test 2 Flashcards
what are antibodies/immunoglobulin
Circulating proteins produced by vertebrates in response to foreign antigens
-Primary mediators of humoral immunity against all classes of microbes
what are the two types of antibodies?
- Membrane-bound on B lymphocyte surface: Function= antigen receptor
- Secreted antibodies in circulation and tissues: Function= Prevent entry and spread of microbes and neutralize toxins
Plasma and serum proteins are generally separated into:
albumins and Globulins
- Antibodies are generally found in gamma globulins
describe the common structure of antibodies
- symmetric core structure composed of two identical light chains and two identical heavy chains that both contain a series of repeating units that fold in a globular motif that is called an ig domain which contains two layers of beta-pleated sheet
- Both heavy chain and light chains have terminal variable regions that participate in antigen binding C regions
- The C regions of the heavy chains mediate effector functions (regions interact with effector cells or molecules)
- two antigen binding sites
- disulphide bonds link the Ab molecule together
- Fc receptors bind antibody constant regions
heavy chain vs light chain C region and variable region
heavy chains:
V region- one Ig domain
C region- 3-4 Ig domains
light chains:
V region- one Ig domain
C region- one Ig domain
what are proteolytic fragments of an igG molecule
-the unfolded hinge region between the domains of the heavy chain are the most susceptible to proteolytic cleavage by enzymes
Fab: fragment, antigen binding- these two fragments retain the ability to bind antigens
Fc: Fragment, crystallizable- can self-associate and crystallize into a lattice
where is most of the sequence and variability differences among different antibodies confined to
three short stretches in the V region of the heavy chain and three stretches in the V region of the light chain
- Each stretch ~10 amino acids
- Variability in antigen binding is generated by hypervariable segments/regions that form loops that act as fingers and constitute the antigen binding region
- Hypervariable regions can also be called complementarity-determining regions (CDRs)
what are isotypes
distinct classes of antibody molecules divided based on differences in the structure of the heavy and light chain C regions
ex IgA, IgD, IgE, IgG, IgM
IgA and IgG can be subdivided into closely related subclasses
-the heavy chain C regions of all antibody molecules of one isotype have essentially the same amino acid sequence that are different from other isotypes and have different effector functions
what permits antibodies to bind to different arrays of antigens ?
their flexibility that is conferred, in large part by a hinge region permitting molecular motion
- some flexibility of the antibody molecules is due to the ability of each Vh domain to rotate with respect to the adjacent Ch1 domain
- Allows them to bind two antigens
- Some of the greatest difference between isotypes exist in the hinge region- Leads to different shapes
secreted vs membrane-associated antibodies
-differ in the amino acid sequence of the carboxyl-terminal end of the heavy chain C region
secreted: carboxyl terminal portion is hydrophilic
membrane-bound: carboxyl stretch includes 2 segments: a hydrophobic a-helical transmembrane region followed by intracellular positively charged stretch that bind to neg charged phospholipid head
monomeric vs multimeric isotypes
Secreted IgG, IgE and all membrane-bound forms are monomeric
- Secreted forms of IgM and IgA form multimeric complexes (2+ core Ab molecules are joined)
- Secreted IgM can be pentameric or hexameric
- Multimeric Ab bind antigen with more avidly
Injection of Ig molecules from one species into another results in
an immune response to the different Ab because constant regions of antibodies are different between different species
- makes antibodies largely against the C regions of the introduced Ig
- the response often creates an illness called serum sickness
what are allotypes and idiotypes
allotypes: are polymorphic variants in the sequence of antibodies found in an individual of a species where the variant is recognized by an antibody (same species, different alleles)
idiotypes: the differences between the antibody V regions (same species, different Vh and Vl domains)
what are monoclonal antibodies
- produces a lot of antibodies of a single specificity
- from a single activated B-cell (All effector plasma cells generate same antibody)
- Can fuse B cell with myeloma cancer cell to form a hybridoma which makes only one Ig and is are monoclonal
how are monoclonal antibodies used in research
- Identification of phenotypic markers unique to specific cell types
- Separate differentiate cell types (classification of leukocytes) - Immunodiagnosis
- Infectious and systemic diseases diagnosis - Tumor Detection
- Therapy
limitations of monoclonal antibodies
- Most monoclonal antibodies are produced by immunizing mice but patients treated with mouse antibodies may make antibodies against the mouse Ig, called human anti-mouse antibody (HAMA)
- HAMA block the function or enhance the clearance of the injected monoclonal antibody and can also cause serum sickness
Expression of Antibodies
Heavy and light chains are synthesized on membrane bound ribosomes in the rough ER and the protein is translocated into the ER
- the heavy chains are glycosylated during the translocation process’
- Covalent association of heavy and light chains, stabilized by formation of disulphide bonds also occurs in the ER
- after assembly, the Ig molecules are transported into the Golgi complex where carbohydrates are modified and then moved to plasma membrane in vesicles
- antibodies of the membrane form are anchored in the plasma membrane and the secreted form is transported out of the cell
Expression of Antibodies 2.0
the maturation of B cells from bone marrow progenitors is accompanied by specific changes in Ig gene expression, resulting in the production of Ig molecules in different forms (Expression of Ab changes as B cell differentiates)
- Naive mature B cells makes membrane-bound IgM and IgD which serve as cell surface receptors that recognize antigens and initiate the process of B cell activation and differentiation into plasma cells
- this activation is accompanied with changes to antibody production such as an increased production of the secreted form of Ig relative to the membrane form
- the second change is the expression of Ig heavy chain isotypes other than IgM and IgD, called class switching
what is the half-life of circulating antibodies?
