IMMUNO Flashcards
primary and secondary immune organs
Primary- phagocytes, complement system and barriers like skin
Secondary- T cells, B cells, lymphocytes, macrophages, dendritic cells
the ways in which cell types are identified
by: histology (size, shape, stain, nucleus characteristics), enzymes and antibodies that recognize immune cells
antibody and its Fc region
flexible specific adaptor due to a high affinity antigen binding site and Fc region in order to neutralize toxins (prevent binding to receptors) and aid in phagocytosis
*Fc regions: located on the opposite end of the antigen binding site and is a place where effector cells or proteins can bind to
antibody and its Fc region
flexible specific adaptor due to a high affinity antigen binding site and Fc region in order to neutralize toxins and aid in phagocytosis
*Fc regions: located on the opposite end of the antigen binding site and is a place where effector cells or proteins can bind to
opsonins
antibodies that are capable of forming a high affinity bridge to enhance phagocytosis of bacteria found in the extracellular space (opsonization)
complement
serum protein that recognizes bound antibody molecules and can result in cell lysis
CD
“Cluster of Differentiation”: notation system of antibodies that recognize immune cells
examples:
CD3–> mature T-cells
CD4–> T-helper/regulatory
CD8–> T-cytotoxic
neutrophil
the end product of myeloid differentiation that will not divide and contains primary/azurophilic and secondary/specific granules (bactericidal and hydrolytic enzymes of the cell)
make up 60-70% of the circulating white cells (12hrs) and complete its life cycle at the site of inflammation when released from the bone marrow to fight off infection
eosinophil
1-3% of the circulating leukocytes (30min half life and 12 day survival in tissues) with the ability to destroy parasitic worms by releasing granule contents due to Eosinophilic Basic Protein (EBP)
macrophages-monocytes
derived in the bone marrow and function as effector cells at sites of inflammation phagocytosing bacteria (creating phagosomes and phagolysosomes once the bacteria is degraded) but can exist in a resting state when inflammation is not present
- intracellular killing: bacteria, yeast, parasites
- extracellular killing (in vitro): virally infected cells, larger parasites, tumor cells
**binding of bacterial components to signaling receptors induces the synthesis of inflammatory cytokines
pathway of monocyte precursor to immune response initiation
monocyte (precursor)–> macrophage–> T-cell activation–> initiation of immune responses
mast cell
expels parasites by releasing granules that contain active agents such as histamine
lymphocytes
B-cells: expresses immunoglobulin producing antibodies when fully differentiated as a plasma cell (or can become memory cells) and is only able to express a single variable region (idiotype) with the highest affinity to the antigen that undergoes clonal expansion
T-cells (cytotoxic T-cells): helps B-cells through regulating the immune response and acting as an antigen specific effector cell restricted to killing cells with self and foreign antigen limiting target cell types to infected cells and tumor cells (prevents B-cells from attacking our own cells)
- *some can kill non-self cells associated with transplants
- *histology: no granules present in cytoplasm
- *pathogen-reactive lymphocytes proliferate
lymphocytes
B-cells: expresses immunoglobulin producing antibodies when fully differentiated as a plasma cell
T-cells (cytotoxic T-cells): helps B-cells through regulating the immune response and acting as an antigen specific effector cell restricted to killing cells with self and foreign antigen limiting target cell types to infected cells and tumor cells
- *some can kill non-self cells associated with transplants
- *histology: no granules present in cytoplasm
natural killer cells
granular lymphocytes that kill tumor and virally infected cells WITHOUT specificity
what is the pathway that leads to the production of platelets?
hematopoietic stem cell–> myeloid precursor–> megakaryocyte–> platelets
what do each end of immunoglobulin molecules do?
