Wayne State University Dendritic Cells and APC Immunology Questions

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READ THIS FIRST! Please answer the following questions thoroughly. Make sure you answer all parts of the question completely, providing all of the information that is asked for. Provide sufficient detail so that someone who is not your course instructor could understand what you are talking about. If you use any abbreviations (for example, PAMP), please define what they are. You have to demonstrate to me that you know what you are talking about; I am not going to guess. You can use course notes, and any papers that might be online with their source. There is no maximum or minimum length for your answers, but they should be complete, and as concise as possible. Please don’t include information that is not relevant to the question asked.

Question 1: You have developed a strain of mice that are incapable of producing Follicular Dendritic Cells (FDCs). (This is fictional-I have no idea whether or not such a thing exists).

a) What do follicular Dendritic cells do in our immune system? (Your answer needs to include specific answers to: Where are they found? What is their role in that location?

b) Which immune tissues or organs would be affected by this mutation? What would they look like (e.g. what structures would be missing or altered) Be sure to consider all types of immune tissue.

c. What would be the effect of this mutation on the immune system of the mice? Your answer should include whether or not the mice would be immunosuppressed, and a thorough explanation of why or why not, including what cells/functions of the immune system would be altered.

Question 2: You have another mouse strain that is homozygous for HLA-A and HLA-C. It also has a homozygous inactivating mutation in the HLA-B genes.

a) What are the HLA-A, B, and C genes and what do they encode? Are they part of a bigger gene locus, and, if so, what is in the rest of the locus?

b) The haplotype of this mouse shows that the rest of the alleles in this locus are also homozygous. Could this result in any impairment of the mouse’s immune system? Explain your answer, making sure you explain why the HLA genes are usually polygenic and polymorphic.

c) This same mouse strain carries a homozygous inactivating mutation in the gene encoding the TAP transporter. Will this have any effect on the mouse’s immune system? In your answer, describe the function of TAP, and explain (and describe) what functions are impaired because of its loss. Then explain why this would or would not have any effect on the immune system (including of course, which aspects of the immune system would be impaired).

Question 3: Describe the antibody problem. In your answer, you should describe the basic structure of antibodies (secreted is fine) and how this posed a problem when scientists tried to relate structure to the necessary function of antibodies. Without going into detail about Ig genes, describe the solution to the antibody problem.

Question 4) Part 1: You have been jabbed with a sharp pointy stick, and through this jab, are infected with: 1) a dsRNA virus that can infect many cell types including dendritic cells; 2) a type of intracellular bacteria that infects macrophages/dendritic cells; and 3) Streptococcus covinia (an imaginary extracellular bacteria that secrets a toxin). You had never been jabbed with these particular pathogens before, and you were not infected with a lot of any of the pathogens, so you are not sick because your innate response kicked in, and has been busy working at the site of the jab. Thus, we can move our attention to a nearby lymph node, which now contains the three pathogen types and the S. covinia toxin, as well as immune cells.

a. Which of the following are required for TH cell activation? (from the list below, highlight all that apply). Then write a brief description of TH cell activation making sure that your description contains all the highlighted items.

CD3 IL2

Iga B7

CD4 C5

CD8 CD40

granzymes CD28

Question 4, part 2: It is 1 month later, and unfortunately, another pointy stick jabs you with the same three pathogens. You now have plasma cells/antibodies and appropriate memory B cells, TH effector cells, as well as CTL cells, to respond to the entire infection.

b. You have antibodies against the toxin and against the three pathogen types. What are the antibodies going to do to help you from getting sick?

c. Some of the intracellular bacteria have succeeded in infecting some macrophages. How is your immune system going to try to remove this infection?

d. What parts of the adaptive response is going to rid your body of the virus? (Don’t describe the IFN a/b, other cytokine, and NK responses, even if they would help).

