MCB 4211 Exam 1

how to calculate association diversity

#Hc perm x #Kappa perm OR #Hc perm x #Lambda perm

How to calculate the amount of polypeptide combinations possible

#Kappa perm + #Lambda perm + #Hc perm

How to calculate the number of possible Hc and Lc segments

#Kappa segments + #Lambda segments + #Hc segments

How do you calculate how many segments are in a heavy chain?

#V + #D + #J

how to calculate combinatorial diversity

#V + #D + #J

How do you calculate how many segments are in a light chain?

#V + #J

How do you calculate the number of permutations for a heavy chain?

#V x #D x #J

How do you calculate the number of permutations for a light chain (either Kappa or Lambda, not both)?

#V x #J

Parts of spleen

(lymphoid organ under stomach) 1) Red pulp = removes damaged RBCs 2) White pulp = immunologic function

Carl Jensen

-1903 -was able to successfully transplant tumor from albino white mouse closed colony for 19 generations → required genetic similarity provided by excessive inbreeding -first time you were able to grow and share tumors

Loeb

-1908 -Same Experiment as Jensen, but was able to transplant tumors in Japanese Waltzing Mice closed colonies

Tyzzer

-1909 -Transplanted JWM carcinoma into JWM mice (autograft/isograft) -in F1 generation (JWM x common) → success -in F2 (54 mice) & F3 (16 mice) → failure

C.C. Little

-1914 -found that tumor growth was determined by "dominant" genes -frequency of growth equation (f = (3/4)^n) → frequency = estimate of number of genes controlling transplant that are DIFFERENT between the host and donor -repeated Tyzzer experiment, but with more mice and found 14-15 genes that control a 'like' transplant match (~1.6% success)

Kohler & Milstein V-C Integration Experiment

-AFTER the Ig's had been made, they scrambled the V & C regions of Lc and Hc's, but no differences found -suggests that the V-C integration is encoded in DNA translocation/rearrangement and NOT done in the cell

Landsteiner

-Discovered the ABO blood groups -Haptenation experiments (BSA, Tartaric Acids)

restriction endonucleases (REs)

-Enzymes which cut specific, palindromic DNA sequences → can do both blunt and offset (hanging tag) cuts -However, their sequence typically doesn't generate any function and is thus randomly distributed throughout the genome, leading to a wide array of fragment sizes

How would you show there is Ag-binding of an Fab fragment?

-Fab fragments CANNOT make precipitates -HOWEVER, they CAN interfere with bivalent Ab crosslinking that DOES form an immune complex -So to show an Fab's Ag-binding potential, if you add Fab fragments to a precipitated bivalent Ab-Ag immune complex, the precipitate would collapse due to competitive Ag-binding of the Fab fragment

NOD/SCID/G (NSG) mouse

-G = lack gamma common chain for cytokine recognition -no B or TCRs = no response to signals

Kabat & Wu

-Isolated light chains (Bence-Jones proteins) of B cell leukemia tumors that leak out of nephrons due to kidney failure and sequenced their variable regions to look for variability in amino acid residues position-by-position -found 3 "*Hypervariable*" regions where each patient had different aa residues → believed that these hypervariable regions are what contact Ag's and varying them will allow for specificity

NOD/SCID mouse

-NOD = non-obese diabetic → causes Type I diabetes -SCID = severe compromised immune deficiency -RAG1 gene mutation prevents Ig or TCR production = autoimmune

Foxn1 Mutation

-Nude mouse: cannot make thymus or hair -can cause lack of connective tissue under skin -no longer need to surgically remove thymus to study T cells

George Snell Experiment

-Orthotopic Skin Grafts → taking tail skin from inbred mouse and transplanting it onto a different congenic mouse (differs in one chromosomal region) -most mice did not reject transplant, thus that congenic strain had no major genetic difference -some mice rejected transplant immediately, thus the congenic difference was in a major difference gene (Major Histcompatibility Loci) → those that had minor rejection termed Minor Histocompatibility Loci

Pauldane

-TLDR: if tumor & blood from A mouse is rejected from recipient, recipient doesn't only recognize tumor as foreign, but blood too -suggested that rejection was, in part, mediated by Ab's → showed that mixing blood causes crosslinking of cells which yielded aggregation

enzyme-linked immunosorbent assay (ELISA)

-Uses an enzyme-linked indicator antibody to visualize Ag-Ab reactions → enzyme instead of radiolabel -Relies on a solid support such as a microtiter plate that can adsorb the reactions

Cytotoxic Response

-aggressive cellular response -cells can recognize something foreign and secrete molecules to lyse cell or induce apoptosis

Hyperplastic Response

-aggressive cellular response -seen in coral -when two coral cells encounter one another, they try and overgrow each other

Engulfment Response

-aggressive cellular response -seen in molluscs -molluscs recognize foreign cells and phagocytize them like neutrophils

Principle Component Analysis Map (PCA)

-allows for mapping populations of cells based on what sets of genes are shared amongst those cells most commonly and what sets of genes distinguishes two populations -allows for recapitulation of hematopoietic pathway

allotype

-antigenic shapes unique to one person or family that distinguishes it from another person or family → *all*o = *all*ele → recall *allo*graft -allows us to look at Ab class inheritance

Draining Lymphoid Tissues/Draining Lymph Nodes

-as immune cells wander through the circulation, they move into tissues, and upon exiting, are collected by draining lymph nodes -example: superficial cervical lymph nodes at jaw to collect throat immune cells

Japanese Waltzing Mice

-autosomal recessive -inner ear defect causes them to run in circles -need to breed heterozygote & heterozygote since mice with defect can't breed

Characteristics of Monocytes

-become macrophages once moved into tissues → big (macro) and eat stuff (phage) -found in circulation -phagocytes → use engulfment to internalized foreign material, process it, and activate immune response

Characteristics of Basophils

-bilobed nucleus -stain blue w/ hematoxylin

Patient-Derived Xenograft (PDX)

-can inject excised patient tumor into NSG mice (xenograft = different species) and it allows for growth -can then test different therapeutic drugs on each mice and determine best drug to use on human = tumor will not evolve with each drug test

Percent Homozygosity

-can plot the probability of an in-cross (% homo) over the generations (i.e. how often you will pick mice with same genetic composition) -for single gene between two F2 mice, 98.5% of the gene are homo after 20 generations

Use of CFUS in identifying cells

-can treat cells with Ab + complement to kill specific cells that the Ab will find, eliminating that population from the injection -i.e.: if you knock out granulocytes and they drop off spleen surface, you can tell that the surface molecule targeted differentiates cell populations

Non-Aggressive, Cellular, Defense Response

-cell sorting -seen in sponges -if you degrade two different sponges into just their cells, they can distinguish themselves as self and re-aggregate

What is the Immunoglobulin Supergene Family?