constant region binds to transmembrane surface and variable region has antigen-binding sites at its tips
proteins that make up immunoglobulin molecules
heavy chains and light chains make up constant and variable regions which are attached by disulfide bonds at hinge region and contains carbohydrates
- variable region is located at Y tips and contains antigen-binding sites
- light chains are the outside of Y branches
proteins that make up immunoglobulin molecules
heavy chains and light chains make up constant and variable regions which are attached by disulfide bonds at hinge region and contains carbohydrates
- variable region is located at Y tips and contains antigen-binding sites
- light chains are the outside of Y branches
IgG
- most common
- longest half life
- transportation across placenta
IgA
- one form is secreted (resists acid hydrolysis)
- highly glycosylated
- monomers/dimers/trimmers
- capable of transportation across epithelium
- opsonizing and agglutinating (sticks)
IgM
- primitive
- potent complement fixation
- no opsonization-immune cells don’t have IgM receptors
- greatest molecular mass
- 4 heavy chain domains without a hinge region
- J chain
IgE
- responds to parasites
- allergic reactions
- few in circulation–>binds to target/mast cells
- mediates release of granule contents from mast cells
- mediates changes in vascular permeability
**proteolytic degradation of IgG
IgG –(protease cleavage)–> 1 Fc + 2 Fab fragments
Fc= crystallizable and made of constant region repeats Fab= antigen binding with the variable region
- –not really important but in the notes—-
- papain digests hinge region
- pepsin degrades heavy chain beginning at carboxy terminal and ending at the region of interchain disulfide bonds
proteolytic degradation of IgG
IgG –(protease)–> 1 Fc + 2 Fab fragments
Fc= crystallizable and made of constant region repeats Fab= antigen binding with the variable region
framework residues and hypervariable regions of the variable region of immunoglobulin molecules
framework residues: same between proteins and contribute to the folding of the V region producing the antigen binding site
hypervariable region: 3 in each heavy and light chain contributing to the specific antigen binding site and forming a continuous surface to complement a specific antigen; regions are distant in primary sequence (on two chains) but are close together in the antigen binding site
monoclonal antibody
bound reversibly to an antigen and the affinity between the two is the sum of all of their interactions expressed by the law of mass action
K= [Ab Ag] / [Ab][Ag]
*have affinity but since they bind to the same epitope, there is no cooperativity and no increase in avidity
why are immunoassays used and what are 4 examples?
a process of measuring specific proteins through their properties as antigens or antibodies
examples: ELISA, immunofluorescence, FACS and Western Immunoblot
how do immunoassays work and when are they used?
?
cross-reactivity
antiserum cross reacts with other antigens besides the specific antigen which may be due to impurities (already present antibody or antigenic contaminating proteins) or common/similar structures (homology) on antigens such as epitopes
could lead to: masking an expected result, false results, lowering of the effective sensitivity of the assay or could have no effect on immunoassay test results
techniques used to eliminate cross reactivities
- absorbtion: using cross reacting material to remove the activity that causes the cross reaction
- affinity chromatography: a method used to purify antigens using an insoluble support (ex: agarose) and mild denaturant (ex: salt) to wash away unbound molecules and elute specifically bound molecules
techniques used to eliminate cross reactivities
- absorbtion: using cross reacting material to remove the activity that causes the cross reaction
- affinity chromatography: a method used to purify antigens using an insoluble support and mild denaturant to wash away unbound molecules and elute specifically bound molecules
*preparation of monoclonal antibodies
- immunize an animal
- isolate spleen cells (B-cells which can only live a few days without myeloma cells)
- fuse cells to plasmacytoma tumor cells (unlimited growth)
- select for hybrids of tumor cells and B-cells
- clone hybridomas so each cell grows independently
- select the individual clone with specificity you need
- production of a single clone of one B-cell
- no heterogeneity (population of antibody molecules identical with the same specificity)
what happens when an immunization if to a specific protein?
the clones have individual specificities to all parts of the molecule (epitopes) and to other molecules that have contaminated the immunization preparation
serum sickness
immune reaction (antibodies) to injected proteins causing a hypersensitivity reaction
*this is why you cannot use mouse monoclonals to treat humans unless B or T cells are eliminated
chimeric monoclonal antibodies
constant regions: human
variable regions: from mouse monoclonal
“-ximab” drugs
human monoclonal antibodies
totally made through molecular biology techniques
“-umab” drugs
ELISA
*Enzyme-Linked InnumoSorbant Assay:
add an antibody to tube filled with antigen and incubate it, wash away unbound antibody, add second antibody with a covalently-bound enzyme and incubate it, wash away unbound antibody, detect amount of second antibody-enzyme complex by adding a chemical reagent that turns a certain color in its presence
Immunofluorescence
used to identify a specific cell type, cellular structure or a pathogen through use of specific antisera, unbound antisera is washed away, a second antibody specific for the first antibody but with a fluorescent molecule is added and binds, the fluorescent molecule emits light when exposed to UV light which can be seen under a special microscope
FACS
*Fluorescence Activated Cell Sorter- Flow Cytometry:
machine using lasers with multiple detectors that scan many cells for size and immunofluorescence detecting several antigens an creating a histogram (x-axis: intensity of fluorescence, y-axis: number of cells) and dot plot (top right: both positive, bottom left: both negative)
- cell sorters: analyzes and sorts based on amount of fluorescence
- flow cytometers: only analyze cells
*Western Immunoblot
electrophoretical separation of mixtures of proteins which are bound to nitrocellulose paper, antibody binds to protein of interest, unbound antibodies are washed away, specific antibodies are detected like the ELISA assay providing information such as: amount of antigen (density of bands), molecular weight (how far bands travel), different forms of antigen
*quantitative and qualitative information
Western Immunoblot
electrophoretically separated mixtures of proteins which are bound to nitrocellulose paper, antibody binds to protein of interest, unbound antibodies are washed away, specific antibodies are detected like the ELISA assay providing information such as: amount of antigen, molecular weight, different forms of antigen
titer
how much you can dilute blood before loss of activity
phases of antibody production in response to an antigen
- lag/inductive/latent period
- exponential increase in antibody concentration
- steady state (peak antibody concentration is reached)
- decay/decline: small amount of antibody is detected
immunologic rememberance response
increase in affinity for IgG which persists in blood and response more quickly when the antigen presents itself again
*toxin vs. toxoid
toxin: will kill you since the amount required for a response is lethal
toxoid: protein is chemically modified so it is antigenically the same but not lethal
Hapten-Carrier
hapten: small molecule that cannot induce an antibody response on its own and is recognized by the B-cell
carrier protein: helps make hapten immunogenic and is recognized by the helper T-cell or B-cell
Hapten-Carrier
hapten: small molecule that cannot induce an antibody response on its own and is recognized by the B-cell
carrier protein: helps make hapten immunogenic and is recognized by the helper T-cell or B-cell
Antigen Presenting Cells (APC)
macrophages and dendritic cells which initiate the interaction with antigen by endocytosis or phagocytosis enhanced with a complement, pre-existing antibody or specific receptors through presentation of antigen as a small peptide (antigen/MHC complex) and a second/co-stimulatory signal (B7 antigen on presenting cell binds to CD28 on T-cell) critical for an immune response (B-cell or T-cell activation and proliferation)
Antigen Presenting Cells (APC)
macrophages and dendritic cells which initiate the interaction with antigen by endocytosis or phagocytosis enhanced with a complement, pre-existing antibody or specific receptors through presentation of antigen as a small peptide
what are two things essential for inducing immunity?
presentation of antigen AND co-stimulation
what contributes to the loss of many T and B cell precursors
most rearranged genes won’t function which is the usual mechanism to bring about diversity in antigen receptors (rearrangement of small pieces of DNA)
why does the concept of one gene one protein do not hold for immunoglobulin molecules?
they have a constant region and a variable region
- in the germ line, the constant regions are not next to the variable regions and there are different configurations for Ig gene material compared to the myeloma
- multiple Vs, Js and Ds (segments which proved that rearrangement occurs) are brought together by signal sequences from enzymes
VJD segments of heavy vs. light chains
heavy chain: V (38-46), D (23), J (6)
light chain: V (34-38), J (5)
- of the two light chain possibilities (kappa and lambda), only one is kept active
- germline DNA undergoes somatic recombination for the segments to be joined (light- VJ joins; heavy- DJ then VDJ joins)
how are V, D and J brought together?
enzymes (recombinase genes, RAG 1 and RAG 2) are induced in developing B-cells- signal sequences determine which segments can be joined through random, independent combinations
*RAG- grabs one end of the immunoglobulin and pulls it around and together causing a piece of DNA to be irreversibly discarded (section is cleaned and ligated)
mechanism of the generation of junctional diversity (pay special attention to what TdT can do)
- RAG complex cleaves the heptamer to yield DNA hairpins
- RAG complex opens hairpins generating palindromic P-nucleotides
- N-nucleotide additions by TdT/terminal deoxynucleotidyl transferase (random additions only in heavy chains of B-cells in the pro B stages until an overlap is achieved); repair enzymes repair
- strands are paired
- unpaired nucleotides are removed by an exonuclease
- gaps are filled by DNA synthesis and ligation to form coding joint
***P (in all joining junctions) and N regions made at edit sites by the random additions by TdT may cause DNA to exceed reading frame and stop encoding for the correct AA (results in incorrect IgM which won’t be able to be inserted into the membrane)
early pro-B cell to immature B cell
heavy chain rearrangement:
(early pro-B) D-J rearrangements on both chromosomes
(late pro-B) V-DJ rearrangement on first chromosome and if nonproductive, then on second chromosome and if nonproductive, then apoptosis
light chain rearrangement:
(pre-B) rearrange K gene on first chromosome and if nonproductive, then on second chromosome and if nonproductive, then lambda gene on first chromosome and if nonproductive, then on second chromosome and if nonproductive, then apoptosis (several changes to rearrange in light chain since heavy chain has already been successful in rearranging)
*immature B cell will express mu and K or mu and lambda signal when rearrangement has ended based on if a productive rearrangement was made with K or lambda gene in light chain rearrangement
what causes the switch from IgM to IgD?