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We are in the lymph node, where our B cells, Dendritic Cells, and T cells are waiting. Remember, naïve B cells were recruited to the cortex, and form primary follicles with FDC. Naïve T (TH and TC) cells were recruited to the paracortex, where they associate with DC and FRC Any antigens/pathogens in the extracellular fluid were also carried with the flow of lymph to the node. Dendritic cells and macrophages secrete a chemokine called CCL18 to attract T cell to them. The T cell will use the TCR to scan MHC-Ag on the DC, to see if the DC is presenting an antigen fragment that has epitopes that the T cell can bind to. If there is binding between the TCR and MHCAg, cell adhesion molecules (the ICAMS in the picture below) will help hold the two cells together. T cell receptor (TCR) complex • • • • • The TCR complex contains the TCR, which binds the Antigen MHC, and additional peptides, which form a protein called CD3. CD3 is required because the cytoplasmic region of the TCR peptides are not long enough for signal transduction. CD3 fills the signal transduction role CD3 is really three heterodimer pairs of peptides. CD3 has long enough cytoplasmic tails for signal transduction The tails contain ITAM motif-there are amino acid sequences that recruit signaling kinases. The TCR/CD3 complex also is associated with either CD4 (TH) or CD8 (TC). These are co-receptors, that bind to MHC. The cytoplasmic tail of CD4/8 associates with the signaling intracellular domain of CD3. We will focus on activation of naïve TH cells for now. Interaction between the TH cells and the APC will send three signals to activate the naïve TH. The first signal, called Signal 1, is when the TCR/CD3 complex recognizes and binds MHC II-Ag. CD4 binds to MHC II. The signaling cascade is shown in a later slide. This signaling cascade starts the activation process. Signal 2 is caused by CD28 on the T cell binding to co-stimulator B7 on the APC. B7 is a dimer of B7.1 and B7.2. (B7.1 is also known as CD80. B7.2 is also known as CD86). This activates more intracellular signaling, and will lead to synthesis in the T cell of IL-2 and the IL2 receptor (among other things). IL-2 stimulates proliferation and survival. Signal 3 is caused by the cytokines secreted by the APC that tell the TH what type of effector cell it should become. Next we will go into each signal in more detail • Signal 1: • The ends of the TCR bind the Ag-MHC with high specificity. We will discuss the binding sites later in the course. If it is a TH cell, it binds MHC II. If it is a TC cell, it binds MHC I. • If there is binding between the TCR and MHC-Ag, cell adhesion molecules will help hold the two cells together. • The CD4 (for TH), or CD8 (for TC) coreceptor binds to MHC II (CD4) or I (CD8). This binding is outside the Ag binding areas. • When CD4/8 binds, it brings a kinase, called p56Lck close to the cytoplasmic tails of the CD3 peptides. p56Lck then phosphorylates the CD3 tails to start Signal 1. • • • • Signal 1, continued: Phosphorylation of CD3 tails by p56Lck creates a docking site for another protein kinase called ZAP-70. Once bound to the phosphorylated CD3 tails, ZAP-70 phosphorylates adaptor molecules that recruit components of several additional signaling paths. Paths include PLCg, Ca++, PKC, MAP kinase. . Pathway can activate transcription factors • The Ca++ release caused by Signal 1 stimulates transport of a transcription factor, called NFAT, into the nucleus, where it binds to specific promoters. • The genes that are activated by NFAT depend of whether or not Signal 2 happens. Signal 2 should happen at the same time as Signal 1. • Without Signal 2, NFAT transcribes genes, such as GRAIL (Gene Related to Anergy In Leukocytes) that will lead to a state called anergy. • What is ANERGY? Anergy is a state of non-responsiveness-the T cells can’t respond to activation signals, and so they just sit there and don’t do anything. They eventually die. • GRAIL disrupts the adhesion between T cells and the APC. (it marks adhesion proteins for degradation-its an E3 ubiquitin ligase, for those of you who like to remember Cell Biology) • Without Signal 2, TH cells are unresponsive. MHC-Ag Signal 1 Nature Reviews Immunology 14, 435–446 (2014) • Signal 2, the co-stimulatory signal, is caused by CD28 on T cells binding to B7 on APC. • B7 binding to CD28 activates additional kinases-including the MAP kinase pathway. This results in activation of additional transcription factors. • Instead NFAT causing cells to making GRAIL, and become anergic, when Signal 1 combines with Signal 2, the two paths converge to activate additional transcription factors, including NF-kB and AP-1, that work with NFAT to turn on the genes for IL-2 and the IL-2 receptor (IL-2R, also known as CD25). • If TCR-CD3-CD4/CD8 paths are activated without the CD28-B7 interaction, the T cells enter a state of ANERGY, which means they can’t proliferate, and are generally non-responsive. • Generation of anergic TH cells should help prevent autoimmunity. B7 is only on antigen presenting cells, and it is increased by signaling from PRRs. If there are no PAMPS, then. There is little B7. No T cell activation. Signal 2 B7 MHC-Ag Signal 1 Nature Reviews Immunology 14, 435–446 (2014) This picture shows some of the adhesion molecules that strengthen the connection between the T cell and APC. This is what GRAIL gets rid of. When both Signal 1 and Signal 2 take place, IL-2 is secreted by the T cell, and then the IL-2 binds to the Il-2R that was also made by the T cell. Consider this to be Signal 2a. TCR-MHC II/Ag, CD4-MHC II activate Signal 1, and CD28-B7 activates Signal 2. IL-2 binds to the IL-2 receptor (CD25). This starts a another signaling path that causes the T cells to enter the cell cycle and to proliferate and begin differentiation into effector cells or memory cells. In addition to inducing the T cells to proliferate, IL-2 is also a survival signal, and at this stage, if there is no IL-2, T cells die by apoptosis. What happens as result of Signals 1 and 2 Signal through a JAK-STAT pathway, interfaces with MAP Kinase (ERK) pathway, which starts the cell cycle, and the PDK-1 path, which affects apoptosis regulators to allow survival. Signal three is the differentiation signal. It is started when cytokines secreted by the APC or other nearby cells bind to specific cytokine receptors on to the T cell. In many cases, the APC secretes specific cytokines that work using the JAK-STAT signaling pathway. The STATs, which are specific for each cytokine receptor, turn on genes encoding key regulatory transcription factors (see next slide), which then turn on the genes specific to a type of TH effector cells, or memory cell. In many cases, the PRR on the APC that were activated by the antigen/PAMPS determine which cytokines are secreted for Signal 3. Signal 3 Cytokine for signal 3 from APC Key regulatory Transcription factor Genes that are turned on-the genes encode these cytokines that are secreted by the effector TH cells. The top row on the figure shows the cytokines that act as Signal 3. Note that some of the Signal 3 cytokines are inflammatory cytokines, which were made by the APC in response to TLR signaling. Naïve TH cells differentiate into a type of effector TH cells (e.g. TREG, TFH,TH17, TH1, and TH2). We will discuss these in later slides and also later in the course. Each effector TH cells is characterized by a distinct regulatory transcription factor, that helps turn on the genes needed for the cell to do its job. Among these gene are cytokines that are secreted by the TH effector cells-each type has its own distinct set of cytokines that it secretes. We will discuss these effector functions in detail a bit later in the course. Right now we need to talk more about Signal 3. O’Neill LA, Bryant CE, Doyle SLTherapeutic targeting of toll-like receptors for infectious and inflammatory diseases and cancer. Pharmacol Rev 61:177-197 This is to remind you of the TLR pathways. IL-12 is another inflammatory cytokine that is made when PAMPS bind to the TLRs. • • Signal 3 for differentiation of TH1 is the best understood. See the upper half of this somewhat confusing drawing. The APC (still in the lymph node, so this is likely a DC) secretes IL-12 for Signal 3, when it engages TH cell (if this is what the PRR/PAMP told it to do). • IL-12 causes a few TH cells to differentiate into TH1 cells. • The TH1 cells then secrete IFN-g, which stimulates the APC to secrete more IL-12, increasing production of TH1. IFN-g also inhibits formation of TH2 cells.( Other effector cytokines such as Il-2 and TNF-b are also secreted-we will talk about those later). • IFN-g can also come from NK cells (and CTL), in response to viral infection. The macrophages and DC near the NK cells will be exposed the IFN-g, and the APC also will go to the lymph node, this time secreting more IL-12. This helps make TH1 which will help get rid of the virallyinfected cells (by helping Tc become CTL) • Signal 3 for differentiation of TH2 (lower half of diagram): • IL-4 causes a few TH cells to differentiate into TH2. The TH2 cells then secrete more IL-4, which stimulates production of still more TH2, and suppresses production of TH1. Where does the IL-4 come from? Not necessarily the APC. Instead, other cells (basophils, eosinophils, or mast cells) can secrete this in response to “invasion” by an allergen or helminth (worm) infection. They also travel to the secondary lymph tissue. Note that there is cross-regulation, to make sure one type of CD4 cell types predominates Embedded question: What is signal two in TH activation and why is it necessary? TREG can be made in thymus, during the process that gives us naïve TH and TC cells. If TREG are made in the thymus, they are called nTREG, for natural TREG. We will discuss that later in the course However, naïve TH cells can be directed to differentiate into TREG cells as well. These are called induced TREG, or iTREG. This usually happens in the gut, in the MALTs, because TREG suppress our inflammatory immune response. TREG help prevent us from attacking our gut microbiome. Without TREG, the DC and macrophages in the MALT and extracellular tissue in the gut would be responding to the microbiome PAMPS and starting localized inflammatory responses all the time. One cytokine that can act as Signal 3 to make naïve TH differentiate into iTREG is TGF-b. When naïve TH cells are exposed to TGF- b, a transcription factor called FoxP3 is turned on, and FoxP3 turns on genes that TREG cells use to do their effector function job (which is to suppress the immune system, and we’ll talk bout effector functions later in the course). Signal 3 for Treg is TGF-b. In our gut, our microbiome (probiotic bacteria) send signals (discussed in next slide) to tell dendritic cells to make IL-10, TGF-b, and Retinoic Acid (RA). These act as a Signal 3 that tells naïve TH cells (called TH0 in this picture) to differentiate into iTREG. Pathogenic bacteria (not part of our microbiome) have PAMPS that tell the dendritic cells to secrete the Signal 3 cytokines that make naïve TH cells differentiate into TH1, TH2, and TH17 cells, which can all increase the inflammatory response. The TREG cells can leave the MALTs and go all around our body, helping to control our immune response. We will talk about that more later. If fed the correct stuff, like dietary fiber, the bacteria in the gut microbiome secrete things like small chain fatty acids (SCFA). SCFA inhibit TLR signaling by sentinel cells, to reduce recruitment of immune cells like neutrophils. This helps the good bacteria in our microbiome evade our immune system. SCFA also induce dendritic cells to make TGF-b, IL-10, and Retinoic acid (RA). These three cytokines ac as Signal 3 for iTreg, and also inhibit differentiation into TH1 and TH17 cells. Anti-inflammatory diets give our microbiome bacteria the right food to make SCFA. Unfortunately, the right food is more like green leafy vegetables and less like chocolate cake. In the MALT, the signal 3 cytokines that cause activated TH to differentiate into TREG can also come from other TREG cells. This means that TREG can convert activated naïve TH cells into iTREG cells. Since I am a nerd, this reminds me of the Borg, which is why that picture is below. Don’t stress about it if you don’t get it. Now we know how naïve TH (CD4+) cells are activated and how they differentiate into the effector cells (TH1, TH2, TH17,TFH, and TREG) (we didn’t discuss TH17 or TFH much, because we do have time limits). We will talk about what the effector cells do later in the course. Virus-infected DC This happens in secondary lymph tissue. How are naïve TC (CD8+) cells activated, and how do they differentiate into CTLs (cytotoxic T lymphocytes)? Il-2R Il-2 The simplest way is for a Dendritic cell to become infected by a virus at the site where the virus enters the body (say, in the lungs, where someone inhaled droplets containing SARS-CoV-2). We will come back to how DC got this infection in a couple of slides. The ribosomes in the infected DC will now start making viral proteins. Some of the viral proteins will be degraded by the Ub-proteasome system, and presented on MHC I using the endogenous antigen pathway. The DC will go to the nearest lymph node (or other secondary lymphoid tissue) and find Tc which, like TH, are in the paracortex. Virus-infected DC Once near the Tc, the infected dendritic cell and the Tc will interact (chemokines from DC attract Tc) Signal 1 is still MHC-1/Ag-TCR/CD3, and MHC I-CD8. Signal 2 is still B7-CD28. The two signals combined activate NFAT, AP1, and NFkB transcription factors to turn on the gene for IL-2 and IL-2 receptor. If you don’t have B7-CD28, only NFAT is turned on, and you don’t get IL-2, you get ANERGY. So far this is just like activation of TH cells. IL-2 is secreted by the Tc cells, and it binds to the IL-2 receptor, causing the Tc to start proliferating. Signal 3, which is not shown in the picture to the left, is IL-12. IL-12 comes from the infected DC, and is made when the viral PAMP activated a TLR . You can see the pathway if you go to the earlier slide that has all the TLR paths. The activated Tc differentiates into a CTL. We will talk about how CTLs work later in the course. This is what one hopes will happen. Sometimes, the Tc does not make enough IL-2 to drive proliferation and survival of the Tc. It depends on what the virus is, apparently. The problem seems to be insufficient B7 on the DC-so signal two is too low. In this case, the Tc needs help (cytokines) from a nearby TH CD4 cell. This is usually a TH1 cell. The CD4 cell is activated by the same DC as the Tc. This can happen because: 1) The DC can phagocytose the virus as well as be infected by the virus. This would allow the viral proteins to be processed by the exogenous pathway and presented on MHC II. 2) The crossover pathway of Ag presentation, which I told you to not worry about. You can go back and look at it if you want. TH cells will bind to the same APC as the Tc (but will bind to MHC II-Ag). They will bind whatever B7 is there. It takes less signal 2 to activate TH cells than Tc cells. Signal 2, in addition to turning on the IL-2 gene, will turn on expression of CD40L, which binds to CD40 on the APC. Binding to CD40 starts a signaling cascade that results in the APC making more B7. It also causes the APC to make 4-IBBL, which is a member of the TNF family and is another co-stimulator of Tc cells. It binds to 4-IBB on the Tc. The extra co-stimulation, will cause the Tc to also turn on the genes for IL-2 and IL-2R. This, combined with the extra IL-2 from the TH cell, drives Tc proliferation and survival. IL-12 from the APC will still be signal 3 to differentiate into CTL. It could also be signal 3 for the TH cells, and cause them to become TH1 cells. TH1 can also help activate Tc. DC-SIGN We have talked briefly about how viruses infect only certain cells because the bind to surface protein receptors that are only expressed on certain cell types. For example, SAS-CoV-2 infects cells that have the ACE2 receptor on their surface. So how can Dendritic cells be infected with viruses to present on MHC I? It turns out that DC have sticky proteins on their surface, called DC-SIGNs. Most viruses can bind to these and use this receptor to infect the DC. This allows DC to present most viral antigens on MHC I, so that CTL that are specific to that virus can form. The virus shown in this picture is HIV, and it also binds to other things. Don’t worry about that right now. Types of effector T cells We have finally made T cells that can DO SOMETHING!! We’ll talk about what they do to keep us healthy right after we activate some B cells. Dendritic cells and macrophages are sentinel cells. Detect pathogen type using TLR, NLR, RLR. Pick up antigen-process. Able to present on MHC II. Binding of PRR and activation of the signaling path causes the Dendritic cells to migrate, carried by the increased amount of fluid to the lymphatic system. They enter the lymph tissue (e.g. lymph node) and the combined action of signaling molecules from the TLR and from the lymph tissue cells causes expression of the genes needed to activate the TH cells (for example B7, called in this picture CD80 and CD86, and increased MHC II. INNATE ADAPTIVE Resident dendritic cells pick up (eat, phagocytosis) the pathogen/antigen, and migrate using the lymphatic system to a nearby lymph node. Some of the pathogen also migrates with the lymph on its own Lymph fluid is increased by exudate that comes from the innate response In the lymph node, the dendritic cell presents antigen to naïve TH cells to start the adaptive response B cells and resident DC or Macrophage can pick up free antigen Dendritic cells are one type of Antigen Presenting Cells or APC. APC’s “eat” antigens, and digest them in lysosomes, and present them on MHC II. Binding of PAMPs to PRRs on Dentritic cell surface increases expression of MHC II. It also increases expression of a cell surface protein dimer called B7 (aka CD80, CD86). This is part of the signal complex that activates TH cells. It binds to a protein called CD28 on the T cells. More detail in the future. The PRR binding also causes the DC to secrete cytokines that help direct TH cell differentiation into the different types of TH effector cells (TH1, TH2, TFH, TH17, Treg. All this happens in secondary lymphatic tissue, which includes lymph nodes, the spleen, and Mucosal Associated Lymphatic Tissue, or MALTS. Lymphatic organs and tissues • Primary lymphatic tissue: where lymphocytes develop from precursor cells – Thymus: T cell development – Bone Marrow (bursa in birds) : B cell development • Secondary lymphatic tissue: Where lymphocytes interact with antigen-each type of tissue is specialized for the pathogen’s route of entry – Spleen: antigens in the blood – Lymph nodes : antigens in the interstitial fluid – MALT: mucosal-associated lymphoid tissue; antigens in the mucosa – WHY?? to maximize chance of immune cells interacting with antigen • Lymphatic system collects all the fluid that leaks out of blood vessels into the interstitial fluid. It drains all tissues. Collection is into lymphatic vessels. • The fluid is eventually returned to the blood through the thoracic duct, which empties into the left subclavian vein near the heart. • Lymph nodes are located at junctions of lymphatic vessels. Embedded question: name three types of secondary lymphatic tissue, and say what route of antigen entry they are linked to. Lymph nodes • Three “layers”, but with dynamic movement of cells • cortex-contains follicular dendritic cells (FDC) and B-cells-organized into primary and