-collection of genes al sharing domain-like structure -have the ability to amplify gene segments via gene duplication

Non-inducible, Humoral, Defense Response

-constitutive -ex: snail agglutant that clumps together foreign molecules

complement

-constitutively present in sera -only activated in adaptive immune response when interacting with Ab to form MAC

Immune Response Genes (IR Genes)

-drive an organism's ability to recognize certain antigenic forms -IR genes can be found in major histocompatibility complex (MHC)

Define determination

-genetic change -determines fate of cell and its descendants

Diffusion test/assay

-have a well that connects to a semipermeable, diffusible membrane -at start of membrane and well, there is an Ab labeled with enzyme that will capture a specific Ag - at end of membrane is an immobilized Ab that will also capture the AG → also have positive control further along -upon adding nasal secretion, Ag will bind colored-Ab, diffuse down membrane, and potentially bind the immobilized Ab to form a colored line

flow cytometry setup

-have flow cell in which cells pass through one at a time -at a right angle to the flow cell, lasers are concentrated through lens and pass through each cell -the lasers are then collected by different mirrors and bandpass filters and read by the computer -some lasers are diffracted into side scatter while some reman forward and are referred to as forward scatter

CFUS Assay (Colony Forming Unit of Spleen)

-hematopoietic cells can migrate to spleen and will form colonies on surface -you can inject specific hematopoietic cells into an immunodeficient animal and see if they were able to migrate to spleen, supporting functionality -can titrate number of cells injected and see how many end up on surface and how many don't

What are cytokines?

-immune hormones that are soluble mediators of cell activation -can be made by many cell types -can stimulate immune cells AND other cell types

Kohler & Milstein Main Experiment

-immunized mice with Sheep RBCs (SRBCs) and took the mouse's splenic B cells (can produce anti-SRBC Ab's) -they then made hybrid cell lines from the fusion between mouse myeloma cells and the splenic anti-SRBC cells -then tested those cells using IEF and Plaque Assays

Characteristics of Eosinophils

-important in allergic reactions -stain red w/ eosin

Till & McCullough experiment

-inject hematopoietic cells from mouse A into tail vein of irradiated mouse B -remove spleen and looked for colonies of those cells on spleen surface -observe how much irradiation is takes to completely remove all immune function

Humanized mouse

-inject human bone marrow cells into NSG mouse, you can develop a mouse with the human immune response -allows you to look at human disease and complete hematopoietic pathway EXCEPT for MALT tissues

How were previous monoclonal B cell tumors made before Kohler & Milstein?

-injected Balb/c mice in peritoneal cavity with either mineral oil or turpentine as chemical irritant → yielded MOPCs and TEPCs (PC = plasmacytoma) -they were B cell tumors made as a consequence of chronic inflammatory stimulation -HOWEVER, could not predesignate what mAb's those tumors would make before generating them

Why are vaccinations beneficial?

-innately, there is 1 in a million Ab's circulating the blood that is specific to one antigen, which is not nearly enough to fight the infection -pre-exposure to the antigen greatly increases the proliferative capacity of the B cells producing that specific Ab -by increasing the amount of B cells, you greatly increase the odds of one of them randomly encountering the first virus you inhale, increasing the response speed

Abbey Lathrop

-kept many mouse strains -First to be able to produce Japanese Waltzing Mice strain -did so with closed colony

Immune Complex

-large complex formed by the non-covalent interactions between antibodies and antigens with each arm of the antibody holding an antigen -complex becomes so large that it is no longer soluble and will precipitate out of solution

Characteristics of pluripotent stem cells

-least differentiated & least determined -exist in bone marrow -influenced by extracellular conditions

Lymphatic Vessels

-like blood vessels, but exclusively for leukocyte transport -locations vary from person to person and are wherever they want in the body -vessels are anchored by the lymph nodes -collect lymphatic fluid/leukocytes from draining lymph nodes

Characteristics of (Im)Mature Dendritic Cells

-long cellular processes/projections of membrane → for capturing of floating material in blood → internalize that material, process, and activate like macrophages

Characteristics of Mast Cells

-molecule on surface called FC-epsilon that releases histamines -involved in allergy response

Characteristics of Neutrophils

-multi-lobed nuclei (could carry apoptotic cues) -largest fraction of granulocytes -earliest arrival in infection -activates innate response -dies soon after migration to wound site

Heterogenous Inducible Response

-need multiple molecules -ex: vertebrate antibodies

closed colony

-no import of genetic material from the outside -slowly reduces genetic heterogeneity

Flatworm Response

-no memory -only recognize xenogeneic difference

Homogenous Inducible Response

-only need one type of molecule -ex: lobster bacteriaciden

Major Histocompatibility Complex (MHC)

-originally discovered by Dausset, Snell, & Benacerraf -major genes involved in recognition/rejection

Define differentiation

-phenotypic change -cell can express new molecule or become a different shape

Radioimmuno Assay

-radio=radioactivity (not radial) -when you radioactively label you Ag or Ab, you can measure the potential radioactivity of the formed precipitate to see if the radioactive Ag or Ag bound to its appropriate counterpart

Electrophoresis of serum proteins

-separates into 5 blobs, with the first and last being larger than the middle 3 -first to last: Albumin, alpha-1, alpha-2, beta, and Gamma -the blobs of proteins are globular proteins and in the gamma band of the profile → thus "Gamma Globulins" -Once they realized the gamma globulins contained Ab's → called the "immunoglobulins, Ig's"

DeGeorge Syndrome

-similar to mouse Foxn1 mutation -lacks T cell compartment, but still has functional B cell compartment

Earthworm Response

-some memory -recognize both xenogeneic and allogeneic differences

Antigen definition

-some molecule that is recognized as foreign -contextual definition, doesn't actually need to be foreign to the body

Mouse Defense Response

-strong memory -exquisite specificity -recognize both xenogeneic and allogeneic differences

Potential for tail end of some soluble Ab's

-tail end of soluble Ab's CAN be picked up by a receptor on other cells and connect as a receptor to activate those cells or capture a cognate Ag -binding of the cell receptor to the tail end of the Ab will propagate a structural change through the cell

De novo pathway of DNA synthesis

-take existing fundamental building blocks to make NTs for synthesis -blocked by drug aminopterin

Why is the Line of Equivalence actually a line between the two samples and not a smear in a radial immunodiffusion assay?

-the closer to the Ag, you have too much Ag for any immune complexes to form, and vice versa for Ab → immune complexes are dissolved in sample excess

If you add extra Ab to the Ab well in a radial immunodiffusion assay, what happens to the Line of Equivalence?

-the line moves closer to the Ag well to escape the high concentration of Ab -however, the line does not smear as the increased Ab concentration collapses the old immune complexes via competitive binding

Why are any autoreactive cells eliminated in the primary lymphoid tissues?

-the primary lymphoid tissues are never supposed to be exposed to Ag's -thus, if ANY form of Ab stimulation occurs in these tissues, it is classified as auto-reactive, and the response will be eliminated via apoptosis or anergy

Imagine that a radioactively labeled Ag is a viral protein and there is an Ab is specific for that viral protein. If you take a patient sample and add it to that mix and it's virally infected with that same virus, what would you expect the effect to be on the amount of radioactivity of the precipitate?

