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Intro Lecture
Immunology - the study of the body’s defense against infection Historical Roots of the Study of Immunology: ● 1798 Jenner: cowpox immunization ○ Recognized milk maids were more resistant (showed immunologic memory and immunologic specificity) ○ vacca meaning cow ● 1891 Koch: DTH vs Tuberculin Ag ○ Studied Delayed Type Hypersensitivity ○ Ex. poison ivy b/c it takes time for cells to migrate towards exposed area ○ Ex. TB vaccine b/c you get exposed to a little protein and a reaction occurs later if you have the disease ● 1895 Bordet: Complement + Antibody + bacteria = lysis ○ Complement is a component of serum that acts in conjunction with antibodies to destroy pathogenic bacteria ● 1901 Landsteiner: ABO blood groups, if antibody A clumps blood, then you have Type A ● 1914 Little: genetic theory of tumor transplantation (same as organ transplants) ○ Showed determination of self vs. non-self ● 1936 Gorer: identification of MHC antigens ● 1939 Kabat and Tselius: antibodies as gamma globulins ○ Antibodies- proteins that have affinities for other specific proteins
2 - Immune Response
Suppression on inflammatory response gives relief When you upregulate the immune system, it could cause an autoimmune disorder Psychoneuroimmunology - studies the relationship between immunity, the endocrine system, and the central and peripheral nervous systems Pavlov conditioning has been shown to work in the same way that the immune system can work Autism, Parkinson's → do they have inflammatory components to it? Antibodies can get into the brain, disproving that nothing can pass through the blood brain barrier Lymphatics in the brain meninges, keeps the inflammation out of the brain Xenotransplantation - transplanting donor tissues from other species to people who have defective tissues ● A way of replacing necessary tissues (hearts) for which we do not have enough a supply for Design an antibody to act as an enzyme in stabilizing the intermediate stage ( abzymes ) ● Abzymes → antibodies with a variable region possessing enzymatic activity Hematopoiesis - the formation of blood (in bone marrow) → start with pluripotent (stem cells (PSCs) and gives rise to either 1) Common Myeloid Progenitor (CMP) or 2) Common Lymphoid Progenitor (CLP) ● Lymphopoiesis → produce lymphocytes
Common Myeloid Progenitor (CMP) → Erythrocytes, Thrombocytes (from megakaryocytes), Mast Cells, Monocytes (Macrophages, Dendritic), Granulocytes (Basophils, Eosinophils, Neutrophils) → ALL Leukocytes
- Myelopoiesis – RBCs, Thrombocytes, Granulocytes, Monocytes (myeloid cells) ● Granulocytes: ○ Neutrophils (most abundant) – polymorphonuclear neutrophils (PMN) (i.e. irregular shape), die quickly after arriving to scene (~ 3 days) ■ Multi-lobed, arrive earliest in most sites of infection, doesn’t require amplification, largest fraction of granulocytes ■ Organize lamellapod to chase bacteria and engulf it ○ Eosinophils – 2-5%, stains pink with eosin, helps respond to allergies (endoparasites) ○ Basophils – 0.1% of WBCs, stains blue with hematoxylin, IgE binds to Fc changes upon differentiation receptor, helps respond to allergies (ectoparasites) (ex. tics) ● Monocytes: largest type of WBC, differentiates into macrophages or myeloid lineage dendritic cells, part of the innate immune system but influences adaptive immune system, matures upon entering tissue, involved in antigen-processing/presentation by engulfing an antigen and presenting it to other cells to activate a cascade (tissue macrophages) ○ Dendritic cells pinocytose the surrounding environment to scan for intruders. Will phagocytose intruder and present the antigen, then they migrate to lymph nodes and activate adaptive immune response ● Thrombocytes: clotting, vasodilation to allow cells to leave vessel and go to tissue, make complement-activating factors, make chemokine (immune hormones that regulate immune response) ● Mast Cells: molecular on the surface of FC epsilon receptor (for IgE), when activated causes the release of histamines, specifically activated by things like pollen ○ Important in allergic reactions (histamine causes vasodilation and vessel poration) Common Lymphoid Progenitor (CLP) → T-lymphocytes (T-cells), B-lymphocytes (B-cells), NK cells