selective splicing of primary RNA transcripts made of both IgM and IgD constant regions
*splicing control also determines whether a cell synthesizes membrane bound Ig (2 extra exons) or secreted Ig
**AID
*Activation-Induced Cytidine Deaminase
enzyme that attacks regions allowing for another editing inducing mutations and allowing section of DNA being cut and goes to gamma chain (causes mutations that produce antibody diversity)
- makes mutations DURING the immune response across DNA causing single base changes
- responsible for class switching and somatic hypermutation
class switching
a variable region is joined to a new constant region (except in IgD) which brings about more diversity only with the help of T-cells and during an immune response
- since the variable region stays the same, class switching does not affect antigen specificity
- the antibody retains affinity for the same antigens, but can interact with different effector molecules
- daughter cells can produce antibodies of different isotypes
somatic hypermutations
targets rearranged gene segments encoding the variable region (gives it the capability to respond to new threats- alters affinity)
*only in immune cells and not transmittable to offspring
**B-cell vs. T-cell receptor formation
- most of the mechanisms for their formations are the same except T-cell receptor does not undergo somatic hypermutation
- also, T-cell will NOT change during the immune response
regulation of T-cell proliferation in the thymus
T is selected for the elimination of strong self-reacting T-cells (tolerized to self antigens) which occurs in order to prevent autoimmune diseases (also related to transplantations)
- T-cells become MHC-restricted (peptide is presented by MHC molecule for TCR recognition) and lineage committed since the thymus provides the appropriate microenvironment
- selection occurs at several stages (positive and negative selection)
what would a deficiency in RAG 1 or RAG 2 lead to?
inability to make T and B cells leading to immunodeficiency
which MHC molecules do CD4 and CD8 lineage T ells recognize?
CD4- beta 2 of MHC II
CD8- alpha 3 of MHC I
DiGeorge’s Syndrome
human T-cell immunodeficiency due to a loss of a TF required for thymic epithelial differentiation
embryonic thymic development
thymus is formed from the fusion of the 3rd pair of pharyngeal pouch (endoderm) and the cleft (ectoderm)
what are the major regions of the thymus and what happens with age?
regions: cortex, medulla and cortical-medullary junction
* function decreases as age increases
migration of T-cells to and from the thymus
- T-cell precursors (prothymocytes which are not completely committed yet) leave the bone marrow and enter the thymus via blood vessels at the cortico-medullary junction
- mature T-cells leave the thymus and enter secondary lymphoid tissues (GALT, spleen, lymph node) through venules in the medulla
- chemokines and sphingosine 1-phosphate signal traffic for cell movement
- stage is identified by cell surface markers
2 major lineages of T-cells
gamma:delta and alpha:beta
fate of immature thymocytes
death by apoptosis if MHC is not recognized
pre-T cell receptor
2 chains (heterodimer)–> heterodimer with itself (superdimer- more complex)–> signaling components (pre-T cell receptor)–> TCR
synthesis of T-cell receptor alpha and beta chains
- alpha: alpha chain locus can sustain many attempts at a functional rearrangement (usage of upstream components to try and create a functional product)
- its rearrangement will always eliminate the linked delta-chain locus
-beta: two attempts can be made to achieve a productive rearrangement of the beta chain locus
early T-cell development mechanism
progenitor cell–> proliferation–> double negative T cells commit to T lineage–> rearrange beta genes–(check-point for pre-TCR)–> proliferating double negative pre-T cells–> immature double-positive cells rearrange alpha genes–(checkpoint for TCR)–> mature double positive cells (FULLY FUNCTIONAL TCR)
*all steps occur in cortex except proliferation of progenitor cells which occurs in the medulla
double positive thymocytes recognition of self peptides presented by MHC
receptor binds self-peptide:self-MHC class I--> CD8 receptor binds self-peptide:self-MHC class II--> CD4
negative selection of alpha:beta T-cells
- by dendritic cells, macrophages and other cells in the thymus in which tight binding will initiate apoptosis and moderate binding allows the cell to live
- prevents too strong of a recognition
ligands
self peptide/MHC complexes expressed on stromal and hematopoietic cells
- self peptides are derived from endogenous (self) sourcses
- Aire TF plays a key role in regulating the expression of some tissue specific antigens in medullary epithelial cells in the thymus
engagement of the TCR by self-MHC:peptide complexes
can lead to either further maturation or to cell death
Avidity model of T cell selection
depends on the affinity of the TCP-peptide/MHC interaction and the density of the peptide/MHC on the thymic epithelial cell
- avidity determines the total strength of signal delivered which dictates the outcome of the signaling event
- balance of avidity and signal intensity for positive selection whereas too much avidity= negative selection and too little will cause death by neglect
**T-cell repertoire
sum of all of the specificities of the mature T cells produced by the thymus and this differs between individuals
*shaped by thymic selection and modified in the periphery throughout life by the encounters with antigens
what is needed for suppression of autoreactive T cells by regulatory T cells?