-the radioactivity of the precipitate decreases due to competition → the patient's non-radioactive Ag virus will compete for the Ab, decreasing the radioactivity

Congenic Strains

-through selective breeding, have inbred strain with a section of the chromosome containing donor material surrounded by passenger loci from another strain -allows you to attribute phenotypic differences to donor material differences

Limitations of Cell/Tumor Lines

-use fetal bovine serum to provide basic growth factors/nutrients, but not nuanced -cells need constant movement/circulation to avoid being near waste accumulations → modern mechanisms can mimic circulation, but still cause cell shearing/mechanical damages **this is why using immunodeficient mice is good for immune work since it provides the circulation and nuanced factors without mounting a response**

George Snell

-used congenic strains to map transplant rejection seen in Little & Tyzzer experiments to specific genetic region differences -termed the Major Histocompatibility Genes

Salvage pathway of DNA synthesis

-uses hypoxanthine and thymidine and modifies them to use as NTs to synthesize DNA -pathway requires HGPRT enzyme

Complement-mediated cytotoxicity assay

-utilizes the formation of MAC when combining complement, cells, & Ab's -take maternal antiserum that expresses both paternal and maternal HLA Ab's, combine with complement and another patient's cells -if the other patient carries Ag's that are recognized by the HLA Ab's, the MAC will form, lysing the patient cells -can then measure cell death by adding trypan blue

Kabat & Wu Plot

-variability on y-axis and aa position (from amino termini to carboxy termini) on x-axis -aa residues that were consistent were given a variability of 1 -the aa residues that stayed around 1 were called "framework" residues

FC Receptor (FCR) Ab Binding

-when FCR is on cells, pick up Ab's bound to Ag's that mediate the cell's ability to engage the extracellular environment -ex: FCR-Epsilon on mast cells binds IgE and when IgE is bound to specific allergen, the mast cell is signaled to de-granulate, releasing histamines that cause allergic reaction/response

What is the benefit of using IEF over SDS gels (especially for Kohler & Milstein)?

-you can separate two proteins of the same size if they have a different aa sequence → i.e., heavy chains all have typical sizes, but their aa sequences can be variable, thus allowing for actual separation/detection in IEF

Clonal Selection Theory

-you have the ability to take genetic building blocks and assemble genes and then select from those genetic alternatives the specific Ab's that are useful → here self-Ab's are produced, but eliminated by some mechanism

3 Types of Immune Responses

1) Activation → increases vigor 2) Apoptosis → signal cells to die 3) Anergy → biochemical state to not respond to stimulus (e.g., allergy response suppression)

2 Types of Cellular, Defense Response

1) Aggressive 2) Non-aggressive

Landsteiner Experiment

1) Antibodies were produced and yielded from 3 rabbits, each injected with BSA molecules haptenated with levo-, dextro-, and mesotartaric acid enantiomers 2) replaced BSA with hemoglobin and combined with each Ab and looked for precipitation 3) found cognate reactions and some cross-reactivity

4 cell types derived from common lymphoid progenitors

1) B cells 2) T cells 3) NK cells 4) ILC (innate lymphoid cells)

2 Types of Immune Defense Reactions

1) Cellular 2) Humoral

3 Progenitors Derived from Pluripotent Stem Cell

1) Common lymphoid progenitor 2) Common myeloid progenitor 3) Immature Dendritic Cell

3 Mechanisms of Adjuvant Help

1) Depo Effect 2) Nonspecific Stimulation of Phagocytosis 3) Nonspecific Stimulation of Lymphocytes

What two processes allow for hematopoiesis progression?

1) Determination 2) Differentiation

3 Types of Cytotoxic, Aggressive, Cellular, Defense Response

1) Flatworm 2) Earthworm 3) Mouse

Characteristics of Kohler & Milstein's Hybrid Lines (4)

1) Grow in HAT selective media 2) The lines contain metacentric chromosome present in one of the parental myeloma lines 3) Karyotype after 4 months in culture is smaller than parental, but larger than BALB/c (not just fusion of spleen cells) 4) secreted Ig's contained MOPC21 protein

2 Types of Inducible, Humoral, Defense Response

1) Homogenous 2) Heterogenous

What are the 5 forces that regulate Ab-Ag binding interactions?

1) Hydrogen Bonding 2) Electrostatic Forces 3) Hydrophobic Forces 4) Van der Waals 5) Cation-pi Forces

3 Types of Aggressive, Cellular, Defense Response

1) Hyperplastic 2) Engulfment 3) Cytotoxic

5 Classes of Ig's

1) IgG 2) IgM 3) IgD 4) IgA 5) IgE

2 Types of Humoral, Defense Response

1) Inducible 2) Non-inducible

3 Tartaric Acid enantiomers

1) Levo 2) Dextro 3) Meso Chemistry is all consistent, only change is the ORIENTATION of the hydroxyl and hydrogen groups around the carbon backbone

What are the 5 types of granulocytes

1) Neutrophil 2) Eosinophil 3) Basophil 4) Unknown Precursor of Mast Cells (Lymphocyte) 5) Monocyte (Macrophage)

Is thymus the only site for T cell differentiation? How do you know?

1) No, there must be a secondary site 2) If you keep mice alive after neonatal thymectomy, you can see low levels of T cells appear

Characteristics of IgE class (4)

1) Part of allergy response 2) monomeric structure 3) EXTREMELY low concentration (100x less than IgD) 4) Epsilon Hc

How does recombinase excision and ligation occur?

1) RSS's fold the protein into a loop to bring themselves closer in proximity 2) the recombinase cuts the sequence at the RSS's 3) the recombinase then ligates the two sequence fragments together as well as the cut loop into a circular fragment 4) this results in a shorter DNA fragment that brings two genes together and an okazaki fragment designated as waste

4 Forces for Residual Heterozygosity

1) Random Chance 2) Homozygous Lethal genes → only Hz offspring will be viable 3) Male vs Female intrinsic Differences 4) Mutation Rate

Characteristics of IgA class (4)

1) Secretory Ig (MALT) 2) monomeric OR dimeric structure 3) high concentration 4) Alpha Hc

Domains in Ab chains

1) Variable Domains at amino termini (NH2) 2) Constant Domains that extend toward carboxy termini (COOH) → labeled from amino termini to carboxy termini found in both BCR Ab's and TCR Ab's

What antibodies are yielded when injecting a rabbit with a haptenated BSA molecule?

1) anti-BSA 2) anti-hapten

What Ab's can be made against Ab's themselves?

1) anti-Hc 2) anti-Lc 3) anti-idiotype

T cell function in immune response

1) become helper T cell to produce cytokines 2) become cytotoxic T cell to induce cell apoptosis

Characteristics of IgM class (6)

1) can fix complement to activate the MAC cascade 2) monomeric OR pentameric structure 3) 150-900 kDa (depends on monomer vs pentamer) 4) Best agglutinant due to having many hands → is already at 900 kDa even before crosslinking 5) high concentration in body 6) Mu Hc

Characteristics of IgG class (5)

1) can fix complement to activate the MAC cascade 2) monomeric structure 3) 150 kDa 4) high concentration in body 5) gamma Hc

Functions of Ab's (4)

1) cause precipitation 2) cause agglutination 3) bind FCR 4) activate complement to form MAC

How might one dissociate an Ab-Ag interaction?

