Immunology Exam 2 LO

11. List and explain mechanisms for regulating cytokines and give an example of ways these have been used for therapy

- There were four things: o Receptor antagonists- molecule binds receptor but there is no induction. Then the true molecule can't bind that receptor (IRAT was the example) o Soluble receptors- the body uses this- ex: soluble receptor for RNF used in arthritis. o Cytokines have opposite effects- one can turn off another. Not used in therapy o Deceptor or fake receptors- the body puts out fake receptors and if the cytokine binds no signal is transduced.

7. List three mechanisms that CTLs use to kill target cells. For each, include the molecules and functions involved - for binding and killing

1) Perforin and granzymes pathway: CTL release their granule contents of perforin and granzyme at point of contact w/target cell, perforin monomers polymerize & insert into target cell membrane. Granzymes enter the target cell through the pore formed by the polymerized perforin. Remember the MAC complex from complement and the defensins can also poke a hole in the membranes! 2) Fas/FasL receptor mediated killing (the death receptor): activated CTL express fas Ligand (FasL) in their membrane, CTL recognition of MHC1/peptide and Fas/FasL binding leads to caspase cascade and apoptosis. This mechanism induces apoptosis in the thymus during T cell development 3) TNF receptor/TNF death signal: kills target similar to Fas/FasL - the extrinsic apoptotic pathway, receptor mediated activation of caspase cascade & apoptosis

1. Review the structure of the antibody molecule by drawing a stick figure and labeling all the domains

110 AA in each domain

2. Compare and contrast the BCR of a B cell making IgM and a clone of that cell that has switched to another isotype. (Don't forget about the light chain)

2. Compare and contrast the BCR of a B cell making IgM and a clone of that cell that has switched to another isotype. (Don't forget about the light chain)

3. Describe with drawings how the DNA changes as a B cell develops and matures, include: a) Germline B cell DNA for the heavy chain, b) Mature naïve B cell DNA for the heavy chain, and c) Memory B cell DNA for the heavy chain

3. Describe with drawings how the DNA changes as a B cell develops and matures, include: a) Germline B cell DNA for the heavy chain, b) Mature naïve B cell DNA for the heavy chain, and c) Memory B cell DNA for the heavy chain see image

2. Describe what antibody affinity maturation is, and explain the mechanisms that result in the population of antibody increasing an affinity

Antibody affinity maturation is w/time the antibody population binds more tightly b/c of somatic mutations that allow tighter binding site at antigen binding site to the antigen so the B cells that have BCR that tightly binds are selected for and undergoes mitosis

1. Compare and contrast the meaning of a positive antibody test and a positive antigen test

Antibody tests: detect the antibody of heartworm in the blood. Can only tell if you have been exposed to it. Antigen test - used in vet clinic used to test antigen after infection. If antigen positive they are infected. If they have a positive antibody test can occur if they have been treated for heartworms. If the antigen is present the dog has the heartworm parasite, the dog could have the disease, was treated, no longer has the parasite but antibody is still present.

2. Explain the differences between apoptosis and necrosis

Apoptosis: active process of programmed cell death. There is not much tissue damage, membrane bound blebs are phagocytosed and digested and individual or cluster of cells that break apart. Necrosis: it is violent, uncontrolled, the membrane is degraded, cytoplasmic contents released, enzymes, inflammation, and there is a larger area involved.

1. Explain how the structure of the lymphoid tissues maximize opportunity for lymphocytes to meet antigen and interact with each other

B & T cells are close enough in lymph nodes and a concentrated amount that have different receptors for antigens. Free antigen in the tissue enters the lymph system and drains to a lymph node. Once it's present in the node, chemokines and adhesion molecules change to slow the movement of the lymphocyte that recognizes that antigen. Lymph node structures are made up of: • Subscapular sinus - reticular dendritic cells & macrophages receive the lymph from afferent lymphatics which contains any subcutaneous antigens • Paracortex (parafollicular area): T cells predominate along w/dendritic cells • Follicle: B cells predominate and arrange in nodules (or follicles), follicular dendritic cells found here • Medulla -efferent lymphatics: reticular cells, lymphocytes, or macrophages

3. Explain why the T and B cells are non-functional but NK cells and native immunity is functional. (in CID)

B & T cells are not formed and cannot populate the lymphoid tissue but NK cells are functional because they are from the lymphoid lineage and do not depend on DNA rearrangement

2. Explain why this disease is called combined immunodeficiency (CID)..

CID - combined immunodeficiency, SCID - severe combined immunodeficiency. It is an autosomal recessive inherited condition commonly from asymptomatic carriers

2. Explain how T and B cells are identified.

Can't histologically see the difference. Look at the surface molecules by using antibody w/fluorescent molecules.

. Please review the data in a technical bulletin for veterinarians for Intervet's Vista vaccine. Describe the basics of the experiment that was done to generate the data. Explain how and why the data on page 5 (shown below) support the statement that the vaccine provides CMI protection? Why would CMI protection be important for infectious bovine rhinotracheitis (IBR) virus. (The technical bulletin is posted in the Worksheet folder).