a measure of how long those antibodies remain in the blood after secretion from B cells (or after injection)
- it is the mean time before the number of antibody molecules is reduced by half
- different antibody isotypes have different half lives
Half-life of Ab is dependent on isotype:
IgE - 2 days in circulation, very short
IgA - 3 days in circulation
IgM - 4 days in circulation
IgG - 21-28 days, very long due to its ability to bind to a specific FcR called the neonatal Fc receptor
what is an antigen and give examples
any substance that may be specifically bound by an antibody molecule or T cell receptor:
Complex carbohydrates Phospholipids Nucleic acids Proteins Metabolite Hormones lipids sugars (T cell receptors mainly recognize peptides) -All antigens are recognized by specific lymphocytes or by Ab however only some can activate lymphocytes
Immunogens
molecules that stimulate immune responses
- some are antigens
- Only macromolecules (proteins, polysaccharides, nucleic acids) can stimulate B cells because B cell activation requires linking of multiple antigen receptors to elicit T cell help
determinant or epitope
the portion of the macromolecule in which the antibody binds
- Macromolecules or microbes usually have several molecular epitopes (polyvalency or multivalancy)
- This can induce clustering of B cell receptors and initiate B cell activation
the spatial arrangement of different epitopes on a single protein molecule may influence the binding of antibodies in several ways…
overlapping: when two determinants are close to one another and the binding of antibody to the first one may cause steric interference with the binding of the Ab to the second one
non-overlapping: when determinants are well separated and two or more Ab molecules can be bound to the same protein antigen without influencing each other
Linear determinant
Ig can bind to accessible determinant whether or not the protein is denatured but can only bind to the inaccessible determinant when the protein is denatured
-Epitopes of several adjacent amino acids
Conformational determinant
- formed by amino acid residues that are not in a sequence but become spatially juxtaposed in the folded protein
- determinant is lost of protein is denatured
- *Epitopes not separated by sequence but by conformational structure
Neoantigenic determinants
Epitope only exposed after proteolysis of post-synthetic modifications (PTM)
-altering the structure of the protein can produce new epitopes and be recognized also by specific antibodies
the recognition of antigen by antibody involves
Van Der Waals forces Hydrophobic interactions Electrostatic forces H-bonding Non-covalent -Strength of binding is called affinity -Some soluble Ab can bind multiple epitopes (same) and the overall strength of attachment is called avidity -Much stronger than single epitope binding (many low affinity interaction (up to 10 per IgM molecule) can produce a high avidity interaction
immune complexes
polyvalent antigen mixed with a specific antibody form immune complexes
- large complexes are formed at concentrations of multivalent antigens and antibodies that are termed the zone of equivalence
- the zones are smaller in relative antigen or antibody excess
cross-reaction
when an antibody produced against one antigen may bind to a different but structurally related antigen
features related to effector functinos
most effector functions of Ab’s are mediated by the Fc regions of the molecules and antibody isotypes that differ in these Fc regions perform distinct functions
distinct effector functions of antibody isotypes
- IgG coats microbe and targets them for phagocytosis by neutrophils and macrophages
-Macrophage and neutrophils have FcR for γ heavy chain - IgE binds Mast cells and triggers their degranulation
- IgG or IgM bound by C1q complement activation
-Two or more antigen bound Ab are required to
trigger effector function: Need adjacent Ab Fc regions
(Prevents free Ab from triggering response and limits wastefully and inappropriately triggering effector responses)
Activated B cell clones vs naive B cell antibodies
-Activated B cell clones produce single or different isotypes (within one cell) with Identical Variable domains
-Naïve B cells only produce membrane-bound IgM and IgD
-Activations induces isotype (class-) switching, C region produced changes
-Produces isotype best able to eliminate the pathogen:
Bacteria and Viruses- IgG to promote phagocytosis
Helminths- IgE for mast cell degranulation
CHAPTER 6 STARTS HERE
Features of MHC-Dependent antigen recognition by T cells
- most T cells recognize peptides and no other molecules bc only peptides bind to MHC molecules
- T cells recognize linear peptides and not conformational determinants of protein antigens bc linear peptides bind to clefts of MHC molecules and protein conformation is lost during generation of these peptides
- T cells recognize cell-associated and not soluble antigens because MHC molecules are membrane proteins that display peptides
MHC restriction
a single T cell can recognize a specific peptide displayed by only one of the large number of different MHC molecules that exist
what are co stimulators
the membrane bound molecules of APCs that serve to activate T cells and function together with antigen to stimulate T cells (cause the second signal)
- APCs display antigen-MHC complexes for recognition by T cells AND provide stimulatory molecules (costimulators) to stimulate full T cell response
- Most important for DC naïve T cell interaction
what enhances APC antigen-presenting
function
Exposure to microbial products
-Reason why immune system responds better to microbes than harmless environmental nonmicrobial substances
-DC and Macrophages have TLR that signal
increased production of MHC molecules and
costimulators
-Activated DCs express cytokine receptors
that help them migrate to sites where T cells are present
what are adjuvants
either are products of microbes or they are microbe mimics that enhance co-stimulator and antigen presentation
-required for optimal T cell induction against purified
antigens in vaccines
T cells also send signals to APC to enhance their antigen presentation function.