1) decrease pH conditions 2) increase salt concentrations

What 3 cell types are derived from the common myeloid progenitor?

1) granulocyte / macrophage progenitor 2) megakaryocyte progenitor 3) erythrocyte progenitor

steps in an ELISA (sandwich ELISA)

1) immobilize Ab on well 2) add Ag to well and wash away excess 3) add Ab-enzyme conjugate to the well and wash away excess 4) add chromogenic substrate to the well Color formation indicates presence of the immune complex

Two forms an Ab may take

1) in plasma membrane = receptor 2) in circulation = soluble molecule

In somatic hypermutation, what happens to a mutation that increases Ab affinity for Ag and what happens to the mutation that decreases the affinity?

1) increase = affinity maturation 2) decrease = no stimulation, functionally eliminated

Components of neutrophil seen in chase video

1) lamellipodia at front edge → no granules → extends in direction of bacterial secretions 2) europod @ back end

Characteristics of the Secondary Immune Response (5)

1) larger response vigor 2) shorter lag phase 3) has memory (AKA amnestic response) 4) high specificity 5) gradual vigor drop-off (gives better immunity)

Characteristics of IgD class (3)

1) monomeric structure 2) 4th lowest concentration 3) Delta Hc

What types of RSS's are there?

1) one-turn 2) two-turn (in regard to alpha helix turns)

Primary Lymphoid Tissue: origin, timing, fate, surgical removal, examples

1) origin = endoectodermal junction 2) timing = early embryonic stages 3) fate = involution into acellular connective tissue after puberty 4) effect of surgical removal = unresponsive to Ag's 5) thymus, bursal equivalents (bone marrow, fetal liver, yolk sac)

Secondary Lymphoid Tissue: origin, timing, fate, surgical removal, examples

1) origin = mesoderm 2) timing = fetal development 3) fate = persists throughout life 4) effect of surgical removal = modest decrease in immune activity 5) examples = lymph nodes, spleen

Radial Immunodiffusion

1) petri dish with layer of agar that you cut holes in and drop your sample into 2) the sample will diffuse out radially to potentially interact with other samples in the system

Process of an equilibrium dialysis experiment

1) place a dialysis tubing sausage containing free Ab in a solution containing radiolabeled free Ag (Ag*) → the Ag* must be smaller than the MWCO 2) overtime the free Ag* will move in and out of the tubing to reach equilibrium 3) as free Ag* moves into the dialysis sausage, it will interact with the free Ab to form an Ab-Ag* immune complex 4) eventually the experiment will reach a state where the dialysis sausage has free Ab and Ab-Ag* immune complexes and the free Ag* inside and out of the sausage is in equilibrium

Hozumi & Tonegawa Experiment

1) purified DNA from a myeloma (monoclonal B cell tumor) and other adult tissues (embryonic, brain, kidney) 2) cut DNA with REs and ran the fragments on a southern blot and destroyed and double stranded structures with heat 3) also isolated mRNA encoding the V-C light chain sequence from the myeloma 4) they then cut the V-C sequence to generate another C sequence and radiolabeled both V-C and C sequences with 32P to generate two mRNA probes 5) then used the V-C and C mRNA probes against the southern blot DNA and looked for band 6) In myeloma tissue, both V-C and C probes yielded one band. In embryonic, brain, and kidney tissues, the V-C probe made two bands while the C probe always made one band

Western Blot

1) separate proteins on gel via electrophoresis 2) transfer proteins to surface of a membrane paper (i.e. via electroblotting) 3) block nonspecific staining 4) add primary antibody 5) add conjugated secondary antibody 6) add chromogenic substrate to detect target bands that have immune complex sandwich

Characteristics of the Primary Immune Response (5)

1) smaller vigor 2) longer lag phase 3) no memory (no prior Ag exposure) 4) nonspecific 5) quick vigor drop-off

Why would some of the cells taken up from the first plaque assay be negative in the second plaque assay (Kohler & Milstein)?

1) there could have been other cells nearby that were scooped up from the first assay 2) when the spindle proteins went to pull 40 chromosomes from the 100 present ones, they may not have grabbed the Hc and Lc specific to the anti-SRBC Ab, therefore they wouldn't be able to create the plaque

Ideally, you could inject allergens straight into the thymus or bone marrow to eliminate any allergic response to that allergen. Why don't we do that now?

1) we don't have a thymus anymore, it involuted after puberty 2) the bone marrow serves as both primary and secondary lymphoid tissues, so you run the risk of inducing anaphylactic shock since there are mature cells ready to respond, not just hematopoietic stem cells

Mary Pourpoint

1712 First to observe/try smallpox vaccination after seeing it in Turkey

Edward Jenner

1798 used dried pustules from milkmaids to vaccinate boy with cowpox to prevent against other strains of smallpox

Koch

1891 discovered delayed type hypersensitivity (DTH) using tuberculin Ag

Bordet

1895 Discovered that combining Ab's + Complement + Bacteria = cell lysis

Peter Goren

1936 Discovered Major Histocompatibility Antigens (MHC) and its importance in transplantation (HLA Ag's)

Kabot & Tselius

1939 Started to understand molecular nature of Ab's

Till & McCullough

1950's -CFUS assay -studied effect of irradiation on immune system

How many T cells exit thymus as functional?

2-5%

If you have 30 Hc permutations and 10 Lamda Lc permutations, what is their associational diversity?

30 x 10 = 300

If you have 300 Lambda-Hc permutations and 60 Kappa-Hc permutations, what is their combinatorial diversity?

300 + 60 = 360 → *remember that you cannot have both lambda and kappa so they must be added, not multiplied*

Epitope (antigenic determinant)

A specific region on the surface of an antigen against which antibodies are formed

Disulfide Bridge

A strong covalent bond formed when the sulfhydryls of two cysteine monomers lose their H's and interact

Example of C.C. Little Experiment

AB locus, AA tumor transplant P1 = AA P2 = BB 1) F1 = AB = Success 2)F2 = AA, 2AB, BB = 75% success (not BB) f=(3/4) ^ 1 locus = 75%

Of Ab and Ag in the immune response, which is the signal molecule, and which is the receptor molecule?

Ab = receptor Ag = signal molecule

What is serum?

Ab's in plasma without clotting factors; derived from blood fractioning

If you add Fab to a mixture of intact Ab + Ag where precipitation DOES occur, what would the effect of the Fab added be to the precipitation?

Adding free Fab will decrease the precipitation as the Fab fragments will competitively bind the free Ag, but cannot crosslink like the intact Ab's, bringing the precipitation back into solution → similar to hapten experiments

What phenomenon explains our reason for vaccination?

Affinity maturation -if you introduce a strong Ab-Ag interaction, it will increase B stimulation time period, increasing that B cell's proliferation and Ab production

How do you measure how much free Ab you have in the dialysis tubing?

After calculating the amount of immune complex formed, you subtract that number from the amount of Ab you started with

What does an anti-idiotype molecule remind you of?

Ag

What is the benefit of producing anti-idiotypes?