Cell-mediated immunity is an immune response that does not involve antibodies but rather involves the activation of phagocytes, antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines in response to an antigen. Historically, the immune system was separated into two branches: humoral immunity, for which the protective function of immunization could be found in the humor (cell-free bodily fluid or serum) and cellular immunity, for which the protective function of immunization was associated with cells. CD4 cells or helper T cells provide protection against different pathogens. Cytotoxic T cells cause death by apoptosis without using cytokines, therefore in cell mediated immunity cytokines are not always present. Vista 5 SQ is a modified live viral vaccine containing IBR, BVD type 1, BVD type 2, BRSV, and PI3 for the vaccination of cattle against respiratory disease caused by these five organisms which are part of the bovine respiratory disease complex (BRD). We know that peripheral blood mononuclear cells (PBMC) such as lymphocytes and monocytes are used in this experiment. They were taken up in the blood and plated causing the monocytes to differentiate into macrophages. To test if there was memory in the cells they added specific antigen (IBR) to the plate and had specific markers with fluorescein-labeled monoclonal antibodies (e.g. six color flow cytometry) to the cultured PBMCs so they detect certain structures such as CD4, IL 2R, and IFNγ cytokine all which are considered essential for Th1 production indicating that it is a memory cell. The cells would take up the virus and if there was memory present they would up regulate the IL 2R (which is also considered the CD25 expression). In this experiment we can say that the vaccine provided CMI protection because the CD4, CD8, and γδ levels were all significantly higher than compared to the control indicating that there was memory TH1 cells. CMI protection would be important for IBR because we want the body to quickly respond with the memory cells made from the vaccine so they can secrete cytokines and kill the virus.

7. Define hybridoma and include in your definition the cells that are used to make hybridomas and the purpose of hybridomas

Cells that make a hybridoma are the myeloma cell which is a plasma tumor cell & a B cell. The purpose of hybridoma is to make a lot of the exact same antibody molecule, it is a monoclonal antibody. We use these to get repeatable results since it's the exact same antibodies used in all the assays. TNF receptors is a monoclonal antibody • Monoclonal antibody (ie. Lymphocytes & monocytes/macrophage) production forms hybridoma. Monoclonal antibodies are derived from one cell that clonally expanded, antibodies produced are identical and recognize the same epitope • Hybridoma = a hybrid cell produced by the fusion of an antibody producing lymphocyte with a tumor cell (myeloma or plasma cell tumor) • Myeloma cell (non secreting tumor) added to B cell lymphocyte • Purpose is for selecting one clone of lymphocyte to make large quantities of antibody with a single specificity. Supernatant is the antibody secreted, all of these antibodies are identical. Get the same thing all the time. Like for the heartworm test, there is one epitope they react too.

1. Explain how an immune response to one type of pathogen can inhibit the response to another type of pathogen and give some clinically important examples where this may play a role

Effects and interactions of Th1 and Th2 Cytokines IFNγ produced by TH1 inhibits TH2 response IL 10 produced by TH2 inhibits TH1 response-vaccinating a parasitized animal (in this animal TH2s are high but when you vaccinate you want more of a TH1 response) IL10- suppresses TH1. (Dr. Brown- during the heavily parasitized and you vaccinate- TH2 response so strong the B cells made IgE and not IgG) Heavily parasitized animal w/strong Th2 response and vaccinate it w/a modified live viral vaccine & want a good Th1 response you may not get it b/c the Th2 cytokines will inhibit the response you want from that vaccine. This is influenced on a primary response not so much in a booster. Other example is a person w/bad allergies who gets a viral infection, sometimes they get a relief from their allergies b/c in fighting the virus they get a strong Th1 response which will inhibit the allergy Th2 response.

1. Describe, in general terms, how apoptosis is induced and what happens in a cell that is induced to undergo apoptosis

Either induced by extrinsic pathway where death is receptor mediated or by the intrinsic pathway through mitochondrial pathway and release of cytokine C. apoptosis is organize, the membrane remains intact and blebs form inside the cell. Either through extrinsic or intrinsic the caspase cap A will degrade cell and get apoptotic bodies

9. Compare and contrast the properties of antibodies in fish, birds and mammals

Fish antibodies: IgM is produced as a pentamer and monomer, antibodies have lower affinity, less diversity, and weaker memory than birds & mammals. IgM can be called IgX, IgR, IgW, IgD, IgY Bird antibodies: have IgM in serum, IgG in serum, IgA in serum and bile but no IgE Mammal antibodies: mammals have IgM, IgD, IgA, IgE, and IgM

9. Explain how birds transfer immunity to their young

Hen concentrates IgG from the blood into the yolk, IgA is produced in the oviduct and as the ovum passes through it acquires IgA in the albumin (egg white), as the chick develops it absorbs the yolk sac and the IgG is absorbed into the bloodstream. As the chick develops it swallows the albumin which contains IgA to coat the intestinal tract.

7. List the major cytokines that inhibit T cell responses

IFNγ inhibits Th2 response, IL10 inhibits TH1 production, TGFβ inhibits T cell proliferation.

