalling similar to interferons. -This receptor is critically important. -Dimers and trimers expressed with gp130 subunit. -The more hematopoietic receptors interact with each other, the higher the affinity they will have for the cytokine Interferon (Class II): Type 1: most important. -Small: 18-20 kDa dimers -Antiviral effects -There are α (large family of 20) and β interferons. Type 2: T & Natural Killer cells produce Type 2 interferon dimers. -γ interferons Type 3: not important for this course. Tumor Necrosis Factor (TNF): -Membrane bound cytokines can be found in this category (as well as soluble cytokines). -Not fully understood/defined yet. -These cytokines either lead to cell proliferation or death. Interleukin 17 (IL-17) -These are also pro-inflammatory like the IL-1 category. -They bind more complicated receptors (various arrangements of 5 protein chains). -The binding of IL-17 to receptors induces a cascade that results in the release of more cytokines and recruitment of leukocytes, ultimately leading to inflammation. -You ultimately get the release of NF-κB. Chemokines: -Specialized cytokines involved in directing leukocyte migration. -Recall: leukocytes (white blood cells) are mobile (i.e. neutrophils), so their movement needs to be directed to the location of infection/need. -Small: 7.5-12.5 kDa. -There are six categories of chemokines -They are categorized based on their disulfide bonds. -Chemokine binds to GPCR à G-protein exchanges GDP for GTP (gets activated) à activates various pathways. MHC Class I Presents antigens to Cytotoxic/Killer T-cells All cells in the body that have a nucleus (therefore carry the gene), carry MHC class I This class presents intracellular antigens to T-killer cells. As you know, this is most likely viral infections. But it can be other intracellular infections as well. -It makes sense that all cells carry MHC class I because every cell in our body is susceptible to infection, so they are prepared. -When they present the antigen, they are calling for their death (signal for suicide). -As you can see, MHC I has one α chain (left) and one β-2-microglobulin(right) -The peptide binding groove is at the top (it is seen in both classes of MHC). -This class can bind antigen pieces that are 8-10 amino acids in length. (Very restricted groove size). -Since being a heterozygote is so beneficial, it keeps the polymorphism of this locus very high over time because it is evolutionarily selected for. -Heterozygotes are more likely to survive in an ever changing environment of pathogens than homozygotes. -Antigens get processed into small pieces and then bind to MHC where they can be presented to T-cells. Since this class deals with intracellular antigens, it follows the endogenous pathway to present the antigen to T-killer cells. -The intracellular antigen gets processed by proteasome inside the cell. Keep in mind that proteasomes can change to immunoproteasomes (mostly found in professional antigen presenting cells pAPC) which chop of antigens to a size and sequence best suited to bind MHC. However, a regular proteasome gets the job done too. -Next, the chopped up antigens enter the RER (where MHC I can be found) through TAP proteins. MHC I can always be found close to TAP. -Then a variety of chaperone proteins help the antigen fragments securely bind MHC class I. -Once the antigen fragment binds the peptide groove securely, MHC I is transported to the golgi via a vesicle and then to the plasma membrane where it can be displayed to present the antigen to a T-killer cell. MHC Class II: -Presents extracellular antigen peptides to Helper T-cells -The only cells that have MHC II are professional antigen presenting cells such as Dendritic cells, Macrophages and B-cells -The class II MHC has one α and one β chain that interact to form the peptide binding groove. -This class can bind antigens of any size up to a maximum 15 amino acids long. -As you know by now, Antigens get processed into small pieces and then bind to MHC where they can be presented to T-cells. Since this class deals with extracellular antigens, it follows the exogenous pathway to present the antigen to T-helper cells. -MHC class II molecule is synthesized in the RER and gets transported via a vesicle to the golgi and then goes on its way to the plasma membrane -Meanwhile, an exogenous antigen enters the cell via endocytosis/phagocytosis -The figure above shows that the endocytosis is receptor mediated (it is a B cell receptor in the figure) -The antigen comes in a vesicle that fuses with a lysosome where it gets processed into pieces. -The lysosome containing the antigen bits fuses with the vesicle containing MHC II and the antigens bind MHC II peptide-binding groove. -The vesicle is then taken to the plasma membrane where the MHC II can display the antigen to a T-helper cell. -Remember, MHC class II is also found in RER, but the antigens do not bind to MHC class II because it is basically blocked/plugged by invariant chain. -As shown in the figure above, the invariant chain is synthesized with MHC class II, and remains plugged until it is leaving the golgi towards the plasma membrane where the invariant chain gets chopped