Ag's can be dangerous to inject into patients, but if you make anti-idiotype and inject it, that can make an anti-anti-idiotype that will function similarly to the Ab that would have been made to the Ag originally, thus the anti-anti-idiotype becoming a surrogate Ab for that Ag shape

How many constant domains are in each Heavy Chain of an Ig?

At least 3 constant domains of the heavy chain

Which cells are involved in the adaptive immune response?

B & T cells

How do T, B, & NK cells differ?

B cells: -stay in bone marrow -can be identified as a B cell once they express a B cell Ig on their membrane -adaptive response T cells: -migrate to thymus to receive differentiation cues -Express T cell receptor on surface adaptive response NK cells: -express neither T or B cell receptors -constitutively present and nonspecific -innate response

How to study B cells

Bursectomized chickens: -1960's experiment accident in which chickens missing bursa were immunized, but weren't able to form Ab's -showed that bursa is required for B cells that produce Ab's Our bursal equivalents = bone marrow, fetal liver yolk sac

chemotaxis

Cell movement that occurs in response to chemical stimulus in form of increasing chemokine concentrations secreted by prey

CD molecules

Cell surface molecules expressed on various cell types in the immune system that are designated by the "cluster of differentiation" or CD nomenclature -originally just name tags -can now fluorescently tag them to see what cell it binds to

Cognate vs Cross-reactive

Cognate = same molecule recognizing the same molecule → ex: omicron recognizing omicron Cross-reactive = one molecule being able to recognize a close-relative/slightly different molecule → ex: cowpox recognizing smallpox

Mitogen for T cells

Confonavolin A (Con A) -binds to alpha-methyl-mannoside (a-CH3-mannoside; part of carbohydrates present on a glycoprotein) -binds to T cell surfaces as well as carbohydrate side chains containing a-CH3-mannoside → when proteins are made and carb side chains containing a-CH3-mann are added, Con A binds, triggering T cell proliferation

What did the Tonegawa and Hozumi experiments tell us?

During B cell differentiation, the configuration of the DNA changes → the change must have been an excision or alteration to the RE cut site that would typically separate the V region from the C region, so the VC migrate together

Mucosal-associated lymphoid tissue (MALT) OR Gut-associated lymphoid tissue (GALT)

Ex: Peyer's patches: -specialized lymph nodes in body of intestinal wall or other mucosal membranes -important in sensing of antigens in these tissues

Which is the only Ig fragment from either Pepsin or Papain digestion is able to successfully precipitate a cognate Ag?

F(ab')2 as it is the only fragment that has both Ag-binding arms (bivalent) and can thus crosslink Ag's to bring them out of solution

What can contribute to junctional diversity?

Frameshift mutations induced by: 1) improper recombinase cutting that is not in triplets 2) enzymatic addition of extra NT's that are not triplets

Kohler & Milstein Fig. 2: Plaque Assay

Fused anti-SRBC & myeloma cells, grew hybrid line on HAT media, then introduced to plaque assay: → Assay placed hybrid cell population in bottom soft agar layer and overlaid an agar layer containing SRBCs w/ complement from guinea pig → Hybrid cells secreting anti-SRBC would diffuse through soft agar and form MAC when interacting w/ SRBCs and complement, causing cell lysis & plaque

Benacerraf

GA & GT polymer injections showed that one polymer was immunogenic in strain 2 but not strain 13, and vice versa

Compared to initial foreign Ag exposure, what is the vigor of the immune response to a second exposure?

Highly increased due to memory of first molecule

Name for MHC in humans?

Human Leukocyte Antigens (HLAs)

MW of Immunoglobulin & Each chain

Ig = ~150 kDa Hc = ~50 kDa (2 each) Lc = ~25 kDa (2 each) Already pretty big itself, once you begin crosslinking into an immune complex, precipitates quickly

Quiz: Which Ig class dimerizes as a secretory Ab?

IgA

Which Ig class is thought of as a secretory molecule associated with MALT?

IgA

Which Ig class has the lowest concentration in the body?

IgE

Which Ig class is associated with the allergy response?

IgE

Which Ig classes can bind and activate the complement cascade?

IgG & IgM

Which Ig classes have high concentrations in the body?

IgG, IgM, IgA

Which two Ig classes have structures other than monomers and what structures are they?

IgM = pentamer IgA = dimer

Which Ig class is the best agglutinant?

IgM due to pentameric structure having multiple hands and being of a high MW itself

Why would you imagine some molecules and antibodies will have cross-reactivity and some will not?

Image 3 antigens: one that matches the Ab exactly, one that has a protrusion in the epitope, and one that has an indent/recess 1) same shape = perfect fit, thus strong reaction 2) protrusion = physically prevents the closeness of the two molecules, thus blocking the interaction completely 3) recess = two molecules can still be close except for a portion of the surface, thus allowing for a reaction, but a much weaker one

How did Kohler & Milstein fuse their cells/cell lines?

Inactivated Sendai Virus → we now use polyethylene glycol (PEG) due to the mouse-lethality of Sendai

Innate vs Adaptive Immunity

Innate: -less specific -no memory Adaptive: -highly specific -has memory

Kohler & Milstein Figure 1: IEF

Isoelectric Focusing (IEF): -separates proteins based on isoelectric point (pH at which they have no net charge) -when they tested their parent chains and initial cell lines, cell lines had complex band pattern, but after disulfide reduction, saw same amount of final product Significance: New Ig's were mix of Hc & Lc of parents and can express both parental variable domain idiotypes and heavy chain isotypes

Domain-like structural significance

It is a molecular motif that joins all the genes that belong to the "Immunoglobulin Supergene Family" → i.e., all genes that have the domain-like structure w/ cysteine disulfide bridges holding the ends of the domains together

Which scientist's work aligns with the phenomenon behind affinity maturation?

Landsteiner's work with cognate and cross-reactive haptenation interactions

Mitogen for B cells

Lipopolysaccharide (LPS) -component of Gram negative cell wall -binds to TLR-4 receptor, causing mitosis (proliferation) of B cells

How do you measure the amount of immune complex in the dialysis tubing?

Measure the radioactivity within the tubing and subtract the outside solution radioactivity from that number as the amount of free Ag* inside and out of the tubing should be equal

Name a cell that is part of lymphoid progenitors/response that is NOT a part of adaptive immune response

NK cells

Are all Antigens Immunogens?

NO

Can both parental chromosomes undergo rearrangement at the same time?

NO, only one chromosome ever rearranges to form the Hc and Lc loci due to *allelic exclusion*

Are the locations of the cysteines in Ab's constant?

NO, the precise cysteine locations and the # of disulfide bridges vary Ab to Ab

How to study T cells

Neonatal thymectomy: -surgically remove thymus from newborn mouse and look at immunological phenotype -when removing thymus, lost all T cells, showing necessity of thymus in T cell existence

Jerne Hypothesis

Network Hypothesis -believed that anti-idiotypes, once made, will turn off the B cells making Ab's with the idiotype, and then anti-anti-idiotypes are made and turn off the anti-idiotype Ab's.... -essentially, each idiotype stimulates the production of a new Ab that shuts off the original B cell

Does class switching affect antigen specificity?