5. List major cytokines that influence B cell isotype switching

IFNγ promotes B cell switching to C' fixing, IL4 promotes B cell switching to IgE. TGF beta influences cells to make IgA

6. List major cytokines important in B and T cell growth and differentiation

IL 2 - lymphocyte proliferation, induces T & B cell mitosis clonal expansion IL4 - produced by antigen presenting cells & signals TH0 cell to become TH2 IL 10 - influences macrophage to become M2 macrophage IL 13 - b cell to become IgE secreting plasma cell TGFβ - tells them to isotype switch to IgA IFNγ - promotes B cell switching to C' fixing

9. Review role of IL1 in immune response, e.g. when is it secreted, what cells secrete it, and some of its effects (see lectures 5 and 6 for review)

IL1 is a proinflammatory from the innate defense cytokines, it is not antigen specific, it responds to an invasion

9. Describe how an IL1 receptor antagonist may aid in managing arthritis

IL1Ra binds to the IL1receptor but does not stimulate the cell to release damaging enzymes

1. Explain the difference between immature and mature dendritic cells.

Immature: • Purpose is to capture antigen, has a lot of Fc receptors • Immature cells are found in the tissues and are efficient in antigen uptake and processing • Low surface MHC2 expression • High intracellular MHC2 ready for expression • High Fc receptor (for binding to antibody that has bound antigen) • Low co-stimulator molecules • Low cytokine production Once the dendritic cell contracts antigen and danger signals then it rapidly matures and migrates to lymphoid tissue in the T cell area. They secrete cytokines that attract T cells Mature: • Efficient antigen processing and presentation in lymphoid organs • High surface MH2 - presenting a lot of peptides • Low Fc • High co-stimulatory molecules • High cytokine production • Once it gets the antigen it matures - professional antigen presenting cell Dendritic cells- C3b and Fc receptors. Antigen that is opsonized can be easily phagocytosed and taken up by dendritic cells.

9. Explain what immersion vaccination in fish is and list several factors that influence the efficacy of immersion vaccination

Immersion vaccination is when fish are put in a tank that has vaccine in it for a bit of time so they can take up vaccine through their gills. Factors influencing antigen uptake include: • Antigen concentration, length of immersion • Water temp, pH, salt concentration • Size of fish - vaccination based on size vs. age • Stress levels influence response • Anesthetics, adjuvants, physical state of antigen (soluble, particulate) Better to have soluble b/c if particulate the water needs to be agitated so the antigen doesn't fall to the bottom.

1. Explain what makes the red line in a positive Heska Solo Canine Antigen test and what a positive result means

In the cassette is a nitrocellulose pad, embedded in the pad is antibody to the D.immitis uterine antigen. This antibody has a red bead covalently bound to it (AB-R). The sample is added and mix well so that they all "wick" through the pad. If antigen is present it will bind to the red bead. At the test pad area there is a monoclonal antibody to the D.immitis covalently bound to the pad. If antigen is present it will bind to the monoclonal antibody on the pad. The red beads will form a red line indicating antigen is present. If antigen is not present only a blue line from the control part of the test will be present.

12. Explain what is meant by the cytokine milieu.

Milieu means mixture so a cytokine milieu is a mixture of cytokines in solution

7. Explain how the CTL and NK cell are complementary, include timing, and targets, and the benefit to the host to have both cell types

NK cells can be used for innate defenses while CTL can be used for adaptive defenses, they both protect the host from intracellular pathogens. They will act on a cell that is not presenting MHC1. CTL will recognize cells on MHCI, then it will kill it.

2. Compare and contrast the immunoglobulin made by a B cell and the plasma cells differentiated from its clones

Only difference is a transmembrane portion, when it becomes a plasma cell it doesn't stick in the membrane and its secreted out, this is at its RNA level

2. Explain why there is a lack of IgM but IgG levels are ok in a day-old CID foal that received colostrum. (in CID)

Passive IgG is obtained from the colostrum and prevents infections early on, there is a lack of IgM because the B cells cannot be stimulated to make antibody.

6. Understand the role of IgM and IgD in B cell development and when they are expressed

Phase 1: generation of BCR in the bone marrow; it's antigen independent but interacts w/ stromal cells. Ig rearrange and result in BCR in the form of cell-surface IgM that can interact with the environment. Phase 2: negative selection in the bone marrow--IgM only on the B cell surface. Immature B cells with the IgM BCR that are strongly stimulated by antigen at this stage will die or change receptor (receptor editing. Negatively selected because they react with self-antigens. Phase 3: B cells migrate to peripheral lymphoid organs. After selection process then they get IgD. Surviving immature B cells go to periphery and mature and express IgD on surface along with IgM (mature naive B cell has IgM and IgD). IgD is a marker of maturation. Phase 4: antibody secretion and memory cells in bone marrow and lymphoid tissue; from differentitation of B cells with IgM and IgD. Can now be activated by specific foreign antigen in the secondary lymphoid tissue. When activated they clonally expand (proliferate) and differentiate into plasma cells and long-lived memory cells.

1. Haemophilus influenza B conjugate vaccine (diphtheria toxoid-conjugate) is a vaccine approved for use in people 18 months and older. The vaccine consists of Haemophilus b capsular polysaccharide covalently linked to diphtheria toxoid. In clinical trials the conjugate vaccine showed greater immunogenicity than the polysaccharide only vaccine. More than 90% of the children vaccinated with conjugate vaccine responded with antibody levels considered to be protective, whereas less than 50% of the children vaccinated with polysaccharide vaccine had such a response. Please explain the improved immunogenicity of the conjugate vaccine over the polysaccharide alone. Include the immune response a) what you would expect from the polysaccharide alone vaccine, b) what you would expect or get from the conjugate vaccine, and c) the reason for the difference.