- Lymphopoiesis – production of lymphocytes in lymphoid tissue (bone marrow for B-cells, Thymus for T-cells), NKs ● CLP in bone marrow migrate to either:
- Thymus (immune response organ above heart) ● CD8+ glycoprotein on immune cells with T-cell receptor ● CD4+ with T-cell receptor (more abundant & killed by HIV)
- Bursal Equivalent (source of antibody-producing B cells). B-cell differentiation occurs in fetal liver, yolk sac, bone marrow, spleen
- Natural Killer (NK) cells don’t have T or B cell receptors, but are large, granular leukocytes with lots of vesicles. They get rid of defective cells (ex. cancer) and don't go through thymus, but come from CLP! ● T-Cells: activated to become part of the adaptive immune response (less specific)
● Delicate process and can have a lot of different choices Determination- genetic change FMLP, experimental peptide for chemotaxis Till + McCullough 1950s ● Took a mouse (A) took the bone marrow out, which had hematopoietic cells, put it into another mouse (B) into the tail vein, then waited 9-11 days ○ Mouse B was irradiated to cause DNA damage to eliminate intrinsic immune system to leave room for transplantation ○ Strain of donor and recipient mouse were the same (near identical twins) so there's no tissue rejection ● Then took out the spleen from the mice (lymphoid organ), and found that the hematopoietic cells migrate to the spleen causing a little colony ○ Spleen has red pulp for rbc removal from damage, and white pulp the immunological part of it ○ Look like little pimples, colonies of different stem cells ● Wanted to know how much radiation was need to kill the new stem cells, so they gave them different doses of radiation ● CFU-S assay ○ Colony forming units of the spleen ○ CSFs stimulated these factors Surface molecules as name tags → way to identify a molecule was their ● Rabbit anti-mouse brain antiserum ○ Bound to thymus ○ RAMB → theta → Thy- ○ T200, B220, LCA → cluster of differentiation or their CD number ■ Became CD ○ CDs allowed for more efficient cross talk between labs
Lecture 3
Myeloid Lineage - blood cells that includes all leukocytes except lymphocytes Mast Cells - large, granule-rich cells found in connective tissues throughout the body (most abundant in submucosal tissues and the dermis) that store and release bioactive molecules (histamines). Crucial in allergic reactions. Has a molecule called Fc, facilitates phagocytosis Cytokines - proteins made by a cell that affect the behavior of other cells, especially immune cells, they are made by lymphocytes, often called interleukins (IL) Neutrophils → included in the early reaction to S. aureus infection as they are usually the first cell at the site of infection (innate immunity) Primary Immune Response (IR) - the adaptive IR that follows first exposure of an antigen. Baseline is positive. Encounters a foreign antigen → small response (small bump). We vaccinate in order to activate this response
Secondary Immune Response (IR) - IR to second exposure to an antigen & generated by the reactivation of memory lymphocytes. Begins sooner after exposure, produces greater levels of antibodies & produces class-switched antibodies. Re-exposure to the same antigen creates a much more robust IR with a longer duration and shorter lag time than the Primary IR Innate IR uses Myeloid cells Adaptive IR uses lymphocytes ● Cell-mediated immunity uses T-cells as the effector mechanism ● Humoral immunity uses B cells (antibodies) as effector mechanisms. Antibodies are found in blood plasma and extracellular fluids Monocytes are professional antigen-presenting cells ● Tissue macrophages → monocytes in circulation can become macrophages when it enters tissue ● Dendritic cells → bone marrow-derived cells found in most tissues, including lymphoid tissues. Monocytes in circulation can become dendritic cells when it enters bone marrow or surrounding tissues (looks like a sea urchin) Principal Component Analysis (PCA): ● Axis are not a single gene but characteristics of a group of genes ● The 2 axis tell us the major set of genes that are within the cells types ● If there are lines between the populations, that is a transition state between the populations