secondary follicles • Paracortex: T-cells, fibroblast reticular cells (FRC), dendritic cells • medulla-differentiated plasma cells Note: Follicular dendritic cells are not blood cells, and do not differentiate from the hematopoietic stem cell. They are more like fibroblasts. These are not the DC we already know and love IN Antigens/pathgens and Dendritic cells come intot he node through the afferent vessels, in the lymph. OUT Lymphocyte homing • Lymphocytes (T and B cells) enter the nodes from the blood., by the process of extravasation. It is similar to what we learned for neutrophils; the process uses cell adhesion molecules on lymphocytes and blood vessels to slow lymphocytes, then there is activation which causes a change in integrins, and then arrest/adhesion, and extravasation. In lymph nodes and some other secondary lymph tissue, this happens in specialized areas of post capillary venules called HEV’s (high endothelial venules). Cells in these HEV’s have the correct cell adhesion molecules 1 x 104 lymphocytes leave blood through HEVs into a lymph node every second. Where do the chemokines come from? FDC and FRC Selectin is on the lymphocyte. Mucin-like CAM is on the HEV cell. This is different from neutrophils B cell chemokine is CXCL3 T cell chemokines are CCL19, CCL21 Chemokines come from FCD, FRC in the nodes • • • • • After B cells go through the HEV, chemokines (CXCL13) from the FDC attract them to the cortex by binding to the CXCR5 receptor on B cells. In cortex, the primary follicles are loosely organized naïve B-cells and FDC these B-cells are looking for antigen (remember, B cells can bind free antigen.) They will not stay in this node for long (1 hour if not stimulated) MRC are marginal reticular cells-they are part of the network (stroma) supporting the node-they, and FDC, are thought to make conduits for antigen flowing in with the lymph T cells come in through the HEV, and migrate to the paracortex, guided by chemokines (CCL19, CCL21) from the FRC . T cells have receptors for the chemokines (CCR7). Dendritic cells also are recruited by these chemokines. T cells crawl around the FRC network, interacting with dendritic cells that came into the node with the lymph, and are attached to the FRC. • Antigens in interstitial fluid are picked up by lymph, carried to nearest lymph node • antigen may have been picked up, processed by dendritic cells, also carried in by lymph • Antigens (Ag) trickle through node • Ag interact with B-cells in Primary follicle; B-cells get primed (discuss in detail later in the course) • Ag get ingested by dendritic cells (in tissue or in node), is presented on MHC II. • APC present to TH in paracortex • If a TH is activated, it differentiates (TH1, TH2, etc). TH and primed B cells move to the periphery of the paracortex, where the B cells can interact with the TH cells. • B-cells will then proliferate and differentiate into plasma cells and memory cells, forming a secondary follicle. • plasma cells move to medulla, into lymph (leave using the efferent lymphatic vessel) and finally enter the blood Cortex paracortex • Secondary follicles are more organized than primary • They contain areas of dividing B-cells and FDC in the germinal center, surrounded by densely packed lymphocytes (B cells, memory cells), called a mantle. • Secondary follicles form after antigenic stimulation • Memory cells stay in contact with FDC-will discuss more later in course Spleen- a filtering organ • • • • • • There are two types of tissue in the spleen-red pulp and white pulp. Blood comes in through blood vessels that empty out into sinuses containing macrophages (which is the red pulp) The macrophages eat old/damaged red blood cells. They also eat opsonized bacteria bacteria in the blood. The blood could contain antigens. If so, the macrophages will eat them too. Dendritic cells can enter the spleen this way too. white pulp is the lymphoid tissue-B and T cells. In the white pulp, the T and B cells are separated into two distinct regions: the marginal zone, and the peri-arteriolar lymphoid sheath (PALS) Macrophages and Dendritic Cells are in both red and while pulp. Macrophages recognize dying RBC (changes in membrane lipids act as “eat me” signals), but this does not activate the immune system, because they are “self”-no PAMPS. Macrophages can eat free antigen, and can be Antigen presenting cell (APC) to T cells. Dendritic cells remain the most active APC for naïve T cells. • Marginal zone: – – • PALS: – • is between red pulp and PALS contains B cells, macrophages rich in T-cells, like paracortex of lymph nodes There is not as clear a reticular network as is seen in lymph nodes, but T and B cells coming in with the blood find their way to the correct place, using chemokine signals. B cells form primary follicles, organized by chemokines secreted by a splenic version of Follicular Dendritic Cells. T cells are recuited to the PALS by other chemokines. DC go to the PALS to activate T cells, which then move toward the B cells. MALTS mucosal associated lymphoid tissue • • • • • • Found in all mucosal tissue (Gut-associated is GALT; Bronchial is BALT, etc) Can be well organized or very loosely organized. The figure shows a Peyer’s patch, which is well organized. Tonsils, adenoids, appendix-all MALTS MALTS protect mucosal tissue. Quite a lot of pathogens enter through our respiratory and gastrointestinal systems, so this is very important to human health. About 50% of lymphocytes in the body can be found in MALTS Lymph (carrying antigen and Dendritic cells) also flows from intestinal lining and lung tissue to nearby lymph nodes, which we have already covered SED is sub-epithelial dome, Dendritic cells hang out there follicle T and B cells are segregated. They get into MALTS using HEV (High Endothelial Venules), like they do with lymph nodes. Leave blood using extravasation (rolling, activation, arrest, migration). The B cells are organized into follicles, using chemokines and FDC. T cells are outside of the follicles, usually in the spaces between follicles. The antigen gets into the follicle from the lumen of the gut by passing through specialized cells, called M cells, in the gut epithelium. (marked (a) in picture). Dendritic cells hang out near the M cells in the SED, and pick up the antigen (Phagocytosis, uses PRR for PAMPS on pathogen. Antigen is brought to lysosome, digested, and presented on MHC II.) DC present antigen to TH cells (b) and activate them. They differentiate to TH1 or TH2, depending on signal the DC gives them (which depends on PAMPS). B cells in follicles can also pick up antigen from M cells, and process it. T cells can either activate a B cell in the same patch of MALT, or go to another site (via lymph) to find a B cell. • • • • Specialized cells (M cells, or microfold cells) in the epithelial cell layer transport antigen from the lumen of the gut to the MALT. M cells have “pockets”, where lymphocytes and DC can sit in less organized MALT tissue. M cells are specialized cells that pass antigen (undigested) across epithelial layer Both figures show essentially the same thing • B-cells in the MALT can be activated by T cells, and form secondary follicles where they differentiate into plasma cells that migrate along submucosa. • The plasma cells secrete IgA, which is an immunoglobulin that can be secreted into the lumen of the gut. There IgA binds antigens and prevents them from infecting us. We will discuss Ig A and other immunoglobulins later in the course. In the lymph nodes (and MALTS, and spleen) the antigen is taken up by an Antigen Presenting Cell (APC). APC process antigen and present it to TH cells on MHC II. The major APC are dendritic cells, macrophages, and B cells. DC are the ones that are best at activating naïve TH cells. These three are called Professional APC. Other cells types can be stimulated (for example, by inflammation) to be nonprofessional antigen presenting cells. Professional APC are special for 4 reasons: 1) They can take up antigen by endocytosis or phagocytosis 2) The can express MHC II. Not all cell types can. 3) They express MHC II either all the time (constituitive) or when induced 4) They can express the co-stimulator B7. To activate T cells, the APC must express both MHC II and co-stimulator B7. In macrophages, TLR signaling can turn on (induce) MCH II signaling, and so can cytokines (IFNg) from TH1 cells. Nonprofessional cells can be induced to express these, mostly when there is prolonged inflammation (inflammatory cytokines, including IL-12) All cell types are able to express MCH I to some extent, so any cell can process and present antigen on MHC I to TC cells. However, not all cells go to the lymph node, so in the lymph node, Dendritic cells are mostly the ones that present to Tc. DC can be infected with most viruses, even though viruses generally have specificity to specific cell types. (For example, coronavirus binds to ACE2 receptors, which are expressed by epithelial cells in the lung, and to some extent, in the GI tract). Once Tc are activated in the secondary lymph tissue (which is something we’ll go over later in the course), they differentiate into CTL, and go out into the rest of the body, where the infected cell can present on MHC I (altered self) to CTL and cause the CTL to kill the altered self cell. Despite the label on this picture, the term “APC” generally refers to cells that present antigens to TH on MHC II. A brief summary: Antigens from viruses are cytosolic. They are made when the host ribosomes in infected cells translate viral proteins. Cytosolic pathogens are degraded by the Ubiquitin-proteasome system, and the degradation products are displayed on MHC I. The presentation is by Dendritic cells to TC in the lymph node, and by infected cells to CTL in the periphery. . Viruses, cancer? Antigen made on host ribosome Mycobacteria, Listeria most bacteria Antigen made on pathogen ribosome A brief summary: Antigens from both intracellular and extracellular bacteria are made on the pathogen ribosomes. They get into Dendritic cells, macrophages, or B cells by phagocytosis or endocytosis. They are degraded by lysosomes, and the degradation products are displayed on MHC II to TH in the lymph node. Viruses, cancer? Antigen made on host ribosome Mycobacteria, Listeria most bacteria Antigen made on pathogen ribosome This is an overview of antigen processing (degradation) and presentation (attachment to MHC). Endogenous antigens are made on host ribosomes inside the host cell (e.g. viral proteins). They are processed using the endogenous pathway. Exogenous antigens are made outside the cell or in endocytotic vesicles, and their synthesis does not use host ribosomes (e.g. extracellular or intracellular bacteria). They are processed using the exogenous pathway. One interesting point is that it might be possible for exogenous antigens to “crossover” to the endogenous pathway, so that MHC I could present a bacterial (exogenous) peptide. We won’t really discuss that, but its on the figure so I mentioned it. We’ll focus on the endogenous path for MHC I processing first. The antigen is made by host cell ribosomes The MHC I peptides (there are two, see the picture on the right) are made on ribosomes attached to the Rough ER. The MHC I achain is an integral membrane protein in the ER membrane. The b-chain is called b2microglobulin. MHC I The Ubiquitin-proteasome pathway degrades the non-self antigen. The proteasome will also degrade normal (self) proteins. This happens in the cytosol. Note that because the antigen is digested, epitopes that are internal on the antigen are also going to be presented to T cells. The antigen fragments have to bind the MHC I while it is still inside the cell (not on the surface). The MHC I antigen binding site is on the a chain, which is inside the the Rough ER, because that is where proteins destined for the Plasma membrane are made. The antigen peptides are transported into the RER by a transporter called TAP (Transporter associated with Antigen Processing). Energy for transport is provided by ATP hydrolysis. The newly synthesized MHC I a chain is an integral membrane protein in the RER membrane. With help from the chaperone protein calnexin, it folds correctly and associates with b2 microglobulin. (Chaperones are proteins that assist in folding and assembly of other proteins). Calnexin is released, and another chaperone, calreticulin, binds to the MHC I along with tapasin, which brings the MHC I close to the TAP. ERp57 is a protein that first acts to stabilize the complex until MHC I binds a peptide, and then causes release of the MHC I-Ag from the rest. The MHC I-Ag then goes to the cell surface.. It won’t go if it does not have a peptide; it will get degraded because the cell sees it as incompletely folded. Any protein digested by the proteasome, including self peptides, will bind to MHC I. MHC I • MHC I protein has two peptides. • The a peptide binds the endogenous antigen peptide • The b peptide, b2-microglobulin, is structural. • MHC I is not complete until it has the a and b peptides joined together, AND has bound a peptide fragment. • Incomplete proteins are not transported out of the ER. • If MHC I is on the surface, it must be bound to a peptide. • If the cell is not infected MHC I is bound to a SELF peptide • The Thymus removes most developing Tc cells that do not bind MHC, or which react with MHC I bound to self peptide. Binding the antigen fragment changes the shape of the MHC in the region that interacts with T and NK cells. For CTL to recognize infected cells, the infected cell must present non-self antigen on MHC I. All of these viral evasion strategies decrease MHC I expression of viral antigen, which allows the viruses to evade to immune system for a while. (However, as we discussed earlier in the course, NK cells are more likely to kill cells with low MHC I. But, without CTL recognition, the virus infection is unlikely to be removed). https://www.youtube.com/watch?v=VPvCekgPwRI This is a link to a pretty good video about MHC I assembly and presentation. https://www.youtube.com/watch?v=soWtpAO1Nr0 This is another one for MHC I https://www.youtube.com/watch?v=TMnkihN6zVM This is a link to a video about MCH II presentation. We’ll focus on MHC II next. Remember, this is happening after the antigen is brought into the cell by phagocytosis or endocytosis by APC. In the Rough Endoplasmic Reticulum, (RER), the MHC II peptide chains (see next slide) are being synthesized. For those of you who have had Cell Biology, you will recall that proteins destined for the surface of the cell are made in the RER. Antigen fragments MHC II • The MHC II protein contains two peptides, an a and a b chain. Both are made in the RER, and they are joined together in the RER. • The antigen binds in a cleft between the two chains. • Binding of antigen changes the shape of the MHC slightly, especially in the region that interact with T cells Antigen binding cleft Overview: The MHC II travels in a vesicle to the endosome/phagosome/lysosome containing the digested antigen fragments. The vesicle fuses to the lysosome. The digested antigen fragments bind to the MHC II and the MHC II-Ag complex gets transported to the surface of the cell. Antigen fragments In the RER, the a and b chains of MHC II are made, and the two peptides are assembled. No self proteins stick to the new MHC II on its way to the lysosome, because the antigen binding site is blocked by a protein called the Invariant chain (in one of the videos, it is called Ii). The invariant chain also helps with folding of the MHC 2 chains, and with sending the MHC II on the correct transport path. The path from the ER is to the Golgi, then to early endosome, late endosomes, and finally to a lysosome, which contain the digested antigen fragments. The proteases in the endosomes/lysosome gradually degrade the invariant chain, leaving a small piece behind called CLIP. CLIP continues to block the antigen binding site. Digested invariant chain Embedded question: What happens to MHC I peptides if they can’t bind antigen? What effect would this have on levels of Tc cells? To remove CLIP, a special MHC II called HLA-DM (MHC II terminology will be described later) is used. HLA-DM does not bind or present antigens-it lives in the endosomes. HLA-DM mediates the exchange of CLIP for a real antigen peptide. The real MHC IIAg goes to the cell surface. Another special MHC II called HLA-DO helps regulate the exchange. If MHC II is on the surface, it must be bound to a peptide. If there is no pathogen present, MHC II is bound to a SELF peptide (but remember, most cells don’t make MHC II, and those that do are induced to do so by PAMPS). A self peptide is a normal cellular protein. The Thymus removes developing TH cells that react with self MHC II or which do not bind to MHC at all. This is a summary slide that puts it all together. • • • • • • Both MHC I and MHC II are encoded by genes that are linked together on a single stretch of DNA (stretching over 4 million bp) on chromosome 6 in humans. It is called the HLA locus or the MHC locus (HLA stands for Human Leukocyte Antigen) In the MHC/HLA locus, there are three classes of genes Class I genes (pink) encode the a chain of MHC I proteins. Note that there are three genes (HLA-A, HLA-B, and HLA-C) that encode the MHC I a chain. MHC I is polygenic. Class II genes (blue) encode the a and b chains of MHC II proteins. Note that there are three gene pairs. MHC II is also polygenic Class III genes encode other things, such as complement proteins, TNF The gene for b2 microglobulin is not in this locus; it is on chromosome 15. a and b chains of MHC II These encode the a chain of MHC I • • • • Because there are multiple genes encoding the MHC I and II proteins, the MHC genes/proteins are polygenic. All the genes (within the MHC I or II group) encode similar proteins, but the proteins have slight sequence and structural differences. All three genes/gene pairs in each group are expressed, and both copies of chromosome 6 are expressed. Within a population, there are many different variants of each of the genes for MHC I a chain and for the two MHC II chains-the genes are polymorphic. So, even MHC proteins encoded by the same gene type (e.g. HLA-A) can have slightly different sequences and structures. All the MHC genes are linked, and are inherited together as a single genetic trait. Humans (diploid) have two copies of the locus, one from dad, one from mom. Note that in the class two region, there are also genes for the TAP transporter, and for subunits of the proteasome. In the class III region, there are genes for complement proteins and for some cytokines, among other things. • • • • • There are three main distinct genes for the MHC I a peptide (polygenic), so human express three slightly different a chains encoded by each class I region. The genes are called HLA-B, HLA-B, HLAC. Each gene is expressed. Among humans, there are many variants (alleles) of these genes (polymorphic). Your parents are unlikely to have the same alleles as each other, and you are unlikely to have the same alleles on both your chromosomes. You are diploid, so you have 6 different genes (One A from mom, one A from dad; one B from mom, one B from dad, one C from mom, one C from dad). You probably have different alleles for each gene. Each gene is expressed, so you probably have 6 different MHC I proteins on the surface of your cells You also have minor (non-classical) MHC 1 genes, which we won’t discuss here. Currently, in humans, there are 6425 known HLA-A alleles, 7754 known HLAB alleles, and 6329 known HLA-C alleles. This number constantly changes as more people and more diverse populations