Polysaccharide capsule - B cell can only see peptides so it doesn't see the capsule. If you want to induce antibody to the capsule (polysaccharide), you can covalently bind the protein to the polysaccharide so you get T cell help in the area where the B cell recognizes the polysaccharide. • B cell binds bacterial polysaccharide epitope linked to tetanus toxoid protein • Antigen is internalized and processed via the exogenous pathway of the B cell • Peptides are presented to MHCII on T cell • TH cell recognizes peptide (epitope) than B cell, and TH receives signals from TCR and co-stimulatory molecules (CD4) so you have a good antibody response • TH secretes cytokines into confined space between the B cell and Th cell • Activates B cell which proliferates • The antibody produced is to the polysaccharide the BCR recognized on the surface of the bacterium. If you don't have the protein and only had the B cell responding to the polysaccharide you are not having T cell recruitment so you don't get good isotype switching b/c its all the cytokines from the helper cells that help it class switch, also you don't get good clonal expansion of B cells since there is no IL2 being secreted by Th1 cells.

2. Explain how antibody diversity is generated.

Random event that requires cutting and splicing of DNA.

2. List the effector function including signature cytokine (s) of each of the TH cell subtypes

Signature cytokines for the different TH subtypes: • TH1 = Signature cytokine: interferon gamma (IFN γ)-MI macropahges(causes them to make more NO and lysosomal enzymes) IgG enhance cell mediated immunity Secretes cytokines that activate M1 macrophages, neutrophils, NK cells. Secrete cytokines that signal B cells to make opsonizing and complement fixing antibodies (IgG) for extracellular bacteria or viruses (systemic) (even though IgM can fix complement you won't get high tittered high affinity IgM) • TH2 = IL 4, IL 5, IL 10, IL 13 influence B cells to make IgE Secretes cytokines that activates mast cells and eosinophils and activate B cells to make IgE. Important in IgE mediated allergies. Defense against helminthes and other parasites and mucosal pathogens. Player for allergies that are mediated by IgE- genetic link- bad allergies tend to make too much IL4 • TH17 = IL 17 A & F, IL 22 results in chemotaxis & reaction of neutrophils Attract and activate neutrophils and monocytes; lead to acute inflammation and important in response to extracellular bacteria and fungi Add on to acute inflammation and innate response. This is the adaptive immune system secreting cytokines to bring more neutrophils to the site. • Treg = immunosuppressive cytokines like TGFβ suppresses the immune response Suppress T cell response by secreting suppressive cytokines or by cell-cell interaction to prevent autoimmunity. Helps prevent reactions to self-peptides. If a naïve T cell recognizes an antigen without any danger signals it may become a T reg cell. TGFB- immunosuppressive cytokine- don't want inflammation in the gut all the time

1. Describe the circulation of mature naïve and memory T and B cells, mainly T cells how is this related to a positive intradermal TB skin test?

T cells in general circulate more than B cells. Once a B cell is a plasma cell it goes wherever it wants to. Naïve lymphocytes circulate between bloodstream and lymph differently than memory lymphocytes. The lymphocytes circulate under the direction of addressin (adhesion molecules) and chemokines. Remember, a naïve lymphocyte is a mature lymphocyte that has not met its antigen Naïve T cell: • Cross wall at the postcapillary high endothelial venules. Use selectin mediated loose binding then integrins allow tight binding on high endothelial venules (HEV) so they diapedese into the paracortex and from there • Go into certain areas by addressin molecules - ie B cells go to the follicles and T cells go to the paracortex region. • The naïve T cell percolate through the organ looking for its antigen • Exit through efferent ducts into the thoracic duct and go back into circulation through the anterior vena cava. Memory T cell: • Will leave the blood in the tissues through the endothelium where the infection is. Do not leave through the lymph node. • Cytokines secreted in result of that and then they are up regulated by adhesion molecule on the epithelium at the high endothelial venules. If the antigen is not there, it will go into the afferent lymphatic's, goes into a lymph node, percolates through, still finds no antigen and goes out through efferent lymphatics back into the thoracic duct and back into the circulation. Effector B cell does not circulate, stays in bone marrow or spleen and doesn't secrete antibody. Naïve B cell circulates the same way as the naïve T cell see page

1. Use your understanding of immunologic response to describe the responses to vaccinations

TCR and CD4 recognize MHCII peptides (APC processes antigens and presents them via the exogenous pathway). APC secretes cytokines (IL1, IL6, TNFα, IL12, IL15) that signals the TH-cell. The specific T cell needed secretes cytokines (IL4, GM-CSF, IFNγ,TNFβ) to bring about immunity in response to the signals from APC. (The live vaccine will stimulate CD8 cells to react quicker Modified live will infect the cells to be process through the endogenous pathway. Modified life intranasal will go through mucosal surface and activate IgA. )

4. List the main Th1 and Th2 cytokines and give some important functions associated with these cytokines

Th1 - IL2, IFNγ, TNFβ, TNFα activate macrophages and neutrophils to be better at killing, T cell and NK cell cytotoxicity, & IgG production for opsonization. Specifically the activation of IL2 will stimulate the nucleus to have clonal expansion to allow more IL2R to have a higher affinity. IFNγ influence macrophages, natural killer cells, neutrophils, CTL. Th2 - IL 4, 5, 10, 13 IL4: influences B cells to class switch to IgE IL 5: used in adaptive immunity - activates eosinophils and stimulates bone marrow production of eosinophils. IL10: immunosuppressive and anti-inflammatory cytokine, ex. Inhibits Th1 formation, inhibits IL2 production, and influences macrophages to become M2 macrophages IL13: similar actions as IL4.