what are the signals and what is enhanced
- CD4+ T cells express CD40 ligand: Binds CD40 on DC and macrophage
- T cells secrete cytokines such as IFN-γ: Binds to their receptors on APCs
Results in increased:
- Ability to process and present antigen
- Increased expression of co-stimulators
- Secretion of cytokines that activate T cells
Bidirectional signaling provides positive feedback loop
routes of antigen entry
microbial antigens commonly enter through the skin and gastrointestinal and respiratory tracts where they are captured by DCs and transported to regional lymph nodes via lymphatic vessels where they become concentrated
-antigens that enter the blood stream are captured by APCs in the spleen
where do DCs present antigens
Respiratory epithelia Interstitial of parenchymal organs GI epithelia Skin epithelia Lymph nodes -Membraneous or spike-like projections Related to mononuclear phagocytes
Two main-types of DCs
Conventional DCs:
- Able to stimulate strong T cell responses
- Most numerous DC in lymphoid organs
- Give rise to resident tissue DCs
- Tissue DC mature and migrate when they encounter antigen
- DC in the epithelia occupy as much as 25% of surface area of epidermis (Only 1% of cell population)
- In GI
- DC projections extend into lumen to capture antigens
Plasmacytoid DCs:
- Similar morphology to conventional DC but found in blood and only small numbers in lymph organs (Principally T cell zones of spleen and lymph nodes)
- Major function = Secretions of large amounts of type I interferon (Response to viral infections)
- Present to T cells
mature vs immature DCs
- Migration of DC from tissue to lymph nodes marks transition from immature to mature DCs
- 0ccurs after the cell encountered an antigen and TLRs and intracellular microbial sensors (or TNF) signal maturation
immature or resting DCs: express membrane receptors such as C-type lectins and capture and endocytose microbes
Mature DC: lose adhesiveness to epithelia and migrate to lymph nodes. express CCR7 (cytokine receptor)
which targets T cell zones of lymph nodes
Properties that make DCs most efficient APCs for initiation of primary T cell responses
- Located at common sites of microbial entry
- Express receptors for microbe capture and microbial response
- Migration from epithelia/ tissue to T cell zones of lymph nodes
- Mature DCs express high levels of:
MHC-peptide antigen complexes, costimulators, and cytokines, all of which are needed to activate T cells
Major Histocompatibility Complex (MHC)
MHC I and MHC II are most polymorphic genes in genome ~3500 alleles in population
Class I MHC CD8+ T cells
Class II MHC CD4+ T cells
-Class I molecules are constitutively expresses on virtually all nucleated cells
-Class II are only expressed on DC, macrophages, B cells and a few others
-Cytokines increase the expression of MHC molecules
During both adaptive and innate response
-Interferons increase MHC I expression on most cell types (IFN-α, IFN-β and IFN-γ). Produced during early innate response to viruses and Stimulates antigen presentation to virus-specific T cells
-IFN-γ stimulates MHC II expression in APCs
Structural Properties
Each MHC molecule has an extracellular
peptide binding cleft, followed by Ig-like domain and transmembrane and cytoplasmic domains
MHC I: one polypeptide chain encoded
in the MHC and One non-MHC encoded chains
MHC II: two MHC encoded polypeptide chains
-3-D structure of both classes is similar
-Polymorphic amino acids of MHC are located in and next to the peptide-binding cleft
-Ig-like domains are non-polymorphic
MHC I
-Two non-covalently linked polypeptide chains
-Extracellular, TM and cytosolic domains
-Peptide binding cleft can bind 8-11 amino acids
-Antigen must be processed
-CD8 binds to α3
-Binding of α chain with β2 chain is stabilized by
antigen binding within the peptide binding cleft
-Antigen, α chains and β2 chains heterotrimer are
required for expression cell surface
MHC II
- Two non-covalently bound polypeptide chains
-Both encoded by MHC genes
-Both are polymorphic
-Amino terminal α and β segments form peptide binding cleft
-Ends of peptide binding cleft is open to accommodate larger antigens (30+)