All lymphocytes are leukocytes, not all leukocytes are lymphocytes The cells are organized into: ● Primary lymphoid tissue ○ Origin: tissue at the endodermal and ectodermal junction ○ Time: during early embryonic stages ○ Fate: involutes after puberty (becomes acellular) ■ Once you reach puberty, they begin to lose their WBC compartment and become connective tissue ○ Surgically Remove: organ becomes unresponsive to the antigen, lose a major population of the cells ○ Ex. thymus → bursal equivalent (bone marrow, liver, yolk sac) ● Secondary lymphoid tissue ○ Origin: tissue at mesoderm ○ Time: fetal life ○ Fate: persists through life ○ Surgically Remove: modest decrease in the immune activity ○ Ex. lymph nodes, spleen Peyer’s Patches → in the body of the intestinal wall ● Important in our sensing of materials in the lumen of the gut ● If we were highly responsive to the things we eat and they caused inflammation, then can cause hypersensitivities Mucosal Associated Lymphoid tissue “MALT”
● Cellular → the cell is affecting the change (differentiation) ○ Aggressive: hyperplastic response where they over grow the other (coral land and grow near one another and when they encounter is they can try to over grow the other, again, recognizing that the other sponge is different ○ Non Aggressive: cell sorting in sponges (can be dissociated into single cells, 2 sponges from different species and mix them, they have the capacity to sort themselves back out won't stick ott he foreign subcell) sorting suggests that they can recognize nonself between surface to surface contact ○ Engulfment: mollusk recognizing something foreign and phagocytosis it (hemocytes) ○ Cytotoxic:” flatworm type” have hemocytes, no memory, can only recognize xenogeneic (diff between species), different from allogeneic (individuals of the same species), and different from syngeneic (genetically identical) ■ “Earthworm type” somememory, can recognize xeno + allo ■ “Mouse type” includes humans, strong memory, xeno + allo ● Humoral → soluble molecular reaction that is found in humans in the liquid fraction of the blood ○ Inducible: ■ Homogeneous: one molecule that does the job ● Lobster bactericidal, turned on in the presence of bacterial stimulation and causes the bacteria to die ■ Heterogeneous: ● Vertebrate antibodies ● Ex. an antibody-mediated response ○ Noninducible: there all the time (constitutive) a snail agglutinin (clumps foreign molecules together) *How would you describe a skin graft exchange between a flatworm and an earthworm? To transplant a tumor, it has to be from the same mouse species to same mouse species
Lecture 4
Cytokine storms can be fatal CD Llama and camel make a stable antibody compared to the human If molecules are similar to one another, then they can be useful to see where they have to same shapes to interact with their receptors Abby Lathrop - kept mice as a hobby because of the coat color differences ● Gold, spotted, no hair, ● Behavioral characteristics ○ Japanese waltzing mice → run in a circle ■ Autosomal recessive gene, to maintain it and sell them to others, need to maintain them by breeding waltzing mice with waltzing mice ■ Closed colony, no import of genetic material form the outside
■ Mating siblings/cousins, because of the same lineage, slowly reducing heterogenic integrity People tried to transplant tumors into mice to get them to grow, so that they could be studied for longer periods of time. This was unsuccessful until they transplanted these spontaneous tumors into animals from the same closed colony (they interbred mice). This created restricted genetic availability, creating a more homogeneous colony, so the tumors will have a genetically more similar composition to the rest of the mouse population. Now, you could transplant a tumor from one mouse in that colony to other mice within the same colony. So the donor and recipient must match in some aspect in order for a successful transplant. ● Transplants carried genetic differences causing a rejection from the host body’s immunity Carl Jensen 1903 → took a white mouse close colony that produced a tumor ● Albino gene was recessive ● Was able to grow in 19 different generations of mice Loeb in 1908 → JWM grown in the same way ● Became