13. Describe a "cytokine storm" and possible outcomes.

That means a lot of cytokines produced all at one time. Pro-inflammatory cytokines that are in low, moderate, or high levels - high levels is a cytokine storm. You could die from this (lecture 7/8) you could get leakage into the lungs, intracellular coagulopathy which leads to death

6. Explain what "isotype switching while maintaining antigen specificity" means

The antigen specificity does not change but the effector function does, ex. Isotype switch from IgM to IgE can result in an effector change of complement fixation to mast cell sensitization

1. Explain what the precipitate is in a precipitation assay and what the equivalence zone means. Describe why there is no precipitate when there is antibody excess or antigen excess.

The zone of equivalence is when you have just the right amount of antigens and antibody causing them to cross-link and form a precipitate. Uses are Coggin's test for equine infectious anemia (EIA) or AGID for influenza antibodies. A single antigen molecule binds to a single site and can be bound to one antibody. So there is no precipitate when there is excess antibody or antigen because there is only one binding site available.

2. Explain how this defect affects T and B cells. (CID)

There is a defect in DNA - protein kinase enzyme, w/out this enzyme functional BCR/TCR cannot form b/c they cannot splice the VDJ regions together to form the variable portion of the receptor.

5. Explain how the complete blood count and lymphoid tissues are affected in a foal with this disease. (CID)

There is a low lymphocyte count but other WBC (neutrophils, monocytes, and eosinophils) are ok b/c the myeloid lineage was not affected. When looking at the lymphoid tissue - thymus has almost all epithelial and stromal components. The lymph nodes lack lymphoid follicles and germinal centers, cellular depletion of paracortex, NK cells, fibroblasts, & macrophages are present. In the spleen there is a lack of germinal centers & periarticular sheaths.

2. Explain the mechanism of action of Type 1 interferons, where they come from, and explain their role in immunity

They induce an antiviral state in neighboring cells by binding to receptors that stimulate cells to make enzymes that block viral replication. They enhance susceptibility to CTL killing by increasing expression of MHCI, also activate NK cells to be more aggressive and push response to TH1 cells. Type 1 (innate immunity) interferons inhibit protein synthesis in all cells (not so good), interferons are produced 12-24 hours after exposure to infection. The name interferon means it interferes w/virus replication. In adaptive immunity you won't see IgM until 7-10 days later. see flow chart

9. List the immune system components of crustaceans and explain the challenge of vaccination of this species

They only have innate immunity so there are memory in innate immunity, the challenge of vaccinating them that since they don't have an adaptive immune system there is not much potential to vaccinate

1. During some viral infections there is a decrease in MHC I expression. Is this an advantage for the host or the virus? Explain.

This is an advantage for the virus because that means it has time to replicate and cause infection in the host cells without being detected by CD8 since there is a decreased MHCI expression.

1. Describe how the thymus changes with age and how that relates to immunity and disease later in life

Thymus involutes w/age most of the clones are formed before puberty or even birth. As you get older the chance of getting disease increase b/c of thymus involuted. Thymus dependent immunity will decrease. Certain diseases such as glucocorticoids can accelerate the thymus to atrophy. Increases cancer, autoimmunity, and diseases.

1. Define titration and explain how a titer is determined.

Titration - measurement of the level of specific antibodies in serum, achieved by testing increasing dilutions of the serum for antibody activity. Titer - highest dilution of the serum that gives a reaction in an immunologic test. see image

2. Compare and contrast Type 1 interferons (alpha and beta) and Type 2 interferon (gamma).

Type 1 interferon = part of native immunity, produced by virally infected nucleated cells. When viruses replicate and spread it induces IFN1 and are inhibited by it (makes cell resistant to infection) Type 2 interferon = important for adaptive and innate defenses. Produced by NK cells and activated by TH1 cells. Ex. IFNγ activates macrophages, neutrophils, and NK cells & promotes more TH1 development, inhibits Th2, promotes B cell switching to C'fixing, opsonizing IgG production, increase MHC expression on APC.

1. Explain T cell education - when, where, how: a. Development of important surface molecules b. Positive Selection c. Negative selection d. Role of thymic epithelial cells

a. Development of important surface molecules T cell precursors go from bone marrow to the thymus and go to the cortex where they mature to get their receptors. Don't have CD4, CD8 or TCR at that time. Develop TCR is in the thymus by rearranging the DNA so now it can have all 3 components. b. Positive Selection TCR, CD4 or CD8 recognizes MHCI or MHCII c. Negative selection If it recognizes the self-peptide in the groove than it dies. You don't want recognition of self d. Role of thymic epithelial cells Purpose is to present self peptides. ex. Liver cell only expresses some of the genes to make that DNA, they get specific to what they express. But thymic epithelial cells will make a lot of proteins from expressing a lot of genes that are put on MHCI and MHCII.