No, it is *independent* of VDJ arranging and thus Ag-binding pocket formation

Does the immune response vigor start at 0?

No, there is innate immunity

How many of each light chain and heavy chain class can one Ig have?

ONE of each → i.e. you can have IgA with Kappa or IgA with Lambda, but NOT BOTH

Trick to remember Papain vs Pepsin

P*a*p*a*in = 2 types of fragments: Fab *AND* Fc Peps*i*n = creates b*i*valent fragment

Pro/Con of Inbred Strains

Pro: -consistent phenotype -can share material between labs Con: -loss of fitness (reverse of hybrid vigor) → small litters → short lifespan → smaller body size → increased disease susceptibility **However, benefit of phenotypic consistency FAR outweighs any con**

B cell function in Immune Response

Produce Ab's to: 1) bind virus to prevent it being taken up/integrated 2) bind virally infected cells and activate complement proteins to lyse the cell

Difference between SDS gel and IEF gel

SDS: -denatures proteins into an ellipsoid shape -adds its own negative charge to the proteins -when electrical current is applied, the proteins move according to their MW and induced charge -*separates based on MW* IEF: -no denaturing or charge application occurs -instead, gel carries ampholytes (ampholines in his talk) that travel to a certain electric field position and set up a pH gradient -the protein samples then move to their isoelectric point within that gradient -*separates based on isoelectric point (intrinsic sequence charge)*

If you have injected a person with a vaccination and then introduced them to a completely different foreign molecule, what would be the vigor of the immune response?

Same as first exposure since there is no memory of the new molecule

Which cadence of antigen exposure elicits the best immune response?

Slow and Steady >>> Fast and Clear i.e. attenuated virus > killed virus

What causes lymph node swelling during infection?

T & B cell proliferation in the lymph nodes?

Quiz: the common lymphoid progenitor cell lineage gives rise to...

T cells

Why did Kohler & Milstein use SRBC's?

T-dependent Ag that could drive Ab production in mice (it was just commonly used then)

T dependent vs T independent antigens

T-dependent: -some Ab's require thymus-derived T-cell interaction to be produced, despite being produced by B cells themselves T-independent: -Ab's that do not require thymus-derived T-cells to be produced

Quiz: a student becomes sick with strep throat and the infection causes swelling of the lymph nodes near the infection. Why do these lymph nodes increase in size?

The T and B lymphocytes needed to combat the specific pathogen are proliferating in the lymph nodes

What allowed the hybrid myelomas to grow on HAT media when the parental myeloma lines couldn't?

The anti-SRBC cells from the mouse spleen were positive for HGPRT, thus insensitive to HAT media

What structure of an Ig makes the molecule the founding member of the Immunoglobulin Supergene Family?

The looped domain-like structures held together by INTRAchain disulfide bridges

What determines the speed at which an Ab-Ag interaction dissociates?

The strength of the bond/interaction

What was different about Kohler & Milstein's hybridoma/myeloma from today's myelomas?

Their myeloma line made its own Ig so that when the hybridoma cells were constructing their Ab's, they might mix and match the Hc's and Lc's of the two Ig types, not just produce the one mAb

Why is being non-covalent important for Ab-Ag interactions?

They are dissociable → can form and fall apart

Kohler & Milstein Fig. 4: Isotype Lysis Assay

They cut holes in petri agar and put hybrid supernatant in cell hole and added anti-Ig's (anti-idiotypes) to surrounding holes, and then overlaid that with agar containing complement and SRBCs → the supernatant anti-SRBCs would diffuse radially, as would the anti-idiotypes → when the anti-SRBC Ab encounters the SRBCs and complement it will create a plaque → if the anti-idiotype against the anti-SRBC Ab encounters that anti-SRBC Ab, then it would precipitate the anti-SRBC Ab, preventing the zone of lysis from reaching the anti-idiotype well → they found that the hybrid line was producing IgM Ab's

Why do most T cells die in the thymus?

They don't meet the specific requirements and undergo apoptosis

How did Kohler & Milstein modify their cells before isoelectric focusing?

They fed the cells radioactive 14C-lysine to allow for their visualization via film

How did Kohler & Milstein know their colonies were of the hybrid cell line?

They plated the colonies on HAT media and left them for an extended period of time: -if the cells were from the parental myeloma line, they would be HGPRT- and could not grow on HAT media -if the cells were from the anti-SRBC spleen cells alone, they would not have the immortality of the myeloma line, thus dying after the extended period of time Thus only the immortal, HGPRT+ cells formed by the fusion would be present on the HAT media

Why couldn't the parental myeloma lines from Kohler & Milstein grow on HAT media?

They were HGPRT negative

How does the Ag binding to the Ab affect the Ab itself?

Thought that the Ag binding will transmit information throughout the Ab molecule and once the change reaches the torso, it can activate complement → it's this part of the Ab that activates the FCR to cause the cell to behave differently

What regions are present on the light chain loci?

V J

What regions are present on the heavy chain loci?

V (variable) D (diversity) J (joining)

Which occurs first: VDJ rearrangement or class switching?

VDJ is rearranged first to form the Ag-binding pocket and then the B cell can change the class type of the Ig based on its cytokine environment

What is complement-mediated cytotoxicity?

When you add Ab + complement, a membrane attack complex (MAC) can form if the Ab is bound to an Ag or cell and then binds complement, the MAC acts as a molecular drill to lyse and thus kill the cell

Xenogeneic vs Allogeneic vs Syngeneic

Xeno = difference between species Allo = difference between individuals Syn = difference within same individual (or identical twins)

Antibody Structure

Y-shaped 4-polypeptide chains → BIVALENT → 2 long heavy chains (Hc) and 2 short light chains (Lc) → disulfide bridges hold Hc-Hc, Hc-Lc, and Hc-Lc → Ag binding site formed between the amino termini (NH2) of the Lc & Hc arms

Are all Immunogens Antigens?

YES

When calculating all the different combinations and permutations of heavy chains and light chains, how would you account for the constant regions?

YOU DON'T! They do NOT add to the Ag-binding diversity encoded by the VDJ rearrangement

How can you tell which chromosome will undergo the Hc-Lc rearrangement process?

You don't, it's entirely random

How do you study haptens/anti-haptens independently of the carrier molecule?

You haptenate a different carrier protein and introduce it to the anti-hapten Ab i.e.: when you yield anti-BSA and anti-hapten from an immunized rabbit, if you add haptenated hemoglobin, you will only target anti-hapten and not anti-BSA

How do you measure free Ag* inside and out of the dialysis tubing?

You measure the radioactivity of the surrounding solution as inside and out should be in equilibrium

What is the formula to calculate the affinity constant (Ka)?