possible to share the tumor Tyzzer in 1909 → took JWM carcinoma and transplanted it into ● Grows in JWM mice, autograft/isograft ● JWMX “common mouse” F1 (unrelated strain) , it grew ● JWM x Common mouse F2 → no growth, but only 54 recipients ● JWM x common F3 → no growth, 16 recipients ● Suggest something related to the individuals ● When you have a virally or chemically induced tumor, you can get antigenic changes that the immune system will recognize ○ Ex. There are 4 genes that influence rejection in a particular pair of mice. What is the predictive frequency of tumors that will grow? → (¾)^ CC Little in 1914 → susceptibility to tumor growth controlled by co- dominant genes ● ABO blood groups ● For the F2, the frequency of the growth is equal to f = (¾)^n where n is the # of genes that differ between P1 and P2, f = frequency of tumor growth ● Little and Tyzzer → 3/183 = 1.6%, suggests that there are about 14-15 genes that are segregating between mom and dad, possibility of generating a like match AB Locus ● AA x BB → AB for F ● Tumor AA with AB, there is nothing different in terms of the locus ● AB x AB → AA, 2AB, BB, for AA to be transplanted, only can occur in 3/4 of the offspring, just not the BB cross ● Estimate of the number of genes that control transplantation ● Collect blood from the mice, can cause clumping by grabbing hold on them BXS inbreeding ● Mating of offspring over time (siblings) ● Probability of an pincross, % homozygosity of the animals over years ○ Asymptotic activity after 20 years ~ 98.5%
○ After 8/9 generations, the wiggling away has randomly eliminated those end of the chromosome and you selected each generation for a heterozygote that has + and a ● Inbred strain with consistent phenotypes, their is a loss of fitness ○ Smaller litters, shorter lifespans, smaller in size, increase is disease susceptibility Artificial Systems: ● Immunodeficient animals ● Nude ● SCID (Severe Combined Immunodeficiency) Mouse - outcome of this mutation is the same no matter the location of the mutation. Profound change in immune capacity. Unlike nude mice where you only lose T cells, here you lose T cells and B cells. No adaptive immune response so very susceptible to disease. Good recipients for allografts and xenografts. You can further change these mice by adding another mutation creating NSG mice ● NSG mice - have 3 different effects. Lack NK cells, an innate component of the IR. Have SCID (no T or B cells), have gamma common chain defect (used by a lot of cytokine receptors so they do not have a response to some of the important immune hormones (cytokines) that regulate how the IR responds. You can transplant anything into these mice and it's gonna grow ● PDX (patient derived xenograft)→ therapeutic approach to human disease ○ Take NSG mouse and a patient with a tumor, excess tumor, chop it up and put it into a bunch of NSG mice, and the tumor will grow ○ The tumor is evolving and changing so it is not a fair comparison of drugs ○ In PDX mice, the treatment is done in the mice, then use the best drug in the mice in the humans with the tumors ● 2008 → someone took a mouse from the freezer, purified DNA and cloned the mouse
○ If you're frozen and they take your DNA, put it in an embryo and let it grow, is that you? Antigen (Ag) - something recognized as foreign by T or V-cell receptor, may not be actually foreign ● B cell Receptor is a surface antibody = antibody (Ab) Immunogen - something that can stimulate a TcR or BcR mediated activation of adaptive IR ● All immunogens are antigens, not all antigens are immunogens ● Must have an additional capacity to stimulate an IR, which implies that it can be recognized by the IR ● Recognized as foreign, have a certain complexity and a certain size (5-10 kDa) ○ Not all regularly repeating subunits are immunogenic ● Subset of antigens that can elicit and IR because they’re recognized as foreign Antigenic Determinant or an Epitope ● Can be more than one on a specific antigen ● Specific shape of the antigen that is engaged by the TCR or BCR ● Epitope → part of the antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cell ○ Ex. specific piece of the antigen to which an antibody binds BSA and chemically couple small molecules to it, the small molecules can be experimentally added to antigens, when they exist individually, they are not immunogens, not big enough ● They acquires size and complexity of BSA ● Called a hapten and the BSA would be a haptenated BSA ● These antibodies can recognize the haptens on the surface of the antibodies ● Carrier protein Have a rabbit and injected it with BSA triangle haptenated protein, make 2 categories of antibodies, anti-triangle, and anti-BSA ● If we want to study the reaction to the triangle, ● Immunoprecipitation is hidden ● Haptenate a different protein and then …