-β2 segments bind with CD4
-Fully assembled is heterotrimer
-Antigen, α chains and β2 chains heterotrimer are
required for expression cell surface
Antigen Loading
Each individual only has a few different MHC molecules
~6 Class I and 10-20 Class II
MHC molecules have broad specificity for peptide
Each MHC molecule has single peptide binding cleft
Can bind one peptide at a time
-Small number of MHC-antigen complexes can stimulate specific T cells
-Most peptides presented on MHC molecules are self antigens but TCR only looking for foreign antigens
Structural basis of peptide binding to MHC molecules
-Non-covalent binding
-Peptide fit in MHC binding cleft through:
Electrostatic interactions, Hydrogen bonding and Van der Waals interactions
-TCR recognize both peptide and MHC molecule:
-> Peptide specific for TCR specificity
-> MHC specific for class restriction
Antigen Processing (MHC class I pathway)
- Proteolytic degradation of cytosolic proteins
- peptide fragments are transported to ER where they bind to newly synthesized MHC class I molecules
- TAP transports peptide from cytosol to ER lumen
- MHC I-antigen complexes traffic through the Golgi complex and then Transported to cell surface
Antigen Processing (MHC II)
- Antigens for MHC II molecules are produced from endocytosed antigens in APCs
- Endosome merge with lysosomes for degradation through the phagolysosome
- MHC II molecules are synthesized in ER and transported to endosomes
- Endosome fuses with antigen in vesicles
- MHC II-antigen heterotrimer can be transported and expressed on cell surface
CHAPTER 7**
pathway of NF-kB Activation
- important for lymphocyte development
1. canonical NF-kB heterodimers reside in cytosol bound to an inhibitor of NF-kB called IkBa
2. upstream signalling= activation of ubiquitin E3 ligase that adds ubiquitin chain to protein called NEMO and allows IKKB to be activated by an upstream kinase
3. Active IKKB phosphorylates the inhibitory protein bound to NF-kB (IkBa) which tags this protein for ubiquitin
4. polyubiquitinated IkBa is targeted for degradation in proteasome and the NF-kB heterodimer is free to enter nucleus
Lymphocyte Development: miRNA
Gene expression mostly driven through transcription factors but also by miRNA
miRNA:
Small endogenous non-coding RNA molecules
Generated in nucleus
Cleaved in cytosol
21-22 nt
B Cell Development
B cells develop in bone marrow -Progenitors are Ig –negative Immature B cells with membrane-bound IgM Leave bone barrow and mature in spleen -Follicular B cell -Membrane bound IgM and IgD Recirculate and populate peripheral lymphoid organs Pro-B cell: Earliest bone marrow cell committed to the B cell lineage, No Ig production, V(D)J recombination of heavy chain Pre-B cell: Haven’t yet rearranged light chain Express heavy chain on cell surface Immature B cell: Produce complete IgM molecule Mature B cell: -Follicular B cell= most mature, Produce IgD and IgM and Recirculate between lymph nodes -B-1 and Marginal zone B cells= Express limited antigen receptor repertoire and Secrete IgM spontaneously (React with microbial polysaccharides and lipids) B-1 cells secrete IgA into mucus
Maturation of T lymphocytes
Most maturation occurs in cortex of thymus
- αβ T cells mature into CD4+ and CD8+ cells as they leave the cortex for the medulla
- Thymus produces cytokines and chemokines to stimulate T cell development (esp. IL-7)
- 95% of cells produced by single precursor will die through apoptosis before reaching medulla (Improper TCR production, Self-reactivity, Failure of positive selection by MHC molecules in thymus)
Pro T cell:
-Double negative-> No TCR, No CD4 or CD8
->TCR rearrangement
-Double Positive thymocytes/developing T cells
->Express CD4 and CD8
->Express TCR
*Expression of CD4 OR CD8 marks phenotypic
AND functional maturation
Antigen Receptor Generation
Generated through gene rearrangements within individual lymphocytes
=V(D)J recombination
-Exon for each antigen (variable region, (V)) fused to downstream segment on same chromosome
-Ab have similar C-terminal CH and CL BUT- very different N-terminal regions, VH and VL