are examined. There are 3 major gene pairs (polygenic) for MHCII: DQ, DP, and DR. The A gene encodes the a chain, and the B gene encodes the b chain. DO and DM are used for CLIP displacement. The DP gene products pair with each other, as do the DR and DQ products. The final MHC II proteins is called HLADP, -DQ, or –DR. All 3 sets are expressed. Among humans, there are many variants (allele) of each of the genes (polymorphic). Your parents are unlikely to have the same allele. So you probably have 6 different versions of MHC II genes expressed on APC. • • • • • • Within a species, the MHC genes are very polymorphic. What this means is that in a population, there are a bunch of slightly different nucleotide sequences encoding the DPa1 gene, and so on. The different genes are called alleles. There are a lot of different alleles, that can differ in DNA sequence by as much as 10%, and may differ in as many as 20 aa. You have possibly two alleles for each gene; one allele from mom, and one allele from dad. It is possible that mom and dad might have some of the same alleles, but it is extremely unlikely that many of their alleles are the same, unless they are related. You inherit the ENTIRE HLA locus (all 4 million plus bp) as a linked unit. Your set of MHC I and MHC II genes/proteins is pretty rare-hard to find someone who matches you. • 3621 1674 233 1968 279 29 Why so polymorphic? May help bind a diverse set of antigen peptides. Most of the variation among different alleles is in the peptide binding cleft. Since antigens need to be presented, they have to bind to MHC. If all the MHC are the same, then some peptides might not be able to be presented to T cells, and there would be no response. One of the problems with inbreeding-genetic diversity. First noted in inbred mouse strains-a failure to respond to some antigens. Taken into consideration when managing populations of endangered species. Each identified allele has a number (e.g. DP-A60). Your HLA haplotype is the entire set of your alleles in the HLA-region. Your HLA-region on one of your chromosome 6’s will be identical in all alleles to the region on one of your mother’s two chromosome 6s. The HLA region on your other chromosome 6 will be identical to one of your fathers two HLA-regions. You have a ¼ chance of having the same HLA haplotype as one of your siblings. During T cell development, T cells that bind tightly to the host set of MHCs are removed to prevent auto-immune diseases. But, MHCs encoded by heterologous alleles have a slightly different shape than yours, so your T cells see tissues from other people as altered self. If you get a tissue transplant, your T cells will probably attack the transplant unless the MHCs from donor and recipient match. For tissue/organ transplants, usually they look at HLA-A, HLA-B alleles for MHC I (which is on all cell types) , And HLA-DR alleles for MHC II (which is only on APC). Try to get the best match. Also need to match blood type. Here is an example of what tissue typing might look like. Your best chance to get a match is a sibling. In the general population, members of the same ethnic/racial group are more likely to be matches. Can lead to disparities in access to transplanted organs. Antigens • • • • • • By definition, antigens are molecules that bind to antibodies (or Immunoglobulin) However, now we tend to think of them as things that bind to antibodies, or T Cell Receptors (TCR) or immunoglobulins on surface of B cells (mIg) If antigens elicit an immune response they are considered to be immunogens. Some very small molecules can bind to antibodies but do not raise an immune responsethese are called haptens. The part of the antigen that physically makes contact with the antibody protein (or TCR) is called the epitope. Antigens can have multiple copies of the same epitope, as well as a lot of different epitopes. We will go over how the TCR or Ig bind the epitopes later in the course. The epitope of an antigen binds to the ends of the surface immunoglobulin on a B cell. The binding is specific. All the surface immunoglobulins on a single B cell bind to the same epitope. Every B cell has slightly different antigen-binding sites on the surface immunoglobulins, so every B cell binds a different epitope. • Epitopes are parts of antigens. • most antigens have lots of different epitopes-the protein pictured on the right is the surface glycoprotein of Ebola Virus. Shown are 4 different epitopes, each of which are part of a different functional region (mechanism of action, or MoA) of the protein. • Since most pathogens have more than one copy of their surface proteins, the surface of a pathogen will have multiple copies of each epitope. This becomes more relevant when we get to how B cells are activated. This is a picture of the SARS-CoV-1 spike protein (the one that emerged in 2003.) The stuff marked in red are epitopes that are genetically identical (same aa sequence) to ones on the spike protein of the current virus, SARS-CoV-2. Knowing B cell epitopes, and also knowing the Mechanism of Action of those epitopes (for example, the epitopes in the region where Spike interacts with the host cell receptor) help with vaccine development and evaluation. You want to generate antibodies that block the epitopes involved in host binding, since this will prevent infection. doi: https://doi.org/10.1101/2020.02.19.955484 Are the mutations in epitopes with important MoA? Or in epitopes that are more likely to bind to B cells and generate protective antibodies? Although all epitopes have a specific conformation, the amino acids contribution to that conformation can be next to each other (sequential/linear) or folded together as part of the proteins tertiary structure (conformational). This also matters when looking at the mutations of a pathogen, to see if they are going to prevent an immune response Pathogen: SARS-CoV-2 Antigen: Spike protein Epitope: S1-RBD, the amino acids in Spike that bind to the host receptor ACE2. • • • For B-cell antigens: The antigens are usually undigested, and on the surface of a pathogen (unless it is an isolated protein). The epitopes are usually on the surface of the antigen.. For TH-cell antigens: proteins (or entire pathogens) are picked up by antigen presenting cells, and the proteins are digested in lysosomes. Protein fragments, containing the epitopes, are presented on the surface of the APC by MHC II. Because TH cells interact with digested antigen (mostly proteins), the epitopes can either be on the surface of the antigen, or be internal amino acids. The presented epitopes are small fragments, and they usually are sequential Epitopes recognized by T cells must be presented on MHC I or MHC II Antigen binding to B cells; no presentation needed Antigen binding to T cells requires that an Antigen Processing Cell (APC) digest an internalized pathogen, and present it on MHC. For TH, MHC II does the presenting. For TC, MHC I does the presenting. MHC II binds antigen fragments that come into the cell from the outside, and are digested in lysosomes. Only a few cell types can express MHC II. MHC I binds antigen fragments that were made in the cell, and digested by the ubiquitinproteasome For presenting antigen on MHC I. All cell types are able to express MCH I to some extent, so any cell can process and present antigen on MHC I to TC cells. However, not all cells go to the lymph node, so in the lymph node, Dendritic cells are mostly the ones that present to Tc. DC can be infected with most viruses, even though viruses generally have specificity to specific cell types. (For example, coronavirus binds to ACE2 receptors, which are expressed by epithelial cells in the lung, and to some extent, other tissues). Once Tc are activated in the secondary lymph tissue (which is something we’ll go over later in the course), they differentiate into CTL, and go out into the rest of the body, where the infected cell can present on MHC I (altered self) to CTL and cause the CTL to kill the altered self cell. Antigens are either picked up at the site of infection by sentinel cells, or they flow with the lymph to the lymph nodes (or other similar tissue) and are is taken up by an Antigen Presenting Cell (APC) in the node. APC process antigen and present it to TH cells on MHC II. The major APC are dendritic cells, macrophages, and B cells. DC are the ones that are best at activating naïve TH cells. These three are called Professional APC. Other cells types can be stimulated (for example, by inflammation) to be non-professional antigen presenting cells. P rofes s ional AP C are s pecial for 4 reas ons : 1) T hey can take up antigen by endocytos is or phagocytos is 2) T he can expres s MHC II. Not all cell types can. 3) T hey expres s MHC II either all the time (cons tituitive) or when induced 4) T hey can expres s the co-s timulator B 7. Embedded question: name four features that allow cells to be professional antigen presenting cells To activate T cells, the APC must express both MHC II and co-stimulator B7. In macrophages, TLR signaling can turn on (induce) MCH II and B7 expression, and so can cytokines (IFN-g) from TH1 cells. It’s part of macrophage activation Nonprofessional cells can be induced to express MHC II and B7, mostly when there is prolonged inflammation (inflammatory cytokines, including IL-12) The external TLRs, that recognize bacterial PAMPS are not shown, but also activate the NF-kB and MAPK pathways, turning on CD80/86 (B7) and MHC II. A brief summary: Antigens from both intracellular and extracellular bacteria are made on the pathogen’s ribosomes. They get into Dendritic cells, macrophages, or B cells by phagocytosis or endocytosis. They are degraded by lysosomes, and the degradation products containing epitopes are displayed on MHC II to TH in the lymph node. Viruses, cancer? Mycobacteria, Listeria Antigen made on host ribosome most bacteria Antigen made on pathogen ribosome A brief summary: Antigens from viruses are cytosolic. They are made when the host ribosomes in infected cells translate viral proteins. Cytosolic pathogens are degraded by the Ubiquitin-proteasome system, and the degradation products containing epitopes are displayed on MHC I. The presentation is by Dendritic cells to TC in the lymph node, and by infected cells to CTL in the periphery. . Viruses, cancer? Antigen made on host ribosome Mycobacteria, Listeria most bacteria Antigen made on pathogen ribosome Our next infectious agents are helminths (gastrointestinal nemotodes, or worms). Worms are BIG, relative to immune cells. Can’t phagocytose them. Helminth larva infect by passing through the intestinal epithelial cell lining, into the submucosa. The adult worms generally stay outside, unless the cell lining is damaged. So need to try to expel them, and to strengthen the mucosal barrier. The worms have PAMPS, and cause tissue damage, so there are also danger signals. Mucosal Immunology (2018) 11, 304-315; doi:10.1038/mi.2017.113 The epithelial cells of the intestine may detect some PAMPS, and DAMPS, and secrete cytokines that help strengthen the mucosal barrier. Dendritic cells pick up worm antigens, mostly from infecting larvae, and present to naïve TH in MALT or nearby lymph node. TH2 cells are induced because this is what the PAMPS in the worm are directing. TH2 cells can direct B cells to become IgE secreting plasma cells. TH2 leave the node and go to the infected mucosa. TH2 cytokines can activate macrophages (process is called alternative activation since it is TH2 and not TH1 doing it)-these macrophages secrete toxic material, increase inflammation, bringing in neutrophils. The neutrophils try to phagocytose the larvae. Mucosal Immunology (2018) 11, 304-315; doi:10.1038/mi.2017.113 The plasma cells that differentiated produce IgE. The IgE binds to Fc receptors on the surface of mast cells, eosinophiles, basophils, macrophages, and neutrophils, which are located in the sub-mucosa. When the cells with bound IgE encounter adult worm antigens that the IgE binds to, the cells release cytokines to bring in eosinophils and more neutrophils, and also release stuff to increase mucus production, and enzymes to digest adhesions between the worm and the intestinal lining. The epithelial cells are also induced to multiply, thickening the barrier. There is also increased muscle contraction, and this should help expel the worm. If the worms have penetrated the intestine, IgE bound to the worm may activate complement, which can also bind to mast cells and cause degranulation. C3b and IgE bound to larva can act as opsonins and bind to neutrophils which can try to phagocytose the larva, and/or release toxic mediators. Eosinophils can be activated by IgE bound to the worm, or by factors released by mast cells. The eosinophils release the contents of their granules, which are toxic (anti-helminth peptides, reactive oxygen species, other toxic stuff) TH2 cells activate macrophages-phagocytosis, release of enzymes and toxins in granules. IL-4 TH2 This response will hopefully expel or kill any adult worms and will kill larvae so the infection does not get worse. Luckily, there are also effective anti-helminth drugs, but there is always the risk of reinfection or resistance. IL-4 TH2 Immune response to viruses. The recognition and proliferation/differentiation stages happen in the secondary lymphoid tissue, and we have already covered it. Antigen is presented on MHC I. Signal 3 produces CTLs TH1 cells might be needed during the stimulation phase to generate sufficient IL-2 and co-stimulation to activate Tc. • Once the CTL differentiate in the secondary lymphoid tissue, they go off into the rest of the body (through the blood) and look for the altered self cells that will present the specific antigen to the T cell Receptor on the CTL. Remember, each CTL will recognize only a single epitope, so the action of the CTLs is extremely specific. • As it roams the tissues in the body, the CTL non-specifically and weakly binds to cells using cell adhesion molecules. • Remember, all nucleated cells present peptides generated using the Ubproteasome on MHC I. Normal cells present self-antigens. Those will not be recognized by any CTL TCR. If there is no specific interaction through TCR, the CTL will move on. Infected or cancer cells present non-self antigens on MHC I. These get recognized by the TCR, which binds to MHC I/Ag. CD8 binds to MHC I. CD8 and CD3 send a signal cascade in side the cell. Once the TCR makes a specific contact with MHC I/Ag, the CTL will use other Cell Adhesion molecules to make bind tighter to the target cell. This is called conjugate formation, and it is there to prevent the CTL from affecting any nearby cells. It also sends signals inside the CTL. The signals in the CTL cause rearrangement of the cytoskeleton, and granules containing cytotoxic material is brought to the part of the CTL that is next to the target cell. The granules move to the CTL surface, and merge with the plasma membrane, releasing the toxic stuff. All the cell-cell contacts between the CTL and target cell are then broken, and the CTL moves on to the next target. The target cells dies by apoptosis. Some nice pictures of the CTL attacking the target cell. This is what is in the granules, and it’s the same as with NK cells. Drawing of how granules fuse to plasma membrane, releasing perforin which then forms pores in the membrane of the target cell. Perforin has homology to C9 of the complement proteins. An EM picture of what the pores look like • • • • • • • Perforation alone does not kill the cells, even though water should be able to get in. Perforation lets in granzymes, particularly granzyme B Granzyme B can also get in by binding to a mannose-6-phosphate receptor, and endocytosis. The granzyme activates caspase 8, which activates caspase 3, which cleaves cellular components and causes apoptosis. CTL also have FasL on their surface, a protein that that binds to the Fas receptor, activating the death pathway by yet another way. The killing is neat and clean. The cells don’t blow up, releasing their contents all around. Remember, if the cells blew up, there would be DAMPS and alarmins, and this would activate TLR and the inflammasome. With apoptosis, there is little innocent bystander damage, and little inflammation. Macrophages and other phagocytes come around and clean up the cellular fragments left after apoptosis. Once CTL are made, they eventually need to be controlled. This is done using surface proteins similar to CD28 (which binds to B7, this is signal 2), except the action is inhibitory, not stimulatory. One of the inhibitory proteins is PD-1, which binds to PD-L1 or PD-L2 on the non-self cell. This will block the signaling from the TCR/CD3 so the CTL is not triggered to release its granules. In the picture below, this is called inhibition of effector function. PD-1 is made as part of CTL differentiation. The PD-1 response is considered an immunological checkpoint, since it controls the immune system. It helps us from having autoimmune responses. However, tumor cells in many human cancers express high levels of PD-L1 or PD-L2 (PD-ligand 1 or 2). This prevents our CTL from removing the non-self tumor cells. Immunotherapy, in the form of PD-1 blockers, is a newer from of cancer treatment that in some cases is extremely effective. Cells from a tumor can be tested to see if they express high levels of PD-L1/2, in which case this could be a good treatment. Many of the new drugs are designed monoclonal antibodies, which work by the antibody effector function of neutralization. This pretty much finishes up how our immune system responds to viral infections, and tries to deal with cancer. Don’t forget the innate response, with the NK cells and all. Embedded question: name two ways TREG suppress immune responses Finally, what do TREG do? In general they suppress the immune response by preventing other types of TH cells from working. • • • • • • One way TREG cells work is by preventing Signal 2 to in TH cell activation. Remember, CD28 binding to B7.1, B7.2 is required for activation. B7.1,2 can also bind to a T cell surface protein called CTLA-4. CTLA-4 is similar to CD28, but is inhibitory,not stimulatory. CTLA-4 is on TREG cells. It is one of the genes turned on by FoxP3. CTLA-4 binds B7 about 20 times more strongly than CD28 does. Binding to CTLA-4 is inhibitory, because it competes with, and inhibits binding to CD28. CTLA does not interact with the same signaling path as CD28. By preventing the CD28-B7 interaction, TREG can induce ANERGY in TH1 or TH2 cells (GRAIL gets turned on instead of IL2 and IL-2R(CD25). This is what is affected on the TH cell Nature Reviews Immunology 4, 841-855 (November 2004) • TREG also stop TH cells from responding to IL-2, if the TH cells got past the CTLA4 inhibition of B7. • Normally, when B7 binds to CD28, IL-2 and IL-2R are made by the TH cell. IL-2 binds to the IL-2 receptor (CD25). This starts a signaling path that causes the T cells to enter the cell cycle and to proliferate and begin differentiation into effector cells or memory cells. • IL-2 is also a survival signal, so if there is no IL-2, T cells die • In addition to CTLA, TREG cells have a lot of CD25 (IL-2R) on their surface (CD25 is another gene activated by FoxP3). So they can titrate all of the secreted IL-2,preventing the naïve T cell from getting any. The naïve T cells die from lack of IL-2 activation (and immune system is suppressed). TREG cells inhibit the immune system by: • Depleting the local area of IL-2 by making a lot of CD25/IL-2R. Loss of survival/proliferation signal causes the TH cells to die. • Producing TGF-b which converts TH into TREG. • Producing IL-10 which decreases