Lecture 5 (9/8 and on on syl)
Carl Landsteiner → studied the chemical basis of antigens in the early 30s ● Defined ABO blood groups Gorer → studied MHC and is important in the survival of transplanted tissue Hapten- small molecular shape that decorates the carrier protein, to small to be an immunogen ● The carrier protein gives the size/complexity, the hapten gives the antigenic determinant ● Can be recognized as an antigen ● Large molecules are large enough to meet the size ● Stimulate the production of antibodies ● When “fastened” to a protein, the entire complex becomes highly immunogenic Carrier Protein- confer the size and complexity the hapten needs to become an immunogen
● Cognate interactions, everything is haptenated with the same things, but tested against different carrier proteins Antigens (Haptens) → Will decorate the carrier proteins to cause an immune response Immunogens (antiserum against the haptenated proteins) ● Cognate reactions → circled ● Cross reactive interaction → cognate antibody can also recognize something sort of like the original immunogen, antibody doesn’t make a precise decision, but can react to something that is related ● Adding small uncharged molecules may have less of an effect than adding large charged Lock and Key Hypothesis- engage based on the fit of the active site ● If we modify the shape, the binding is compromised because something is now in the way Immune Response Genes - ● “Ir Genes” exist in the major histocompatibility complex ○ Cluster of genes found in all vertebrates, discovered by Davsset, Snell, and Benacerraf ● Discovered when they made congenic mice that differed from the background strain by a single specific region of the chromosome ● Snell did graft exchanges between different congenic partners, where one strain was normal and the other had just a slight regional difference on the chromosome ● Orthotopic skin grafts ○ Take skin from one and then swap them and see if it was rejected, no rejection was found because those parts of the genes did have any histocompatibility portions ● Major histocompatibility complex vs minor ● MHC drove a very rapid tissue rejection, minors much more leisurely ● Poly (G-A) was immunogenic is strain 13 but not in strain 2 guinea pigs ○ 13 → could make a response ○ 2 → no immune response ● Poly (G-T) was the reverse ○ 13 → no immune response ○ 2 → Could make a response ● Shows that there were genes in the … Each B cell makes one kind of antigen, means there's millions of B cells making their own Mitogens for a B cell include lipopolysaccharide (component of the cell wall of gram negative bacteria) ● “LPS” = TLR4 as receptor ● T cells → concanavalin A binds to alpha-methyl mannoside carbohydrate-glycoprotein
○ T cell mitogen (ConA) → comes from jackbeads, lectin, discovered because it agglutinated cells “agglutination” (clumping) T-Dependent vs T-Independent Antigens ● Need T lymphocytes to produce antibodies because they activate the IR and others don’t CD Cluster of Differentiation Antigens ● CD4 binds to T cells Adjuvant → something that enriches the host immune response to enhance it ● Have use in vaccine bc they nonspecifically enhance the response to the vaccine immunogen ● Ex. Alum,
- Depot Effect → gradual release of antigens (ex. Mix an aqueous antigen with mineral oil, emulsify, then inject it, mineral oil will hold the antigen and slowly release it to stimulate the immune response) longer and slower is better
- Nonspecific Stimulation of Phagocytosis → stimulate phagocytes, you stimulate the B cells