[AbAg] ------------ [Ab][Ag]

Radioimmunoassay

a technique for determining antibody levels by introducing an antigen labeled with a radioisotope and measuring the subsequent radioactivity of the antibody component

Class switching

ability of a B cell to produce a different class of antibody when stimulated by a certain cytokine environment

Membrane Attack Complex (MAC)

acts as a molecular drill in that it has an affinity to penetrate the plasma membrane and establishes a pore that prevents the cell from osmoregulation, thus causing all the water to leak out (i.e. cell lysis)

Quiz: A radioimmunoassay (RIA) can measure the amount of viral protein in serum by...

adding that sample to a mixture of anti-viral Ab and a radiolabeled sample of the same viral protein to determine competitive reduction of the radiolabel in the precipitate

what is the result of junctional diversity?

allelic exclusion no longer applies, and the second parental chromosome can undergo rearrangement to produce the necessary B or T cells

flow cytometry

allows us to measure, cell-by-cell, a number of fluorescent and spectral characteristics

Immunogen

antigens that: 1) are recognized as foreign by BCRs or TCRs 2) have a certain complexity 3) are of a certain size (5-10kDa)

Haptens

antigens too small to provoke immune responses; attach to carrier molecules to become immunogens

Somatic hypermutation

as B cells continue to divide, the Ig sequence is hypermutable which can lead to the production of a slightly different Ig → if this mutation increases the affinity of the Ig for the Ag, affinity maturation will apply, and that mutation will come to make up a larger portion of the Ab's produced

What was the only thing Kohler & Milstein could tell from Figure 1?

can only tell that hybridoma was making an Ig that was not encoded by the parental myeloma → they hoped it was using information from the splenic B cell parent

Quiz: Mitogens are experimental reagents that...

cause cellular proliferation

Ab binding Complement

causes cascade of enzymatic changes that produces the "Membrane Attack Complex (MAC)" that will cause cell lysis

Path of leukocyte transport in the blood

circulating blood → tissues → draining lymph nodes → lymphatic vessels → thoracic duct → circulating blood

Benacerraf & McDevitt

combined Benacerraf's work with GA and GT polymers and McDevitt's knowledge of mice and confirmed Snell's discovery of MHC genes and their regulation of the immune response to foreign antigens

What does side scatter measure?

complexity and granularity

Which domain does class switching apply to on Hc's and Lc's?

constant domain

Ab precipitation/agglutination purpose

crosslink antigens to precipitate/aggregate them out of solution to prevent their antigenic capacity

Quiz: The Till and McCullough experiment to evaluate CFU-s numbers by injecting bone marrow into an irradiated recipient was able to

demonstrate that bone marrow contained pluripotent stem cells that could differentiate into many different hematopoietic lineages

Quiz: Primary lymphoid tissues

develop early in embryonic life

Papain digestion of Ig's

digests/cleaves Ig AT the hinge structure, above the Hc-Hc interchain disulfide bridge Produces: 2 Fab fragments & 1 Fc fragment

Pepsin digestion of Ig's

digests/cleaves below the Hc-Hc interchain disulfide bridge Produces: 1 F(ab')2 fragment & peptides

Quiz: an example of a non-aggressive cellular defense is:

disaggregated marine sponge cells that fail to interact with cells from an unrelated sponge, preventing the two sponges from forming an aggregate of both cell types

Cation-pi forces (may not be necessary to know)

electronegative cations interacting with electron clouds

Recombinase

enzyme that cuts recognition signal sequences (RSS's) and ligates the cut ends back together → is responsible for the rearrangement of Ig's

F(ab')2

fragment containing both Ag-binding domains of an Ig as well as the top of the Hc-Hc disulfide linkage → called "ab'" because the length of the Hc's are longer in Fab' than Fab → called ")2" since the fragment is BIvalent

Benefit of gene duplication

generating different bits of DNA that share sequence homology on the same chromosome can allow for mutation in one gene that will not affect the physiological role required since there is a compensatory gene still present/stable → allows you to take beneficial mutations/structures and apply it to different constraints and produce other Ig's over time for the Supergene Family

Gene duplication

generation of extra copies of a gene in a genome over evolutionary time **If duplication (or deletion) is too large, it can be genetically lethal**

Residual Heterozygosity

genes in mouse will continue to segregate due to random forces

Fab fragment

has variable region of Hc and Lc and Hc-Lc interchain disulfide, allowing for continued Ag binding/recognition

What is in HAT media and what is their purpose?

hypoxanthine, aminopterin, and thymidine → aminopterin blocks de novo DNA synthesis → hypoxanthine and thymidine can be used for salvage pathway DNA synthesis, but only if cell has HGPRT enzyme

Gene duplication via inversion

if inversion on one of the chromosomes, you can get homologous pairing, but the second chromosome will be in an omega loop structure → loop allows you to flip partner genes, but that twist results in gene duplication/deletion

mass cytometers

instead of using fluorescence, the cells are labeled with metals of specific molecular weights and passed through a mass spectrometer that will separate them based on size → however, ridiculously expensive

Co-isogenic Strain

instead of whole gene region like in congenic strains, it is a single point mutation within the gene/locus

Intrachain & interchain disulfide bonds

intrachain: between domain loops of a single Lc & Hc interchain: connects Hc-Hc and Lc-Hc

Purpose of irradiation in Till & McCullough experiments

irradiation causes DNA damage in dividing cells (stem cells), thus deleting the intrinsic immune system

What is an anti-idiotype?

it is an Ab that is formed against the Ag-binding site of another Ab → this Ag-binding site serves as its own epitope for Ab's

Fc fragment

it is the nonbinding, crystallizing fragment (ergo F'c') → top portion is glycosylated (carb side chain) to confer stability for use in therapeutics → point where transmembrane region would be added on to make it a cell surface receptor (i.e. BcR)

What is the constraint on the free Ag* in an equilibrium dialysis experiment?

it must be smaller than the MWCO to allow for free diffusion to reach equilibrium

What was the benefit of large endoplasmic reticulum for Kohler & Milstein's hybridoma lines?

large ER = increased Ab production

Lymph Nodes

membrane-encapsulated sacks of lymphocytes located all throughout body

Adjuvants

molecules that enrich/boost the host immune response to an immunogen challenge NONSPECIFICALLY

What is the type of bond that occurs between Ab's and Ag's?

non-covalent

What type of interaction is found in immune complexes?

non-covalent: → bidirectional → dissociable → strong

People are cloning individuals out of induced pluripotent stem cells (de-differentiated fibroblasts). Could this be done with a B cell?

not likely as the de-differentiated cell would lack some of the genetic material from a naive B cell that was cut out during its differentiation → the excision of genetic material is *permanent* and cannot be undone in a B cell

How many constant domains are in each Light Chain of an Ig?

one

Instructional Hypothesis and how it was disputed

one gene makes one Ab that then molds itself around each Ag it encounters to make a Ag-specific Ab → disputed by us not having Ab's against molecules naturally occurring within us

How did Kohler & Milstein separate out RBCs from their splenic mouse cells?

osmolysis → increase water concentration in cells to burst membrane → WBCs have more flexible membranes and could withstand this

Van der Waals forces (may not be necessary to know)

overlap between electron clouds

Nonspecific Stimulation of Lymphocytes Effect of Adjuvants

particularly stimulates T cells, but also B cells → if stimulating T cells nonspecifically, helps T-dependent Ag's more effectively and enrich the Ab production induced by that Ag

Mitogens

polyclonal activator that can stimulate the proliferation of EITHER B cells OR T cells like a surrogate antigen