and the CLIP region remains tight in the peptide groove. The incoming antigen needs to have very high affinity for MHC class II to outcompete CLIP. That way, only peptides that fit very tightly in the groove will end up binding MHC. -Majority of the time you will find normal human proteins in the MHC class II groove. VDJ Recombination: -The first step of the process is having RAG-1/2 Recombinase enzyme nick DNA at a Recombination Signal Sequence (RSS). -One type of RSS has a Heptamer-23bp spacer-Nonamer -Another type of RSS has a Nonamer-12bp spacer-Heptamer -The nick is single stranded -The 3’OH group finishes the nick as a double stranded break, providing us with a hairpin loop -The signalling ends that aren’t hairpin looped actually find each other and bind each other, forming the signal joint. -We break the hairpin loop with the artemis protein, which nicks one strand in the loop which can lead to either overhangs or blunt ends depending on where it cuts. -If we do get overhangs, we use DNA repair enzymes to fill them in and get blunt ends. -The two blunt ends (one of V and one of J) can now ligate together (In the case of heavy chains it would be D and J first then V.) -In heavy chains TdT lymphoid-specific proteins add an additional means of variation. -When artemis protein cleaves the hairpin loop, you can get so nucleotides exonucleated out, or you can get TdT adding random non templated nucleotides to the ends, extending the DNA sequence. Lymphoid Generation: -The first stage is that HSC is a stem cell that is multipotent -So the multipotent nature of HSC allows it to turn into any red blood cell. -At this stage, the stem cells can even self-renew. -The second stage of the cell is referred to as Multipotent Progenitor (MPP) -These cells have lost the ability to self-renew, but they are still multipotent. -MPP’s express CXCR4 which is what keeps them in the bone marrow. -The third stage is Lymphoid-primed multipotent progenitor cell (LMPP) -Remember lymphoid specific proteins such as RAG-1/2 and TdT? Well, they start being expressed in this stage. That’s why these cells are called “Lymphoid primed” because the expression of these proteins makes them more likely to eventually become lymphocytes. -They also express IL-7R receptors and EBF1 which are B-cell specific transcription factors. This leans the cell more towards becoming a Blymphocyte. -Though they are primed to be lymphoid cells, they can still take the route of becoming innate Myeloid cells. -Then we get Early Lymphoid Progenitor Cells (ELP) -We get more RAG-1/2 expressed here -Stem cell factors decrease -Such as c-kit and sca-1 -Some of these ELP’s exit the bone marrow and enter the thymus (T cell precursors) among other cells. -ELP cells still have the ability to become myeloid cells. -Finally, cells that remained in the bone marrow, can now become Common Lymphoid Precursor (CLP) -These can no longer become myeloid cells. -At this stage we can either become B-cells, T-cells or Natural killer cells -This makes sense because myeloid cells are all innate; we still have the ability to become any adaptive immune cell. -In ELP we said some cells leave for the thymus and could possibly become T-cells. But this doesn’t mean we can’t still produce T-cells at the CLP stage either. We can send them off to the thymus later. -The Ig and T-cell receptor chromatin is becoming more accessible to machinery required for recombination. T Cell Development: -When T cells are immature, we refer to them as thymocytes. Notch is specific to this T-cell lineage (like B220 is to B cell lineage) -We first enter the double negative (DN) stage. -During this stage, thymocytes perform V(D)J recombination and synthesize T-cell receptors. -Recall that this leaves them with either a αβ or δλ T cell. -The heavy (β) chain undergoes recombination first -Check if it folds properly via surrogate light chains (β-selection) -The surrogate light chain is Pre-Tα, Surrogate + Heavy chain = Pre-TCR -If it makes it to the plasma membrane ensuring that the heavy chain works, we start to get proliferation. Proliferation leads to a stop in making heavy chains because Pre-TCR downregulates RAG-1/2. Only very little expression of RAG remains. -This also induces the expression of CD8 and CD4 -Light chain recombination (α) begins. -Once our thymocytes have T cell receptors, they begin associating with both co-receptors CD8 and CD4. We call this the double positive (DP) stage. -They go through selection in the thymus (positive or negative), about 95%of these DP T cells actually fail positive selection. This means they do not receive survival signals and die off (Death by neglect) -If the DP T cells in the thymus are just binding things and not inducing signals, it means they are recognizing self-cells so they die (negative selection). -The affinity of the DP thymocytes to MHC are crucial to positive selection. Only when a DP thymocyte binds MHC with lowintermediate affinity, they pass positive selection. -Presenting cells that come into the thymus carrying different peptides from our different organs. And they are used to check if the thymocytes bind these sequences or not since not all tissue