3. Describe where in the body, and explain what is happening during, the following stages of B cell development:

a. Formation of the antigenic binding site: the variable portion of the heavy chain (Vh) is formed by the random selection of one V segment, one D segment and one J segment. The variable portion of the light chain (VL) is formed by the random selection of one V segment and one J segment. b. Negative selection: in primary lymphoid tissue, cell gets second chance to make receptor or told to die.. If a B cell meets an antigen at this time it will undergo apoptosis (negative selection) c. Class switching: occurs in memory B cells • Will change it constant heavy chain part out in the secondary lymphoid tissue d. Affinity maturation: occurs for IgG, IgE & IgA • Somatic mutations (random): high mutation rate (hypermutation) in VDJ region of DNA results in low affinity and these will undergo apoptosis, high affinity ones are selected for which occurs in secondary lymphoid tissue • High affinity binding BCR are preferentially stimulated; the tighter the binding the more stimulation which leads to mitosis and more clones.

9. Explain how IL1 in joints can result in arthritis

chondrocytes & synoviocytes release IL1 and TNF-->IL1 binds to chondrocytes via IL1 receptors-->release of enzymes-->collagen and proteoglycan degradation-->loss of articular cartilage-->osteoarthritis

7. List a few important properties of a vaccine that would stimulate CTLs

o Has to have antigen inside cytosol to get processed and presented by endogenous pathway to be presented to MHCI cells. o Iscomms has a lipid bilayer that fuses w/cell membrane and puts the antigen on the cytosol - it gets loaded onto MHC1

7. Give an example of an infection that CTLs would be important for controlling

o Important for viral infections o Antibody can't get inside of a cell

1. Describe how dendritic cells and follicular dendritic cells are different, i.e. origin, antigen presentation, locations

see page

1. List the different types of lymphocytes and how they recognize antigen

see page

3. Compare and contrast the immune responses to extracellular pathogens/toxins, to cytosolic pathogens, intravesicular pathogens, and helminthic parasites

see page - Extracellular pathogens- (in the vesicle) Will stimulate TH1 and TH17 via MHC II pathway o Exogenous to MHC II o Two effectors TH1- • Help get IgG to fix complement • Secretes IFN which activates macrophages to M1 to kill TH17- • Secretes IL17 which Attracts neutrophils to kill bacteria - Cytosolic pathogens o Endogenous pathway to MHC I to CD8 cytotoxic T cells o Effector: CTL o Outcome: kills infected cell - Intravesicular pathogens- o Exogenous to MHC II o Effector: TH1- is important in intravesicular infections inside the macrophages o Outcome: TH1 secrete cytokines to activate macrophages to be M1 cells that will kill the cell. - Helminthic parasites (in the vesicle) o Exogenous to MHC II o Effector: TH2 cells provide defense against helminthes by secreting IL4, IL5, IL10, IL13 o Outcome: IgE production

1. Give an explanation for why test kit reagents should be at room temperature before running the tests.

why blood has to be at room temp: Because it has to diffuse for antigen and antibody binding they need to be able to incubate at the correct temp or the test will not work. Temperature effects antigen and antibody binding. test kit reagents?

1. Explain what a γδ T cell is and its role in the immune response

γδ T cell is describing the type of T cell receptor it has. They are part of adaptive immunity have characteristics of innate immunity. They respond to stress molecules and secrete cytokines and induce cell killing, they are important upon mucosal surfaces. Recognized intact unprocessed antigens and self surface stress proteins. Found on mucosal surfaces. Help protect epithelial surfaces through cytotoxicity and cytokine secretion. Respond faster than alpha beta T cells (this is why it has innate tendencies)

7. Describe how an NK cell recognizes a cell to kill and describe the killing mechanisms of the NK cell

• Absence of MHC I will cause NK cells to bind to them and kill them using the same mechanisms as the CTL. • It can also kill cells expressing MicA or MicB (stress proteins). • They can kill by ADCC- antibody dependent cell mediated cytotoxicity- if an antibody binds, the NK cell has Fc receptors for the antibody and will kill the cell.

7. Compare and contrast the various killing mechanisms used by the immune system

• Apoptosis: NK & CTL kill them • Oxidative metabolism - kill cells by H202 & NO, done by neutrophils & macrophages • Netosis - neutrophils spew out histones, granular things to trap bacteria. • Lysozomal enzymes - used in neutrophils & macrophages which breaksdown peptidoglycans. • Defensins - that poke holes in membranes

1. List the important surface molecules that distinguish T and B cells and describe their function.

• B cells have BCR which is an antibody molecule w/a transmembrane surface, they are signal transduction molecules for the receptor (Igα & Igβ) • T cells have 2 types of TCR o αβ and γδ and they bind antigen peptide and MHC o CD3 is the signal transduction molecule (pantine marker, general), CD4 binds to MHCII and CD8 binds to MHC I.

5. List the ways tolerance is induced - include central and peripheral tolerance.

• Central tolerance is induced in the primary lymphoid tissue (bone marrow, bursa of fabricius, peyers pathches), they can o Die by apoptosis o B cells edit its antigen receptor to recognize a different antigen o CD4 T cells become T regulatory cell and suppress T cell response to self-antigen • Peripheral tolerance when a mature naïve lymphocyte recognizes self-antigen w/ TCR in secondary lymphoid tissue ( spleen, ln, MALT) in the absence of "danger" signal it will either o Die by apoptosis o Needs co-stimulatory molecules and cytokines around to become activated o Become anergic - unresponsive o Be suppressed, occurs during fetal development

3. Explain the basic concepts of the clonal selection theory and how it relates to tolerance and memory.

• Clonal selection theory: many cells with specificity all have identical antigen receptors on a single lymphocytes the antigen bind and stimulates the specific lymphocyte produces specific antibody to antigen • This occurs in Primary lymphoid tissue o The differentiated effector cell will have the same receptors of specificity as those of the parental cell they derived from - Memory o Lymphocytes that bear self-molecules are deleted at an early stage in development and are absent from the repertoire of mature lymphocytes -Tolerance