interaction with the same antigen-presenting cell
*important for tissue tolerance
what will activate the complement and classical pathways
pathogens–> complement
antibodies–> classical
endotoxin
Structure: heat stable hydrophobic lipid rich end and hydrophilic polysaccharide rich end that is antigenic and is the determinant of the O antigen; epitope: 3-4 repeating monosaccharides
Function: produces the toxic LPS which resides in the Lipid A region (we know this since cleaving the polysaccharide from the lipid A will not affect the activity of the endotoxin) responsible for the body’s response to gram negative bacteria
*gram negative sepsis
endotoxin shock/septic shock due to gram negative bacterial infection causing decreased BP and CO
- is the response to endotoxins
- high incidence of DIC (disseminated intravascular coagulation) causing thrombi (due to an enzyme cascade) and eventual depletion of coagulation factors which then causes hemorrhage and necrosis
systemic introduction of LPS in low quantities and in high quantities
low: *fever, moderate immunostimulation which causes infiltration of neutrophils and macrophages along with increased vascular permeability and swelling
high: *fever, headache, vomiting, endotoxin shock (septic shock)
acute respiratory distress syndrome
associated with septic shock or gram negative insult to the lungs
why is LPS a pyrogen?
it causes fever
assays used to make sure something is pyrogen-free
- USP rabbit pyrogen test
- LAL- Limulus Amebocyte Lysate
pathway leading to survival after LOCAL infection with gram-negative bacteria
macrophages are activates and secrete TNF-alpha in tissue–> increased release of plasma proteins into tissue–> increased phagocyte and lymphocyte migration to tissue–> platelet adhesion to BV wall–> phagocytosis of bacterial–> vessel occlusion–> containment of infection–> antigens are carried to lymph nodes–> survival
pathway leading to death after SYSTEMIC infection with gram negative bacteria (sepsis)
macrophages are activated in the liver and spleen to release TNF-alpha into blood–> systemic edema causes decreased blood volume, hypoproteinemia, neutropenia followed by neutrophilia–> decreased blood volume causes collapse of vessels–> organ failure/septic shock–> death
mediators of the LPS response
outside the intact organism, LPS is not toxic
inside–> proteins (IL-1 and TNF-alpha) produced by LPS-stimulated macrophages serve as the endogenous mediator which induce blood vessels to become more permeable enabling effector cells and fluid containing soluble effector molecules to enter infected tissue causing inflammation at the site of infection
cytokine effects
overlapping effects in the cell in which too much would be bad
IL-1 beta and TNF-alpha
induce blood vessels to become more permeable enabling effector cells and fluid containing soluble effector molecules to enter infected tissue causing inflammation at the site of infection
IL-6
makes heat by inducing fat and muscle cells to metabolize
CXCL8
recruits neutrophils guiding them to infected tissue
IL-12
activates NK cells that secrete cytokines strengthening the macophages’ response to infection
tumor necrosis factor
induces intravascular coagulation and hemorrhage in the tumor serving as an immunoregulatory molecule while mediating inflammatory processes
*stimulates T and B cells along with inducing fevers just like IL-1
anti-TNF-alpha antibodies
block the induction of shock induced by LPS
TLR
toll-like receptors that bind signals inducing signal transduction pathways and production of cytokines
what complex recognizes LPS?
the TLR4, MD2, CD14 complex which is them broken up by a cascade of P that then produces inflammatory cytokines
what does TLR4 homodimer recognize?
gram-negative bacteria and is located on the plasma membrane of macrophages, dendritic cells, mast cells and eosinophils
interleukin-1
cytokine that is secreted by activated and stimulated macrophages that is produces as a pro-cytokine and must be cleaved by caspase 1 (which is activated by the inflammasome) before being released
inflammasome
inflammation sensitive complex that activates caspase 1
*target of anti-inflammatory drugs
superantigen
binds to MHC class II and then T-cell receptor and CD28 in which activation occurs and the production of TNF alpha and IL 1 occur
toxic shock syndrome
produced by staph aureus and causes fever, vomiting, diarrhea, shock and multi-organ dysfunction (due to mediators)