- Nonspecific stimulation of Lymphocytes → T cells but also the B cells ● Adjuvant co-injected with Teflon as the immune stimulus would not work because Teflon is not of sufficient molecular complexity to induce an immune response Antibodies: Structure and Function ● Comprised of 4 polypeptides linked by disulfide bridges ● 2 heavy chains and 2 light chains ○ Heavy = 50,000 Da, light = 25,000 Da → total 150,000 Da ● Held together by disulfide bridges between Cys-Cys ○ # differs per antibody ● NH2 terminals at the top, COOH terminals at the bottom ○ Antigen binding site at the top ends ● Intrachain disulfide bridges and interchain SS bridges hold the loops together ○ Those loops are called “domains” ○ Variable of the heavy chain = VH ○ Variable of the light chain = VL ○ Constant regions → CH1, CH2, CH3, CL ● Immunoglobulin supergene family
Lecture 6:
Immunoglobulin SuperGene Family ● Characterized by the domain structure ○ IMAGE ○ One little circle is a domain (ex. CH2) ● Epitope → particular shape on the antibody where it will engage ● B cells secrete …
○ With pepsin → cleaves under interchain disulfide hinge, so the hinge persists, but the tail end it digested into peptides ■ F(ab’) because the length of the heavy chain in longer, no Fc portion ● Bivalent → F(ab’) 2 ■ F(ab’)2 + antigen → immune precipitate, add Fab → less precipitate because monovalent can’t cross link so more monovalent Fab = competing for available antigens Antigens of Antibodies
- IgM → Pentamer, Largest, First to be produced in response to an antigen. Activates complement system (innate immunity). Very good at precipitating during experiments ● Heavy chain → Mu 900 kDa Cryoglobulin
- IgA → Dimer, most abundant in body secretions. Provides passive immunity to newborns. Binds and stops pathogens externally before they enter circulation. Secretory immunoglobulin ● Heavy chain → Alpha
- IgE → Monomer, antigen receptors are found on Basophils and Mast Cells. If an allergen is present, it binds and triggers a release of histamine from basophils or mast cells, creating an allergic response (lowest concentration) ● Heavy chain → Epsilon
- IgD → Monomer, B Cell antigen receptor site, present on the surface at the same time as IgM ● Heavy chain → Delta
- IgG → Monomer, most abundant in circulation (blood, lymph). ONLY antibody that can cross the placenta to give fetus passive immunity. Triggers opsonization (binds antigen to trigger phagocytosis). Activates complement system ● Heavy chain → Gamma 150 kDa ● All light chains (25 kDa) are kappa and lambda ○ * = highest concentration ● J-Chain - is a linking protein that allows for dimers and pentamers. They also rely on disulfide bonds between the individual monomers ● Disulfide bridges between antibodies that make up dimer & pentamer ● Each antibody is 150 kDa, light chains are 25 kDa and heavy chains are 50 kDa ● Different mammalian species have different subclasses ● If you see anti-IgA1 = antibody that binds to the alpha-1 heavy chain
Kabat & Wu → took patient serum from leukemia patient with B cell tumors and then they took urine bc they have a specific protein called 77 Bence-Jones protein (clonal light chains), and it is a light chain protein ● The sequence the proteins from a large number of leukemia patients and they looked at the first 50 AAs to see how they changed ● Pt #1 → A B D G T ● Pt #2 → A B D G A ○ Plotted the results of the amino position from the amino terminus to the carboxy terminus looking for variability ○ 3 hypervariable regions → regions where the antibody chain contacts the antigen ■ Vary those parts of the sequence that establish the shape of the pocket, and the frameworks hold the hypervariable regions together ○ Think that each of the antibodies was to a different antigen ○ Found 3 hypervariable regions, and they were parts that engaged with the antigen, and were called Complementary Determining Regions (CDRs) ● Anti-H chian ● Anti-light chain ● Antigenic determinant → develop an antibody to the with proper immunization ● Idiotype →the antigenic shape of the antibody’s antigen combining site ● Jerne Network Hypothesis → stimulate the immune response with the first idiotype to make the first anti- ○ Anti-anti idiotypes, and so on ○ Anti-idiotype reminds the body of the antigen ■ Antigen may be dangerous to inject into the patient, the anti- works well