1. Explain and define cytokines - in general: where do they come from, what do they do, and where do they act (autocrine, paracrine, and endocrine)

• Cytokine - induced protein molecules that are secreted by stimulated cells that regulate intensity and duration of innate and adaptive immune responses. • Function - mediate & regulate (enhance or inhibit) innate immune responses, mediate & regulate adaptive immune responses, mediate & regulate hematopoiesis • Where they come from - come from being secreted by an antibody • Where they act - o Autocrine: cytokine that is made by a cell and then acts on itself (IL2 which induces clonal expansion) o Paracrine: act on another cell nearby like IL12 o Endocrine: at a distant location like IL3, GMCSF that act on bone marrow to increase more WBC

10. Describe how some cytokines might be used clinically

• GCSF - stimulates neutrophil differentiation, used in chemotherapy patients

2. List two ways lymphocytes enter the lymphoid tissues from the blood stream

• High endothelial venules: which are in the lymphoid tissue that involve Naïve T & B cells (lymphocytes). • Afferent lymphatics: out through the tissues and enter the afferent lymphatics which brings them to the lymph nodes done by memory B & T cells.

1. Define lymphocyte homing

• Lymphocytes that have met their antigen in a peripheral ln. will home and stay w/in that system. • They have different addressins that send them to specific lymph nodes. The cells end up where they are based on the adderssins that are upregulated and downregulated that provides direction. The types of addressins depends on the cell

1. Describe what you could do to get T cell help to B cells when B cells are responding to a polysaccharide antigen (like a bacterial capsule). Remember TH cells recognize peptide in MHC II molecules

• Polysaccharide capsule - B cell can only see peptides so it doesn't see the capsule. If you want to induce antibody to the capsule (polysaccharide), you can covalently bind the protein to the polysaccharide so you get T cell help in the area where the B cell recognizes polysaccharide. • B cell binds bacterial polysaccharide epitope linked to tetanus toxoid protein • Antigen is internalized and processed via the exogenous pathway of the B cell • Peptides are presented to MHCII on T cell • TH cell recognizes peptide (epitope) than B cell, and TH receives signals from TCR and costimulatory molecules (CD4) • TH secretes cytokines into confined space between the B cell and Th cell • Activates B cell which proliferates • The antibody produced is to the polysaccharide the BCR recognized on the surface of the bacterium. • If you don't have the protein and only had the B cell responding to the polysaccharide you are not having T cell recruitment so you don't get good isotype switching b/c its all the cytokines from the helper cells that help it class switch, also you don't get good clonal expansion of B cells since there is no IL2 being secreted by Th1 cells. (Polysaccharide is a poor antigen to begin with- if you just had polysaccharide capsule it would be taken up by B cells and presented but results in no recognition- the B- cell would then secrete IgM)

1. Define precipitation and agglutination and explain the difference.

• Precipitation: forms a clumping of a soluble antigen and soluble antibody bind together at optimal proportions • Agglutination: the clumping of

1. List the three professional antigen presenting cells and give brief explanation of how/when they present antigen

• Presents on MHC II. If antigen is opsonized it has been done w/ IgG to bind to Fc portion (could also be done w/acute phase proteins or C3b). It would perform phagocytosis by exogenous pathway. The lysosomes break it up and in the endoplasmic reticulum the phagolysosome and clip is then removed. Then loads membrane and puts it on the membrane. • Dendritic cell: can present to naïve T cells, they are the only cells that can do this. It takes more to stimulate a naïve cell than an adult cell. You have signal from the antigen (TCR, CD4 binding), and pantine marker (CD3) causes a signal to the nucleus to trigger a primary response • The co-stimulatory molecules (when taken in and up regulates its co-stimulatory molecules) which send another signal which is receptor mediated and are cytokines. So if you have an IL12 it would become a Th1 b/c of influence of IL12. If IL4 that would form Th2. • B cell & Macrophage: also antigen presenting cells and present to a memory T helper cell (that has met its antigen)

4. Compare and contrast a primary and secondary antibody response, include the basis for the difference in the lag time, titer, antibody class, and antibody affinity.

• Primary response: naïve response is longer for lag time, has a lower titer and lower antibody binding affinity, and B cell differentiate into plasma cells that secrete IgM. • Secondary response: anamnestic (memory) response is more rapid, higher titer and more clones and the antibody binds with higher affinity because of somatic mutation during cloning, they produce memory IgG cells that are stimulated to become effector plasma cells.

1. List the basic principles of antigen/antibody binding used in immunodiagnostics.

• Reversible & go to equilibrium. Remember Ecoli question from the first exam initially a certain amount of E.coli is in solution, antibody serum is added to the sample then it is incubated and a fresh amount of E.coli is added. There is still the same amount of E.coli that there was initially in the sample. They are coded w/the same amount of antibody b/c they go to equilibrium. • Temperature dependent • Antigen or antibody is bound to solid surface such as plastic or beads and will not affect the ability to bind to antigen or antibody. • Labeled antigen or antibody will compete equally with unlabeled antigen or antibody.