expression of MHC and B7 on APC, and decreases the production of IL-12 and IL-23, which decreases TH1 and TH17 differentiation cytokines (signal 3). So TREG inhibit APC from activating TH cells • Inhibit co-stimulation by using CTLA to inhibit B7 from binding to CD28. Will result in anergy and death. Converted to TREG TFH? TH1 IgA is the most prevalent Ig produced in the gut, where many TREG are. Treg? TH2 TH1,H2 We finally have antibody producing plasma cells! Some of the plasma cells are going to circulate for a while, secreting antibodies to get rid of the infection, if the innate system hasn’t already done it. Some will go to the bone marrow, where they live a long time, and produce protective antibodies. Plasma cells made in the MALT tend to stay there, and secrete IgA into the mucosa. How do antibodies help get rid of infections? What type of infections??? Antibodies have 3 major effector functions that help us fight disease: 1) Neutralization 2) Opsonization 3) Complement Activation There are a few other functions that we will hit in a few slides. Neutralizing antibodies prevent the antigen/pathogen from binding to a cell’s surface. For example, an antibody that bound to the SARSCoV-2 spike protein could neutralize it, preventing it from binding to the ACE2 receptor on lung cells. We have already learned about opsonization and complement activation (classical pathways was activated by antibodies), and you will see it represented in pictures here. More about neutralization: In addition to binding to blocking antigens from binding to a cell surface, neutralizing antibodies can also bind bacterial toxins and prevent them from binding to cell receptors. This is how the vaccine against diphtheria and tetanus works. You get injected with inactivated toxoid proteins and your body builds antibodies that will neutralize the toxin if you ever get infected with the bacteria (Clostridium tetani, Corynebacteria diphteriae). Once the antigen is bound to neutralizing antibodies, the stem (Fc) regions of the antibodies can bind to Fc receptors on macrophages, and the antigen is destroyed in the lysosome. Opsonization is similar, except a microbe is coated and ingested, not just an antigen. Neutralization also prevents toxins and pathogens from entering the body through the mucosal layer. The Ig types that usually does this is IgA. Neutralization 3 ways Prevent a toxin from binding to a receptor Prevent a virus from binding to a cell surface receptor Prevent a bacteria from binding to a cell’s surface An effector function sort of related to opsonization is the ability of antibodies to clear antigens complexes from blood or other places in the body. This is related to complement, and is one of the functions of complement we learned about earlier in the course. Ab can bind soluble antigens, and form a larger complex where the Ag cross-links multiple Ab together. In mucosal tissue, (lumen of gut, for example) this will result in elimination. In the blood, there is no obvious exit route for elimination. However, the close spacing of the Ig Fc regions activates complement via the classical pathway, and C3b is made. C3b lands on the antigen/antibody complex. The C3b can bind Complement Receptors (CRs)on red blood cells or on phagocytes. The Fc portion of the Ab can bind Fc receptors on macrophages in the spleen, which remove the complex and the RBC. Immune complexes can also cause problems (Type 2 and 3 hypersensitivity diseases). So clearance of immune complexes is a antibody effector function. Type III hypersensitivity-antibody complex disease Immune complexes can be a problem-causing inflammation. This is called Type III hypersensitivity. It is one of the major pathologies found with some auto-immune diseases. Example-Lupus, Rheumatoid arthritis. In Lupus, apoptosis leads to generation of antibodies that react against self nuclear proteins (this shouldn’t happen). Since we have a lot of self proteins, there is a ton of antigen binding to the auto-antibodies. The antibodies make an immune complex, which should get cleared. But, if there is too much complex, the normal methods of clearing them don’t work. The complexes then bind to blood vessel walls, and activate complement there, which causes inflammation. They also can bind to the basement membranes in the kidney glomerulus (filtering tissue) and activate complement there too. Inflammation. Results in kidney damage. In RA, the immune complexes are deposited on joint surfaces • When immune complexes are localized by having antigens concentrated intradermally, the type III hypersensitivity reaction is the Arthus reaction • It can happen with a booster shot of a vaccine (when there are already circulating antigens). • Mosquito bites are also an example of the Arthus reaction Other effector functions of antibodies: • Antibody-dependent cell mediated cytotoxicity (ADCC). This involves antibodies binding to non-self antigens on a host cell surface (like proteins produced as a consequence of a virus infection, or cancer) or to a pathogen surface, such as a helminth (worm). Effector cells (like NK cells, eosinophils) bind to the Fc portion of the bound antibodies, and if this causes clustering of the Fc receptors, it starts a signaling cascade that causes release of granules from the effector cell. Above, the effector cell is an NK cell, which releases granzymes and perforins to kill the cell by apoptosis, as we learned in the presentation about the innate response to viruses. The effector cell could also be an eosinophil, which releases toxic and inflammatory compounds. • Mast cell sensitization. In this case, an allergen or pathogen (again, this happens with helminths) causes production of antibodies (usually IgE) that bind to Fc receptors on the surface of mast cells. When the sensitized mast cells encounter any copies of the antigen in the body, the bound immunoglobulins bind the antigen, and this causes clustering of the Fc receptors. This sends a signal to the mast cell to release granules containing histamine and other inflammatory substances. This is the basis of the allergy response. The response can be local, or systemic. If the response is systemic, (antigen gets into your blood) then you can get anaphylaxis Engagement question: Name 5 effector functions of antibodies IgE binds to mast cells Each immunoglobulin class is suited for specific effector functions. The table above summarizes which Ig are good at which functions. Remember, it is the Fc portion that differs among the different Ig types. Note that IgA is good at neutralization, not so good for ADCC. This is the Ig type made in MALTs and secreted into the lumen of the intestines. Its job there is to prevent pathogens from getting into the body through the GI tract. IgGs (there are subclasses of IgG) are found in blood serum and are included in the exudate that leaves the blood as part of the innate response to being jabbed with a pointy stick. They are very good at opsonization and complement activation. Our next infectious agents are helminths (gastrointestinal nemotodes, or worms). Worms are BIG, relative to immune cells. Can’t phagocytose them. Helminth larva infect by passing through the intestinal epithelial cell lining, into the submucosa. The adult worms generally stay outside, unless the cell lining is damaged. So need to try to expel them, and to strengthen the mucosal barrier. The worms have PAMPS, and cause tissue damage, so there are also danger signals. Mucosal Immunology (2018) 11, 304-315; doi:10.1038/mi.2017.113 The epithelial cells of the intestine may detect some PAMPS, and DAMPS, and secrete cytokines that help strengthen the mucosal barrier. Dendritic cells pick up worm antigens, mostly from infecting larvae, and present to naïve TH in MALT or nearby lymph node. TH2 cells are induced because this is what the PAMPS in the worm are directing. TH2 cells can direct B cells to become IgE secreting plasma cells. TH2 leave the node and go to the infected mucosa. TH2 cytokines can activate macrophages (process is called alternative activation since it is TH2 and not TH1 doing it)-these macrophages secrete toxic material, increase inflammation, bringing in neutrophils. The neutrophils try to phagocytose the larvae. Mucosal Immunology (2018) 11, 304-315; doi:10.1038/mi.2017.113 The plasma cells that differentiated produce IgE. The IgE binds to Fc receptors on the surface of mast cells, eosinophiles, basophils, macrophages, and neutrophils, which are located in the sub-mucosa. When the cells with bound IgE encounter adult worm antigens that the IgE binds to, the cells release cytokines to bring in eosinophils and more neutrophils, and also release stuff to increase mucus production, and enzymes to digest adhesions between the worm and the intestinal lining. The epithelial cells are also induced to multiply, thickening the barrier. There is also increased muscle contraction, and this should help expel the worm. If the worms have penetrated the intestine, IgE bound to the worm may activate complement, which can also bind to mast cells and cause degranulation. C3b and IgE bound to larva can act as opsonins and bind to neutrophils which can try to phagocytose the larva, and/or release toxic mediators. Eosinophils can be activated by IgE bound to the worm, or by factors released by mast cells. The eosinophils release the contents of their granules, which are toxic (anti-helminth peptides, reactive oxygen species, other toxic stuff) TH2 cells activate macrophages-phagocytosis, release of enzymes and toxins in granules. IL-4 TH2 This response will hopefully expel or kill any adult worms and will kill larvae so the infection does not get worse. Luckily, there are also effective anti-helminth drugs, but there is always the risk of reinfection or resistance. IL-4 TH2 Immune response to viruses. The recognition and proliferation/differentiation stages happen in the secondary lymphoid tissue, and we have already covered it. Antigen is presented on MHC I. Signal 3 produces CTLs TH1 cells might be needed during the stimulation phase to generate sufficient IL-2 and co-stimulation to activate Tc. • Once the CTL differentiate in the secondary lymphoid tissue, they go off into the rest of the body (through the blood) and look for the altered self cells that will present the specific antigen to the T cell Receptor on the CTL. Remember, each CTL will recognize only a single epitope, so the action of the CTLs is extremely specific. • As it roams the tissues in the body, the CTL non-specifically and weakly binds to cells using cell adhesion molecules. • Remember, all nucleated cells present peptides generated using the Ubproteasome on MHC I. Normal cells present self-antigens. Those will not be recognized by any CTL TCR. If there is no specific interaction through TCR, the CTL will move on. Infected or cancer cells present non-self antigens on MHC I. These get recognized by the TCR, which binds to MHC I/Ag. CD8 binds to MHC I. CD8 and CD3 send a signal cascade in side the cell. Once the TCR makes a specific contact with MHC I/Ag, the CTL will use other Cell Adhesion molecules to make bind tighter to the target cell. This is called conjugate formation, and it is there to prevent the CTL from affecting any nearby cells. It also sends signals inside the CTL. The signals in the CTL cause rearrangement of the cytoskeleton, and granules containing cytotoxic material is brought to the part of the CTL that is next to the target cell. The granules move to the CTL surface, and merge with the plasma membrane, releasing the toxic stuff. All the cell-cell contacts between the CTL and target cell are then broken, and the CTL moves on to the next target. The target cells dies by apoptosis. Some nice pictures of the CTL attacking the target cell. This is what is in the granules, and it’s the same as with NK cells. Drawing of how granules fuse to plasma membrane, releasing perforin which then forms pores in the membrane of the target cell. Perforin has homology to C9 of the complement proteins. An EM picture of what the pores look like • • • • • • • Perforation alone does not kill the cells, even though water should be able to get in. Perforation lets in granzymes, particularly granzyme B Granzyme B can also get in by binding to a mannose-6-phosphate receptor, and endocytosis. The granzyme activates caspase 8, which activates caspase 3, which cleaves cellular components and causes apoptosis. CTL also have FasL on their surface, a protein that that binds to the Fas receptor, activating the death pathway by yet another way. The killing is neat and clean. The cells don’t blow up, releasing their contents all around. Remember, if the cells blew up, there would be DAMPS and alarmins, and this would activate TLR and the inflammasome. With apoptosis, there is little innocent bystander damage, and little inflammation. Macrophages and other phagocytes come around and clean up the cellular fragments left after apoptosis. Once CTL are made, they eventually need to be controlled. This is done using surface proteins similar to CD28 (which binds to B7, this is signal 2), except the action is inhibitory, not stimulatory. One of the inhibitory proteins is PD-1, which binds to PD-L1 or PD-L2 on the non-self cell. This will block the signaling from the TCR/CD3 so the CTL is not triggered to release its granules. In the picture below, this is called inhibition of effector function. PD-1 is made as part of CTL differentiation. The PD-1 response is considered an immunological checkpoint, since it controls the immune system. It helps us from having autoimmune responses. However, tumor cells in many human cancers express high levels of PD-L1 or PD-L2 (PD-ligand 1 or 2). This prevents our CTL from removing the non-self tumor cells. Immunotherapy, in the form of PD-1 blockers, is a newer from of cancer treatment that in some cases is extremely effective. Cells from a tumor can be tested to see if they express high levels of PD-L1/2, in which case this could be a good treatment. Many of the new drugs are designed monoclonal antibodies, which work by the antibody effector function of neutralization. This pretty much finishes up how our immune system responds to viral infections, and tries to deal with cancer. Don’t forget the innate response, with the NK cells and all. Embedded question: name two ways TREG suppress immune responses Finally, what do TREG do? In general they suppress the immune response by preventing other types of TH cells from working. • • • • • • One way TREG cells work is by preventing Signal 2 to in TH cell activation. Remember, CD28 binding to B7.1, B7.2 is required for activation. B7.1,2 can also bind to a T cell surface protein called CTLA-4. CTLA-4 is similar to CD28, but is inhibitory,not stimulatory. CTLA-4 is on TREG cells. It is one of the genes turned on by FoxP3. CTLA-4 binds B7 about 20 times more strongly than CD28 does. Binding to CTLA-4 is inhibitory, because it competes with, and inhibits binding to CD28. CTLA does not interact with the same signaling path as CD28. By preventing the CD28-B7 interaction, TREG can induce ANERGY in TH1 or TH2 cells (GRAIL gets turned on instead of IL2 and IL-2R(CD25). This is what is affected on the TH cell Nature Reviews Immunology 4, 841-855 (November 2004) • TREG also stop TH cells from responding to IL-2, if the TH cells got past the CTLA4 inhibition of B7. • Normally, when B7 binds to CD28, IL-2 and IL-2R are made by the TH cell. IL-2 binds to the IL-2 receptor (CD25). This starts a signaling path that causes the T cells to enter the cell cycle and to proliferate and begin differentiation into effector cells or memory cells. • IL-2 is also a survival signal, so if there is no IL-2, T cells die • In addition to CTLA, TREG cells have a lot of CD25 (IL-2R) on their surface (CD25 is another gene activated by FoxP3). So they can titrate all of the secreted IL-2,preventing the naïve T cell from getting any. The naïve T cells die from lack of IL-2 activation (and immune system is suppressed). TREG cells inhibit the immune system by: • Depleting the local area of IL-2 by making a lot of CD25/IL-2R. Loss of survival/proliferation signal causes the TH cells to die. • Producing TGF-b which converts TH into TREG. • Producing IL-10 which decreases expression of MHC and B7 on APC, and decreases the production of IL-12 and IL-23, which decreases TH1 and TH17 differentiation cytokines (signal 3). So TREG inhibit APC from activating TH cells • Inhibit co-stimulation by using CTLA to inhibit B7 from binding to CD28. Will result in anergy and death. Converted to TREG Viruses (SARS-CoV-2) Fungi (yeast) Bacteria (intracellular) Bacteria (extracellular) Parasite (roundworm) The immune system recognizes what has invaded the system, and generates the correct response Types of effector T cells Extracellular bacteria In adaptive immunity, effector T cells work with other immune cells to eliminate infections B cells (antibodies); eosinophils, mast cells, and basophils; macrophages Response to extracellular bacteria: Innate system, Antibodies, TH1, TH2, and TH17 cells Pointy stick introduces bacteria. FIRST EXPOSURE • Sentinal macrophages use TLR, etc to recognize pathogens and in response, secrete inflammatory cytokines. • The stab and the inflammatory cytokines increase vascular permeability. This releases exudate which contains opsonins such as CRP, also complement, antimicrobial peptides. • Complement can opsonize and lyse bacteria. Antimicrobial peptides can lyse cells. • The inflammatory cytokines also cause extravasation of phagocytes (like neutrophils and more macrophages) which eat phagocytose the bacteria and kill them using by lytic enzyme digestion, reactive oxygen and nitrogen mechanisms, release of toxic granules, and NETosis. • Complement products bind to mast cells and and macrophages causing release of more inflammatory mediators, including prostaglandins and histamine, to enhance the response. B cells in cortex (primary follicles) are primed by Ag binding to surface Ig. B cells go to border between cortex and paracortex.and interact with the T cells. Exudate carries Dendritic cells and free antigen to lymph node Dendritic cells activate TH cells. TFH TFH secrete IL-21 which helps primed B cells proliferate and start class switching, in combination with cytokines from TH1 and TH2 cells. TFH stay in the secondary lymph tissue and reside in the germinal centers with memory B cells and FDC. Help survival of memory cells, and differentiation of memory B cells into plasma cells TFH TH1 Treg? TH2 TH1,H2 Extracellular bacteria-what antibody effector functions are needed? Neutralization, opsonization, and complement activation (IgG and IgM-TH1/TH2) Will also need to recruit more phagocytes from the blood to deal with the opsonized and dead bacteria (IL-17-TH17) The antibody response will remove any remaining bacteria, but also protect the next time you get jabbed by the pointy stick with the same type of bacteria. The antibodies will neutralize the bacterial toxin (if any) The antibodies will activate complement-increasing opsonization, and bacterial lysis, as well as increasing inflammation. The antibodies will opsonize the bacteria directly, increasing phagocytosis Embedded question: Name 3 types of TH effector cells used in the immune response against extracellular bacteria TH17 cells do not affect antibodies, but are a part of the adaptive response to extracellular bacteria. Signal 3 for TH17 is IL-1, IL-6, TNF-a (inflammatory cytokines) TH17 contain the transcription factors STAT3 and RORgT. TH17 secrete IL-17 and IL-22. IL-17 and IL-22 cause cells to secrete chemokines attract neutrophils, Cause increased production on antimicrobial