Precipitation vs Agglutination

precipitation = clumping of macromolecules agglutination = clumping of cells

Quiz: What is an important difference between a primary immune response and a secondary immune response?

primary immune responses have a much longer lag phase before they reach the highest vigor than secondary immune responses

What is the pros and cons of increasing the amount of colors detected in flow cytometry or FACS?

pro: higher refinement for phenotyping due to the ability to use multiple markers/restrictions con: the distinction between blue and blue-green can become difficult and thus the results may become ambiguous

Mercaptoethanol (Kohler & Milstein)

reducing agent that breaks disulfide bridges, separating all Hc's and Lc's from each other

What are the granules in granulocytes?

secretory vesicles that can enhance immune response

Qualities of dialysis tubing for equilibrium dialysis experiments

semipermeable membrane but has a specific molecular weight cut-off (MWCO) → greater than MWCO = no transport → less than MWCO = easy diffusion in and out

Depo Effect of Adjuvants

similar to emulsifying a protein in oil before injection: the immunogen will slowly release into the circulation over time, increasing the immune response

FACS (fluorescence activated cell sorting)

similar to flow cytometry, but here the machine can separate the cells into different tubes based on their characteristics by reaching in and moving them → allows you to separate cell populations, not just analyze them

How did they first discover the Ab surface molecule

smashed mouse brains, injected into rabbit, and took rabbit sera containing Ab's and injected BACK into mouse → bound to mouse brain & thymus (but not human thymus)

How to discern T-dependent cells from T-independent cells?

stimulate neonatal thymectomy or congenital athymic mouse with either T-indep or T-dep Ags -if T-indep, Ab's will be produce -if T-dep, no Ab's will be produced

Nonspecific Stimulation of Phagocytosis Effect of Adjuvants

stimulates phagocytes which then stimulate B cells to produce Ab's

What does the results of a flow cytometry experiment tell you?

that you may have different populations of cells based on their spectral, scatter, or fluorescent characteristics → can make histogram with the amount of fluorescence against the number of cells for each fluorescence amount

What contributes to the isoelectric point of a protein?

the aa residue sequences -each amino terminus -each carboxy terminus -each charged side chain residues (some are + and some are -)

Why did Tonegawa and Hozumi have to use the same strain of adult mice as the tumor?

the change could be attributed to allelic differences

What happens if the parental chromosome undergoing rearrangement encounters a mistake?

the concept of allelic exclusion no longer applies and thus the second parental chromosome can undergo rearrangement

junctional diversity

the errors of cuttings and NT-addition that change the length of the chromosomal sequences that induce frameshift mutations

Why tag a cell with a fluorescent molecule for flow cytometry?

the fluorescent molecule will have a known excitation and emission spectra, therefore it will absorb the laser's wavelength and emit it at a higher, predetermined wavelength which can then be detected and recognized by the computer as that fluorescent molecule → if you attach fluorescent markers that recognize certain surface molecules, you can sort cells according to their surface molecules via fluorescence detection

What is hematopoiesis?

the formation of all blood cells via differentiation and determination

affinity maturation

the higher the affinity of an Ab for an Ag, the longer the two will interact, thus allowing for a longer period of stimulation for the B cell to continue proliferating and making that Ab → thus, the Ab's that bind the Ag better than others (cognate vs cross-reactive) will come to make up the majority of the binding population

What made the karyotype of the hybridomas different from that of the splenic B cell or myeloma (Kohler & Milstein)?

the hybridoma cell type carried chromosome karyotypes from both parents (40 + 60 = 100 chromosomes) → note that there are only so many spindles made in mice to pull apart 40 chromosomes, therefore sometimes lose genetic information

What is the Line of Equivalence?

the line of precipitation that forms between radially-diffused samples of Ab and Ag → says that there is a precipitant that has formed and thus the samples are no longer diffusing past that point

okazaki fragment

the non-replicating, circular DNA fragment formed out of the DNA excised and ligated together by a recombinase → will be marked as waste and broken down

What is the isoelectric point of a protein?

the pH at which the protein has no net charge → thus, the protein cannot be pulled in either direction in a gel by an electric current

Quiz: what happens when you add free hapten to a precipitate between haptenated-carrier protein and Ab?

the precipitate decreases

Haptenation

the process of chemically coupling small molecules to larger carrier proteins to confer the necessary size and complexity to the small molecule for it to be recognized as an antigen

What would you predict would happen if you add an excess of free haptens to a solution containing precipitants of the rabbit anti-BSA, anti-hapten serum and haptenated-hemoglobin?

the reaction precipitant would get SMALLER due to COMPETITVE BINDING by free haptens, decreasing the molecular weight of the immune complex to a soluble size

What are framework amino acids (Kabat & Wu)?

the residues that remained consistent (with a variability of 1) within the variable regions of the B cell tumor light chains

What does forward scatter measure?

the size of the cell

Hydrophobic forces (may not be necessary to know)

the tighter the fit, the more water is forced out, forcing them closer together

Why would do you need to re-clone hybridoma cell lines over time?

there are only so many spindles made in mice to pull apart 40 chromosomes, therefore if a hybridoma has ~100 chromosomes, the daughter cells sometimes lose the genetic information they were selected for in the first place

What happens when someone is mutant for RAG?

they can no longer encode the recombinase that is required for both B and T cell differentiation → the Hc and Lc loci rearrangement discussed for B cells also occurs in T cells

How did Tonegawa and Hozumi prove that their results were not the results of a mutation?

they did the same experiment with other REs and saw the same results in the REs that would generate the same phenomenon

Define Lag Phase of the Immune Response

time from exposure to maximum vigor

What is the dominant forming direction in immune complexes?

to the right

What does a line between two cell populations in a PCA map indicate?

transition state between cell populations

allograft

transplants between individuals of the same species driven by *all*elic differences in histocompatibility Ag's

Why use trypan blue in complement-mediated cytotoxicity assays?

trypan blue is usually cleared by living cells, but cannot be cleared by dead cells → allows for direct counting of dead cells

Which domain does combinatorial, associational, junctional diversity, hypermutation, and affinity maturation apply to on Hc's and Lc's?

variable domain

Electrostatic forces (may not be necessary to know)

when +'s and -'s interact and attract each other

If non-covalent interactions are so weak on their own, why is an Ag-Ab interaction so strong?

when an Ab and Ag interact, many of the 5 forces that hold them together are present → many weak bonds induce a very strong bond

Gene duplication via Unequal Crossing Over

when chromosomal sequences are misaligned and unequal crossing over occurs, can lead to duplications/deletions

Hydrogen bonding (may not be necessary to know)

when electronegative atoms share hydrogens

associational diversity

which Hc and Lc associate → multiply the probabilities of the individual segments

combinatorial diversity

which VDJ segments combine to make a heavy chain or light chain

How would you describe a skin graft between flatworm & earthworm?

xenogeneic DO NOT PICK FRET!!!!!

Selection Hypothesis and how it was disputed

you encode and thus make every Ab and then they are selected for → disputed by the fact that we simply don't have enough genetic material for that