Describe high endothelial venules, function, surface molecules, and location

• Special endothelial cells that have a cuboidal shape, under cytokines influence endothelial height and become more columnar • High endothelial venules: postcapillary venules that are by lymph nodes and lymphoid tissue. Not found in the spleen • Lined w/high endothelium that has more space to allow for diapedesis and has selectins and integrins for loose and tight binding. • Bind to the T cell circulating and bring that to the lymphatics

8. List major cytokines from the adaptive immune system that enhance innate immunity

• TH1 makes it a better natural killer • IL5 from TH2 cells will activate eosinophils • TNF from TH1 will activate neutrophils and regulate endothelial cells

List the major signals required to get activation of a T helper cell and factors that determines the subtype of TH cell that predominates in a response

• There are 3 signals received if it is a naïve T helper cell: 1) T cell receptor to bind to the MHCII and the peptide, you need the CD4 to bind to the constant region of MHCII that will cause the CD3 to send a signal to the nucleus 2) co-stimulatory molecules to bind to the ligand on the antigen presenting cell 3) APC cytokines: to bind to the receptor of the T cell to get the 3rd receptor • For a naïve T cell you need all three signals to get them activate It's specific antigen recognition on MHCII- uses its TCR to bind to the peptide and MCH11 and CD4 sees the constant portion of the MHC2 Co-stimulatory molecules- they have a lot of influence on the T- cell response Cytokines These three signals are needed by a naïve cell. Which APC can activate naïve T cell?- Dendritic Cell.

1. Define Langerhans cell

• They are immature dendritic cells present in the skin which pick up antigen then go to the lymph and present antigen to the T cell. They mature and enter T cell rich area of the lymph node and present antigen to T cell.

List major cytokines important in hematopoiesis and describe their function/impact on the immune response

• Thrombopoeitin and IL11 - stimulate platelet production following hemorrhagic event • IL3 - stimulates myeloid progenitor cells to form platelets, basophils, neutrophils, eosoinophils, and monocytes • IL5 - stimulates eosinophil differentiation during parasite infection/allergy response • Granulocyte monocyte colony stimulating factor (GMCSF) - stimulates neutrophil and monocyte differentiation • GCSF - stimulates neutrophil differentiation, used in chemotherapy patients • MCSF - stimulates monocyte differentiation

1. Describe the most conserved immune protective mechanisms present in insects up through mammals.

• Two common things shared between all species toll-like receptors (TLR) and antimicrobial peptides like defensins. • Alternative pathway of complement is not insects but in all other species listed. • Adaptive immune response sharks through mammals have adaptive immune system which includes: T cell, b cell, antibody, classical complement pathway, MHC molecules

A killed vaccine for a virus is given in the subcutaneous tissue between the shoulder blades of a dog never vaccinated before. a) Where would you expect the antigen to stimulate lymphocytes that recognize it? b) What cells will be presenting antigen? c) What signature cytokines do you expect will be (and do you want) secreted from the antigen presenting cell? d) How will a T cell recognize the antigen - diagram how the antigen is processed and presented to the T cell? e) What type of T cells will be activated and what types do you think will provide the best immunity? f) How will a B cell recognize the antigen - diagram how the antigen is recognized by the B cell? g) How does antibody help to protect from infection? The dog returns three weeks later for a booster. What is different about the response to this dose compared to the first dose and why?

• Where would you expect the antigen to stimulate lymphocytes that recognize it? It would stimulate the dendritic cells to pick up antigen, through the exogenous pathway, and bring it to local lymph nodes where the antigen peptide will be presented to CD4 cells that would undergo clonal expansion. • What cells will be presenting antigen? Dendritic cells will be presenting the antigen via the MHC II to the TCR receptors on the CD4 T cell. • What signature cytokines do you expect will be (and want ) secreted from the antigen presenting cell? • Th1 - IL2, IFNγ, TNFβ, TNFα activate macrophages and neutrophils to be better at killing, T cell and NK cell cytotoxicity, & IgG production for opsonization. Specifically the activation of IL2 will stimulate the nucleus to have clonal expansion to allow more IL2R to have a higher affinity. IFNγ influence macrophages, natural killer cells, neutrophils, CTL. Th2 will not be used because certain cytokines such as IL5 are only used in adaptive immunity. • How will a T cell recognize the antigen - diagram how the antigen is processed and presented to the T cell? focus on the exogenous pathway and the presentation to CD4 T cell • What type of T cells will be activated and what types do you think will provide the best immunity? T helper cells are most likely to be activated, most likely the Th1 will provide the best immunity because it will stimulate the innate defenses • How will a B cell recognize the antigen - diagram how the antigen is recognized by the B cell? o B cell binds to the epitope on the viral coat protein using its BCR o It will signal the nucleus to have clonal expansion to make memory IgM B cells and plasma cells - IgM. • How will antibody help protect from disease? Antibody present in the immune system will help stimulate a shorter lag time to stimulate an immune response if exposed to the virus so your body can actively fight it off 3b. The dog returns three weeks later for a booster. What is different about the response to this dose compared to the first dose and why? The booster will ensure that the dog has a good immune response occurring, by having the booster there is a secondary response occurring thus resulting in a higher affinity to the antibody with a short lag time. This will help ensure a strong immune defense against this virus The booster will ensure that the dog has a good immune response occurring, by having the booster there is a secondary response occurring thus resulting in a higher affinity to the antibody with a short lag time. This will help ensure a strong immune defense against this virus