types are in the thymus. -This is all done in the medulla of the thymus -AIRE transcription factors in the APC allow it to express peptides of different organs at low levels. -Once they pass everything, they go on to become specifically CD8 or CD4 cells. -They can also go on to becoming other T cells such as TH¬17 and T regulatory cells. -T-regulatory cells are a type of T-helper cell that express FoxP3 transcription factor only found here. FoxP3 allows T-regulatory cells to: Secrete cytokines, Communicate with APCs, Directly kill other T-cells, and remove cytokines from environment. Lymph Node Chemokines: -B cells go to the follicle section of the lymph node. -The Follicular DC cells in the follicles release CXCL13 chemokines to signal naïve B cells to their location. -The B cells are CXCR5+ -T cells go to the paracortex section of the lymph node. -FRCs in the paracortex release CCL21 and CCL19 to attract naïve T cells to the site. -The T-cells are CCR7+ B Cell Negative Regulation: -There’s a receptor called Fc that is found on naïve B cells. Fc decreases the likelihood of B cell activation when it binds ligand. -A particular Fc called FcγRIIb has a IgG ligand binding site -IgG are antibodies, meaning that can be free-floating and independently bind antigens. If IgG-bound antigens come across a naïve B cell, the Fc receptor will bind the IgG and inhibit the activation of that B cell receptor. -This is beneficial because if an antigen has an IgG bound to it, it means there is already an immune response triggered to get rid of that antigen/pathogen. So we don’t need an excess response from B cells. T Cell Activation: -Need Antigen + Costimulatory -The costimulatory molecule found on the T cell is called CD28 binds with the costimulatory molecule of the antigen presenting cell (dendritic cell) called CD80 or CD86 (B7-Complex). -Co receptors such as CD3 also bind -Once these have bound, you get IL-2 secretion, inducing proliferation. -Also have negative costimulatories, like CTLA-4 -appears 24 hours post-activation -bind CD80/86 and have a higher affinity for it than CD28. This means that it prevents any more CD28 binding CD80/86 resulting in no more activation -CD8+ T-killer cells require more costimulatory response than T-helper cells did. Done via T-helper cells, TH1 or TH17 cells signal dendritic cells to produce more co-stimulatory receptors by releasing IL-2. In this state, we refer to the dendritic cells as Licensed APCs. Results in 2 CD8 subsets: -TC1: Secrete IFN-γ. induce death via perforin or Fas -TC2: Made when there is IL-4 present, secretes IL-4 and IL-5. Induce death only via perforin B Cell Activation: -B cells can either be activated with the help of T-cells (T-cell dependent) or without the help of T-cells (T-cell independent). -We get membrane spreading to cluster (oligimerize) the receptors -Because B-cells are also categorized as professional antigen presenting cells we do endocytosis, chop up the antigen further, and present it on our surface via MHC. -Once the B cell is presenting the antigen, they go to the T-cell zone. They actually start expressing CCR7 which is what gets attracted to the CCL19 and CCL21. -B cells can go to the borders of the T-cell areas and from primary foci and differentiate into plasma cells (antibody producing cells) (these are the Bcells that don’t go back to the follicle/germinal centre). -They don’t really have surface Ig’s. They secrete Igs (usually in the form of IgM) -ALSO you will see somatic hypermutation. This gradually decreases the affinity of the receptors and so it slows down and eventually stops immune response over time. -The B-cells that do go to the follicle actually require more help from Tcells. Follicle is now germinal centre. -At the germinal centre, we start to see class switching (IgM/IgD change to other isotypes). -AID (activation induced cytidine deaminase) is now being expressed in the germinal centre. This is what mediates somatic hyper mutation (SHM) and class switch recombination. B Cell Development: -Before proB cells we have Pre-Pro B cells. -This is mostly in the LMPP stage where we are getting expression of EBF1 (which was the b-cell specific transcription factor) as well as the expression of RAG-1/2 and TdT. -What really makes the pre-pro b cells lean toward becoming a B cell and not a T cell or Natural Killer cell is the expression of B220 which is a marker specific to B cell lineage. Also we get more expression of EBF1 in b-cell precursor. -We also get expression of the IL-7R receptor, which also helps it become a B-cell because the activation of this receptor enhances production of EBF1. -So why is EBF1 so important for becoming a B-cell? Along with E2A, EBF1 binds to the heavy chain gene locus on DNA and opens it up so that it can make the D and J loci accessible for recombination. -After pre-pro B cell stage you have Early Pro-B cells followed by Late Pro-B cells: -Both Pro-B stages express PAX5 (helps contract the gene locus to bring the V together with the DJ.) -Early Pro-B stage is when D and J come together. -Late Pro-B stage is when V and DJ come together. -1st Checkpoint : Now that we have our heavy chains, we use a surrogate