peptide (b-defensin) Activate macrophages Extravasation This is basically a summary of the entire course! TH1 are also involved in more than helping B cells. When TH1 travel to the site of the infection, they secrete inflammatory cytokines like TNF-a, and also cytokines that activate macrophages, like IFN-g, so the macrophages are more active phagocytes. The activated macrophages also can recruit more neutrophils, and secrete more inflammatory substances. The adaptive response to fungal infections is similar to that for intracellular bacteria. APC recognize PAMPS and go to nodes to generate TH1 and TH2 cells, which help B cells develop into plasma cells that secrete opsonizing, and some neutralizing antibodies. TH1 cells travel to the site of infection and activate macrophages to increase phagocytosis and secrete things that kill fungal cells. TH17 cells are also generated in the lymph nodes, and these also travel to the site of infection and recruit neutrophils and stimulate epithelial cells to secrete antifungal peptides. How does our immune system fight intracellular bacteria? • Our response starts out by the usual route-an APC eats the bacteria by phagocytosis. The macrophages and DC detects the bacteria using PRR, and start an innate response. A dendritic cell eats the bacteria and enough of it gets digested and presented on MHC II to activate and generate TH1 cells in the lymph node. • At the site of infection, macrophages will have eaten the bacteria, but intracellular bacteria don’t allow much fusion of the phagosome with the lysosome, so the killing mechanisms are not activated and the bacteria aren’t killed. /A bit of digestion happens, enough to put MHC II-Ag on the cell surface. The TH1 cells leave the lymph node and end up in the blood. The activated vascular endothelium at the site of infection will allow the TH1 to extravasate at the site of the infection, where the infected macrophages are. The infected macrophage bind the TH1 cell using TCR/MHC II-Ag, CD4/MHC II, and this causes the TH1 cells to secrete cytokines (IFN-g and TNF-b) that will further activate the macrophage, presumably upping the reactive oxygen and nitrogen killing mechanisms. This is cell-mediated cytotoxicity, and it could be enough to kill the intracellular bacteria. Or not…. The chemokines will recruit more immune cells. • In some cases, the bacteria is not killed (such as M. tuberculosis, which is resistant to the cell-mediated killing mechanisms of the macrophage). However, the TH1 cell and the now highly activated infected macrophage send out signals to recruit more TH1 cells and more macrophages. These surround the original infected cell. • The mass of activated macrophages and TH1 cells are called a granuloma, or in the case of tuberculosis, a tubercule. The When the original release of toxic substances from the antigen/pathogen can’t be infected macrophages and recruited removed, this is called neutrophils can cause a lot of tissue Delayed-type, or type IV, damage in the surrounding area. hypersensitivity. • TH1 also secrete cytokines that will cause apoptosis, which is supposed to to kill the If it is not a pathogen, it is infected cell and release the trapped called contact dermatitis. bacteria, so that the surrounding macrophages can try again to kill by phagocytosis • The granuloma also traps the bacteria so they can’t escape and cause a systemic infection. • . Poison ivy makes a compound that binds covalently to skin proteins. When the proteins shed, they are ingested by APC, which then activate TH1 cells. The TH1 cells go to the affected tissue, and activate macrophages which try to get rid of the affected skin cells by releasing inflammatory and other toxic compounds. Results in the red itchy rash. Can be caused by metals as well as other things. The rash can happen a week or so after exposure, but is more likely to occur by activation of memory cells after the second exposure. TFH? TH1 IgA is the most prevalent Ig produced in the gut, where many TREG are. Treg? TH2 TH1,H2 We finally have antibody producing plasma cells! Some of the plasma cells are going to circulate for a while, secreting antibodies to get rid of the infection, if the innate system hasn’t already done it. Some will go to the bone marrow, where they live a long time, and produce protective antibodies. Plasma cells made in the MALT tend to stay there, and secrete IgA into the mucosa. How do antibodies help get rid of infections? What type of infections??? Antibodies have 3 major effector functions that help us fight disease: 1) Neutralization 2) Opsonization 3) Complement Activation There are a few other functions that we will hit in a few slides. Neutralizing antibodies prevent the antigen/pathogen from binding to a cell’s surface. For example, an antibody that bound to the SARSCoV-2 spike protein could neutralize it, preventing it from binding to the ACE2 receptor on lung cells. We have already learned about opsonization and complement activation (classical pathways was activated by antibodies), and you will see it represented in pictures here. More about neutralization: In addition to binding to blocking antigens from binding to a cell surface, neutralizing antibodies can also bind bacterial toxins and prevent them from binding to cell receptors. This is how the vaccine against diphtheria and tetanus works. You get injected with inactivated toxoid proteins and your body builds antibodies that will neutralize the toxin if you ever get infected with the bacteria (Clostridium tetani, Corynebacteria diphteriae). Once the antigen is bound to neutralizing antibodies, the stem (Fc) regions of the antibodies can bind to Fc receptors on macrophages, and the antigen is destroyed in the lysosome. Opsonization is similar, except a microbe is coated and ingested, not just an antigen. Neutralization also prevents toxins and pathogens from entering the body through the mucosal layer. The Ig types that usually does this is IgA. Neutralization 3 ways Prevent a toxin from binding to a receptor Prevent a virus from binding to a cell surface receptor Prevent a bacteria from binding to a cell’s surface An effector function sort of related to opsonization is the ability of antibodies to clear antigens complexes from blood or other places in the body. This is related to complement, and is one of the functions of complement we learned about earlier in the course. Ab can bind soluble antigens, and form a larger complex where the Ag cross-links multiple Ab together. In mucosal tissue, (lumen of gut, for example) this will result in elimination. In the blood, there is no obvious exit route for elimination. However, the close spacing of the Ig Fc regions activates complement via the classical pathway, and C3b is made. C3b lands on the antigen/antibody complex. The C3b can bind Complement Receptors (CRs)on red blood cells or on phagocytes. The Fc portion of the Ab can bind Fc receptors on macrophages in the spleen, which remove the complex and the RBC. Immune complexes can also cause problems (Type 2 and 3 hypersensitivity diseases). So clearance of immune complexes is a antibody effector function. Type III hypersensitivity-antibody complex disease Immune complexes can be a problem-causing inflammation. This is called Type III hypersensitivity. It is one of the major pathologies found with some auto-immune diseases. Example-Lupus, Rheumatoid arthritis. In Lupus, apoptosis leads to generation of antibodies that react against self nuclear proteins (this shouldn’t happen). Since we have a lot of self proteins, there is a ton of antigen binding to the auto-antibodies. The antibodies make an immune complex, which should get cleared. But, if there is too much complex, the normal methods of clearing them don’t work. The complexes then bind to blood vessel walls, and activate complement there, which causes inflammation. They also can bind to the basement membranes in the kidney glomerulus (filtering tissue) and activate complement there too. Inflammation. Results in kidney damage. In RA, the immune complexes are deposited on joint surfaces • When immune complexes are localized by having antigens concentrated intradermally, the type III hypersensitivity reaction is the Arthus reaction • It can happen with a booster shot of a vaccine (when there are already circulating antigens). • Mosquito bites are also an example of the Arthus reaction Other effector functions of antibodies: • Antibody-dependent cell mediated cytotoxicity (ADCC). This involves antibodies binding to non-self antigens on a host cell surface (like proteins produced as a consequence of a virus infection, or cancer) or to a pathogen surface, such as a helminth (worm). Effector cells (like NK cells, eosinophils) bind to the Fc portion of the bound antibodies, and if this causes clustering of the Fc receptors, it starts a signaling cascade that causes release of granules from the effector cell. Above, the effector cell is an NK cell, which releases granzymes and perforins to kill the cell by apoptosis, as we learned in the presentation about the innate response to viruses. The effector cell could also be an eosinophil, which releases toxic and inflammatory compounds. • Mast cell sensitization. In this case, an allergen or pathogen (again, this happens with helminths) causes production of antibodies (usually IgE) that bind to Fc receptors on the surface of mast cells. When the sensitized mast cells encounter any copies of the antigen in the body, the bound immunoglobulins bind the antigen, and this causes clustering of the Fc receptors. This sends a signal to the mast cell to release granules containing histamine and other inflammatory substances. This is the basis of the allergy response. The response can be local, or systemic. If the response is systemic, (antigen gets into your blood) then you can get anaphylaxis Engagement question: Name 5 effector functions of antibodies IgE binds to mast cells Each immunoglobulin class is suited for specific effector functions. The table above summarizes which Ig are good at which functions. Remember, it is the Fc portion that differs among the different Ig types. Note that IgA is good at neutralization, not so good for ADCC. This is the Ig type made in MALTs and secreted into the lumen of the intestines. Its job there is to prevent pathogens from getting into the body through the GI tract. IgGs (there are subclasses of IgG) are found in blood serum and are included in the exudate that leaves the blood as part of the innate response to being jabbed with a pointy stick. They are very good at opsonization and complement activation. We will focus on activation of naïve TH cells for now. Interaction between the TH cells and the APC will send three signals to activate the naïve TH. The first signal, called Signal 1, is when the TCR/CD3 complex recognizes and binds MHC II-Ag. CD4 binds to MHC II. The signaling cascade is shown in a later slide. This signaling cascade starts the activation process. Signal 2 is caused by CD28 on the T cell binding to co-stimulator B7 on the APC. B7 is a dimer of B7.1 and B7.2. (B7.1 is also known as CD80. B7.2 is also known as CD86). This activates more intracellular signaling, and will lead to synthesis in the T cell of IL-2 and the IL2 receptor (among other things). IL-2 stimulates proliferation and survival. Signal 3 is caused by the cytokines secreted by the APC that tell the TH what type of effector cell it should become. Next we will go into each signal in more detail • Signal 2, the co-stimulatory signal, is caused by CD28 on T cells binding to B7 on APC. • B7 binding to CD28 activates additional kinases-including the MAP kinase pathway. This results in activation of additional transcription factors. • Instead NFAT causing cells to making GRAIL, and become anergic, when Signal 1 combines with Signal 2, the two paths converge to activate additional transcription factors, including NF-kB and AP-1, that work with NFAT to turn on the genes for IL-2 and the IL-2 receptor (IL-2R, also known as CD25). • If TCR-CD3-CD4/CD8 paths are activated without the CD28-B7 interaction, the T cells enter a state of ANERGY, which means they can’t proliferate, and are generally non-responsive. • Generation of anergic TH cells should help prevent autoimmunity. B7 is only on antigen presenting cells, and it is increased by signaling from PRRs. If there are no PAMPS, then. There is little B7. No T cell activation. Signal 2 B7 MHC-Ag Signal 1 Nature Reviews Immunology 14, 435–446 (2014) Signal three is the differentiation signal. It is started when cytokines secreted by the APC or other nearby cells bind to specific cytokine receptors on to the T cell. In many cases, the APC secretes specific cytokines that work using the JAK-STAT signaling pathway. The STATs, which are specific for each cytokine receptor, turn on genes encoding key regulatory transcription factors (see next slide), which then turn on the genes specific to a type of TH effector cells, or memory cell. In many cases, the PRR on the APC that were activated by the antigen/PAMPS determine which cytokines are secreted for Signal 3. Signal 3 Cytokine for signal 3 from APC and local environment Key regulatory Transcription factor Genes that are turned on-the genes encode these cytokines that are secreted by the effector TH cells. The top row on the figure shows the cytokines that act as Signal 3. Note that some of the Signal 3 cytokines are inflammatory cytokines, which were made by the APC in response to TLR signaling. Naïve TH cells differentiate into a type of effector TH cells (e.g. TREG, TFH,TH17, TH1, and TH2). We will discuss these in later slides and also later in the course. Each effector TH cells is characterized by a distinct regulatory transcription factor, that helps turn on the genes needed for the cell to do its job. Among these gene are cytokines that are secreted by the TH effector cells-each type has its own distinct set of cytokines that it secretes. We will discuss these effector functions in detail a bit later in the course. Right now we need to talk more about Signal 3. Now we know how naïve TH (CD4+) cells are activated and how they differentiate into the effector cells (TH1, TH2, TH17,TFH, and TREG) (we didn’t discuss TH17 or TFH much, because we do have time limits). We will talk about what the effector cells do later in the course. Virus-infected DC The activation and differentiation happens in secondary lymph tissue. How are naïve TC (CD8+) cells activated, and how do they differentiate into CTLs (cytotoxic T lymphocytes)? Il-2R Il-2 The simplest way is for a dendritic cell to become infected by a virus at the site where the virus enters the body (say, in the lungs, where someone inhaled droplets containing SARS-CoV-2). We will come back to how DC got this infection in a couple of slides. The ribosomes in the infected DC will now start making viral proteins. Some of the viral proteins will be degraded by the Ub-proteasome system, and presented on MHC I using the endogenous antigen pathway. The DC will go to the nearest lymph node (or other secondary lymphoid tissue) and find Tc which, like TH, are in the paracortex. Once in the paracortex, the infected dendritic cell and the Tc will test interactions (chemokines from DC attract Tc) Virus-infected DC Signal 1 is MHC-1/Ag-TCR/CD3, and MHC I-CD8. Signal 2 is B7-CD28. The two signals combined activate NFAT, AP1, and NFkB transcription factors to turn on the gene for IL-2 and IL-2 receptor. If you don’t have B7-CD28, only NFAT is turned on, and you don’t get IL-2, you get ANERGY. So far this is just like activation of TH cells. Il-2R IL-2 is secreted by the Tc cells, and it binds to the IL-2 receptor, causing the Tc to start proliferating. Signal 3, which is not shown in the picture to the left, is IL-12. IL-12 comes from the infected DC and is made Il-2 when the viral PAMP activated a TLR . You can see the pathway if you go to slide that has all the TLR paths. The activated Tc differentiates into a CTL. We will talk about how CTLs work later in the course. This is what one hopes will happen. O’Neill LA, Bryant CE, Doyle SLTherapeutic targeting of toll-like receptors for infectious and inflammatory diseases and cancer. Pharmacol Rev 61:177-197 This is to remind you of the TLR pathways. IL-12 is another inflammatory cytokine that is made when PAMPS bind to the TLRs. Sometimes, the Tc can’t be activated without help. It depends on what the virus is, apparently. The problem seems to be that some viruses do not stimulate B7 production enough in the DC, -so signal two is too low. In this case, the Tc needs help (cytokines) from a nearby TH CD4 cell. This is usually a TH1 cell. The CD4 cell is activated by the same DC as the Tc. This can happen because: 1) The DC can phagocytose the virus as well as be infected by the virus. This would allow the viral proteins to be processed by the exogenous pathway and presented on MHC II. 2) The crossover pathway of Ag presentation, which I told you to not worry about. You can go back and look at it if you want. TH cells will bind to the same APC as the Tc (but will bind to MHC II-Ag). They will bind whatever B7 is there. It takes less signal 2 to activate TH cells than Tc cells (or they coud already be TH1 cells and not need much activation). Signal 2, in addition to turning on the IL-2 gene, will turn on expression of another signaling protein called CD40L, which binds to CD40 on the APC. Binding of CD40L to CD40 starts a signaling cascade that results in the APC making more B7. It also causes the APC to make 4-IBBL, which is a member of the TNF family and is another co-stimulator of Tc cells. It binds to 4-IBB on the Tc. The extra co-stimulation will cause the Tc to also turn on the genes for IL-2 and IL-2R. This, combined with the extra IL-2 from the TH cell, drives Tc proliferation and survival. IL-12 from the APC will still be signal 3 to differentiate into CTL. It could also be signal 3 for the TH cells and cause them to become TH1 cells, if they are not already differentiated. DC-SIGN We have talked briefly about how viruses infect only certain cells because the bind to surface protein receptors that are only expressed on certain cell types. For example, SAS-CoV-2 infects cells that have the ACE2 receptor on their surface. So how can Dendritic cells be infected with viruses to present on MHC I, if they don’t have the specific viral receptor?? It turns out that DC have sticky proteins on their surface, called DC-SIGNs. Most viruses can bind to these and use this receptor to infect the DC. This allows DC to present most viral antigens on MHC I, so that CTL that are specific to that virus can form. The virus shown in this picture is HIV, and it also binds to other things. Don’t worry about that right now. Types of effector T cells We have finally made T cells that can DO SOMETHING!! We’ll talk about what they do to keep us healthy right after we activate some B cells. •Effector TH (usually TH1 or TH2) cells are now in the secondary lymph tissue-ready to help the B cells. •Now it’s time to activate B cells and have them differentiate into a plasma cells that secrete the correct type of antibody, and memory cells •The process is called antigen-dependent B cell maturation •The Antigen-independent phase of B-cell maturation is the development of the naïve B cell in the bone marrow, and we’ll discuss that later in the course Three signal...
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View attached explanation and answer. Let me know if you have any questions.hello , hope you are doing well. This is the final answer. Please if you need any revisions or corrections or further explanation don’t hesitate to ask me.