light chain (VPre-B + λ 5) to check if everything fits together, forming the Pre-BCR. -Now the pre-BCR cell enters a proliferative state. At this proliferative stage, we call the cells Large Pre-B cells -Now that the pre-BCR cells no longer divide due to the downregulation, we call it Small Pre-B cells. You no longer inhibit RAG-1/2 so now you can use it for making light chains. -So once you have a proper light chain and it binds to your heavy chain and makes it to the cell surface successfully, you have an IgM (immature B cell). This is the second checkpoint because we have to make sure the light chain works and when it does we get allelic exclusion of all other light chain alleles. Hypersensitivities: Type I Hypersensitivity: -IgE antibodies usually cause type 1 hypersensitivities. -IgE can be produced in excess, and the excess can bind to mast cells. -Mast cells have both FcγRIIB (inhibiting) Ig receptors and FcεR1 (activating) Ig receptors. -When IgE binds FcεR1receptors on mast cells, an allergen can bind to the IgE bound to the mast cell. This activates the receptor and triggers the mast cell to undergo degranulation. The granules are pro-inflammatory such as histamine. These will cause the allergy symptoms. -Allergy shots try to switch the B cells that are activated that produce the IgE to switch to producing IgG so that we stop this reaction. IgG binds to the inhibitory receptor FcγRIIB Type II Hypersensitivities: -IgG or IgM cause these hypersensitivities. -ABO blood types all have the same galactose sugar group but different antigens on our blood cells. -We are all born with antibodies to the other blood groups. The introduction of any blood type different from yours will be seen as foreign antigen. This triggers hyperacute rejection. -Pregnancy issues with mismatched blood between mother and fetus: -In addition to ABO blood groups, we can either be Rh (+) or (-). -A pregnant mother can be Rh (-) while her fetus is Rh (+). During delivery, fetal blood does come into contact with the mother’s blood. This introduces Rh to the mother and the mother undergoes an immune response and creates Rh memory cells. -During any subsequent pregnancies, another Rh+ baby can be in great danger. The mother has memory cells to attack Rh this time. So the mother’s IgG anti-Rh from memory cells attacks the fetus’ red blood cells and causes erythroblastosis fetalis. This puts the baby in danger to survive. Type III Hypersensitivities: -Soluble antibody mediated hypersensitivity. -Associates with IgG mainly. IgG is involved with the complement system. -When you launch too much complement, it can collect in the blood vessels and trigger damage to the vessels as well as kidneys, etc. Type IV Hypersensitivities -This is T-cell mediated hypersensitivity and it is delayed type hypersensitivity (DTH). Here, we see Inflammation 24-72 hours later -For example, when you touch poison ivy, you get the red rash the next day (delayed). -The effects may be caused by other cells such as macrophages, which is why it takes so long. -There is a sensitization phase and an effector phase. In the sensitization phase you have antigen (from the allergen) being presented to helper T cells on MHC II. T helper cells get activated. Effector phase is when activated T-cells release cytokines, etc. they cause some effect. This effector phase of DTH is similar to acute stage of transplant rejection. Transplant Rejection: Hyperacute Stage: -Happens within minutes to hours due to pre-existing antigens to ABO blood groups &/or donor MHC. Acute Stage: -Week to months post-transplant -Detection of foreign MHC (major histocompatibility complex). -T-helper cell activation à activates t-killer cells, macrophages, Nk cells, etc à attack transplant. -During the Hyperacute stage, T-helper cells are being activated. Then, a week+ into the organ transplant, we actually get the t-helper cells starting to cause an effect. This marks the acute stage. -We are starting to recognize foreign MHC. When you get a transplant, it is impossible not to get at least a few blood cells from the donor enter the patient. Among these cells can be antigen presenting cells which have MHC. The difference in acute vs Hyperacute for recognizing MHC is that in Hyperacute we have pre-existing antibodies for the foreign MHC. But when we get a transplant, even if we didn’t have pre-existing antibodies, our bodies do recognize that the MHC is foreign so we begin a response. And in a week or more, you will start to see your adaptive immune system activated which is the acute stage. -Antibody activation is very rare here. It is T-cell dominated not B cell. Chronic Stage: -Months to years post-transplant -Detection of minor histone-compatibility antigens (any protein that is a polymorph to patient’s own protein) -Activation of humoral response (lots of antibodies) -Minor histocompatibility antigens are due to normal proteins that are in themselves polymorphic in a given population. Even when a transplant donor and recipient are identical with respect to their major histocompatibility complex genes, the amino acid differences in minor proteins can cause the grafted tissue to be slowly rejected.