Question 1 :
a) Dendritic cells (DCs) are a type of APC (=antigen presenting cell) that are different from
FDCs. FDCs (=follicular dendritic cells) are found in the follicles of any secondary
lymphoid organ (spleen , Peyer’s patches , B cell area of lymph nodes , tonsils and
mucosal associated lymphoid tissue). The secondary lymphatic tissue is the location
where lymphocytes interact with an antigen. It is important to note that FDCs are not a
blood cell and do not differentiate from a hematopoietic cell unlike DCs ; they are
stromal cells (mesenchymal origin). In the lymph node, FDCs are located within the
cortex , in the central region of primary follicles and in the light zone of germinal
centers(B cell area not in T area). FDCs play an important role in B cell activation
process. The way they do this is not by internalizing the pathogen (they are not
phagocytic) but by binding to a native antigen and keeping it in B cell follicles.
Consequently, FDCs are important to save all the B cell antigens within the secondary
lymphoid organs playing a crucial role during affinity maturation. In addition , FDCs
secrete CXCL-13 attracting chemokine to recruit B cells expressing CXCR-5 (the
receptor) toward the B cell follicle. Plus, FDCs produce a cell activating factor called
BAFF to maintain survival of germinal center B cell. Additionally, FDCs present the
antigen as immune complex (IC) via CR1 without the need of CMH to the B cell in
germinal center (via their BCR = B cell receptor ). B cell internalize the antigen , degrade
it into peptide and present it by CMH class 2 to a T helper and the stimulation continue
(humoral response). Many B cell in the germinal center fail to to bind to the IC presented
by FDCs and do not receive T helper cell signal. B cell eventually die by apoptosis
initiated by the FDCs ; autoimmunity prevented.
b) When FDCs are absent or defective , germinal center formation will be affected and even
its formation interrupted. Usually the FDCs are needed to generate a B cell activation and
thus formation of high affinity antibodies and are important for germinal center formation
and maintenance. FDCs deficiency is found to be related to immunodeficiency and
autoimmune diseases. Since germinal centers are located within the secondary follicle in
the cortex of the lymph node , in the white pulp of the spleen and in the payer’s patches
these parts will be affected and we will not be able to see secondary follicles anymore but
only primary ones.
c) If we consider that the mouse is unable to produce FDCs , the living organism will be
immunosuppressed. Since FDCs are necessary for B cell activation, clonal expansion and
affinity maturation of antibodies and thus humoral immune response, the capacity of
producing specific antibodies will be affected. In that mice, clonal expansion is deleted.
When this happens, B cell will be produced with less antibody – antigen affinity and the
the memory cell (during secondary immune responses) will not be effective resulting in
recurrent infections .
Question 2 :
a) HLA or human leukocyte antigen system is controlled by a gene located on the short arm
of chromosome 6 (6p21).It codes a cell surface molecule (MHC ; major
histocompatibility complex) to present an antigen to TCR (T cell receptor). On the same
locus, we can distinguish 3 regions : class 1 region contains the classical HLA -A, HLAB, HLA-C genes coding for the class I molecules (expressed on the surface of almost
every nucleated cell) . There are 3 genes that codes for MHC class I alfa chain. In the
class 2 region, we find the HLA – DR (A and B), HLA- DQ and HLA-DP genes. These

families are responsible for coding both alfa and beta chain of MHC class II (expressed
on the surface of B cells, APCs and activated T cells). Finally, the class 3 region does not
code for a HLA molecules but contains genes that codes for various proteins involved in
the immunity such as complement factors , TNF (tumor necrosis factor) and many other
cytokines . HLA genes are inherited as haplotype (as one).
b) There are three different genes coding for MHC I alfa chain : HLA-A,HLA-B,HLA-C. So
each one will express three slightly different alfa chains of the MHC molecule. All 3
genes are expressed. Similarly, there are also three major genes coding for the MHC II
alfa and beta chain molecule :HLA-DP,HLA-DQ,HLA-DR. The resulted proteins (from
all the genes) will have only a very small difference. By definition, a polygenic trait is the
one whose phenotype is influenced by a combination of 2 or more genes. This is the case
of HLA proteins/genes. At a population level, every gene (for example HLA-A) has
different variants or options that can be expressed. These options are called alleles.A
gene is said to be polymorphic if the gene’s locus is occupied by more than one allele.
This is also the case of HLA genetic system known to be polygenic and polymorphic.
Normally, in a diploid human (2n), one will have 6 different genes (for example one A
inherited from the mother and another A from the father so 2 x 3 = 6). Because each gene
express an allele and because all the genes are expressed, the human cell will carry 6
different proteins ver...


Anonymous
Excellent resource! Really helped me get the gist of things.

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