Exam II Flashcards
what is antigen processing?
series of intracellular events in which antigen presenting cells make antigen available for T cells
Antigen processing involves
- Uptake of antigens (proteins)
- Degradation to peptides of MHC I or II
- Transport to the cell surface
what is antigen presentation?
Presentation of MHC peptide complexes on the cell surface for the stimulation of T cells
Typically done by antigen presnting cells (APC)
1. Dendritic cells
2. macrophages
3. B cells
Types of APC?
Dendritic Cells
Macrophages
B cells
T cell receptor binds to both
peptide and MHC molecule
T cells must have
MHC in order to be able to respond to an antigen
Cytotoxic T cells
CD8
CD8 complex consists of
alpha and beta
Helper T cell
CD4
CD8 T cell, their T cell receptor binds to
alpha3 domain of MHC class I
CD4 T Cell, their T cell receptor binds
Beta2 domain of MHC class II
Endocytic processing pathway
Exogenous (antigens from outside the cell) or MHC Class II processing pathway
Bound to MHC Class II and taken into the cell
Cytosolic
Endogenous (proteins that exist in the cytoplasm) or MHC Class I processing pathway
Endocytic Pathway
- Antigen is taken up from the extracellular space into intracellular vesicles
- In early endosomes of neutral pH, endosomal proteases are inactive
- Acidification of vesicles activates proteases to degrade antigen into peptide fragments
- Vesicles containing peptides fuse with vesicles containing MHC Class II molecules
The MCH class II binds to the peptide.
lower pH, more degradation
MHC II molecule is synthesized in
lumen of ER
How MHC class II gets to the processed peptides
- Invariant chains blocks binding of peptides to MHC class II molecules in the ER
- In vesicles, invariant chain is cleaved, leaving the CLIP fragment bound
- CLIP blocks binding of peptides to MHC Class II in vesicles
- HLA-DM facilitates release of CLIP, allowing peptides to bind
MHC Class II - endocytic
presents peptide antigens
Invariant chain - endocytic
directs class II away from secretory pathway to endocytic pathway and blocks peptide loading in the ER
HLA-DM -endocytic
acts as a chaperone or catalyst to facilitate exchange of CLIP with antigenic peptides
pH - endocytic
low pH and degradative environment facilitates denaturation of antigenic proteins
proteases - endocytic
cathepsins and other degradative enzymes chew up antigens into peptides
Cytoplasmic pathway (antigens are in the cytoplasm)
proteasome - degrades the antigen protein into peptides,
- class I is heavy chain is stabilized by calnexin until B2-microglobulin binds
- Calnexin is released and the heterodimer of class I heavy chain and b2m forms the peptife loading complex with calreticulin, tapasin, TAP, ERp57, PDI
- A peptide delivered by TAP binds to the class I heavy chain, forming the mature MHC class I molecule
TAP (I and II)- transport port through the ER membrane.
- The class I molecule dissociates from the peptide loading complex and is exported from the endoplasmic reticulum
MHC class I protein
alpha chain (3 domains) - transmembrane protein
beta 2 microglobulin - associated with all MHC 1 - stabilizes it.
peptides that are produced in the cytosol are transported into the ER
Peptides re generated in the cytoplasm and then transported through TAP into the ER and associate with MHC class I
MHC class I - cytosolic
presents antigenic peptides to T cells
proteasome - cytosolic
multi catalytic enzyme complex that degrades proteins into peptides
TAP - cytosolic
transport that shuttles peptides from cytosol to ER
peptide loading complex - cytosolic
calnexin, calreticulin, tapasin, Erp57: stabilize MHC class I and facilitate association with TAP to enable peptide loading
site where MHC II gets loaded with peptides
endosome
site where MHC I gets loaded with peptides
ER
Cytosolic pathogen
- Degraded in:
- peptides bind to:
- Presented to:
- Effect on presenting cell
Degraded in - cytosol
Peptides bind to - MHC I
Presented to - effector CD8 T cells
Effect on presenting cell - cell death
Intravesicular pathogens
Degraded in:
- peptides bind to:
- Presented to:
- Effect on presenting cell
Degraded in: endocytic vesicle (low pH)
Peptides bind to: MHC class II
Presented to: Effector CD4 T cell
Effect on presenting cell: Activation to kill intravesicular bacteria and parasites
How T cell receptors bind to MHC
They do directly bind to MHC molecule -> Polymorphic reside of MHC and T cell contact reside of peptide
But
they also have direct contain with the peptide bound to MHC. T cell contact reside of peptide
- “pocket of MHC”
Major histocompatability complex (MCH)
refers to the complex of genes that encode molecules on the cell surface that:
- mediate T cell reactivity to pathogen infection -> present peptides derived from pathogens to T cells)
- Compatability of organ transplants
- susceptibility to certain autoimmune diseases
Known as HLA in humans and H-2 in mice
divided into three groups: MHC class I, II and III
HLA
Human Leukocyte Antigen
MHC class I structure
Peptide binding cleft: alpha 1+ alpha 2
1 Transmembrane domain
Associates with beta2Microglobulin (always)
CD8 binds to alpha3
MHC class II structure
Peptide binding cleft: alpha 1+ beta 1
alpha 2 and beta 2 are distal from the binding cleft
2 transmembrane regions
CD4 binds to beta 2 domain.
Difference in structure of MHC I and II
I - beta chain is not a transmembrane protein
II - beta chain is a transmembrane protein
Both MHC I and II consists of a
base of beta pleated sheets
and walls are formed of 2 alpha helices.
MHC class I - restricted
strict binding site, allows peptides of 8-10 aa in length
MHC class II - flexible
flexible peptide binding site allows peptides of 10-24+ aa in length
Where is the variablility?
MHC I - peptide binding cleft on both beta pleated sheets and alpha helices
MHC II - majority is in the beta chain. both alpha helix and beta pleated sheets
MHC is both
polymorphic - every gene locus, more than one allele
polygeny - many different MHC molecules/genes expressed
genetic polymorphism
variants or alternative forms of a gene present in a population at a stable frequency
allele
one type of variant
homozygous
having two identical alleles of the same gene
heterozygous
having two distinct alleles of the same gene
haplotype
the collective set of MHC alleles present on an individual chromosome
Human types of MHC class I isotypes
Highly polymorphic
HLA - A
HLA - B
HLA - C
Human MHC Class II isotypes
Polymorphic
HLA - DP
HLA - DQ
HLA - DR (Oligomorphic + highly polymorphic)
Which HLA is the most polymorphic
HLA-B
Which MHC class is most selective?
MHC class II
BCells, macrophages, dendritic cells only. but these cells are also expressed by class I
MHC Class I expressed by every cell except
erythrocytes
MHC Class I overview
expressed by “all cells”
expression constitutive, but can be up-regulated by Type I interferon ( alpha or beta)
MHC class II overview
expressed by antigen presenting cells: B cells, DC, macrophages – Can be upregulated on these cell types by IFN-gamma / type II interferon
expression inducible above basal levels: interferon gamma and CIITA ( transcription factor)
CIITA
transcription factor that upregulates the expression of MHC Class II
MHC genes are expressed
co-dominantely
constitutive -> Immunoproteasome
constitutive proteasome ( 19S cap + beta chains)
IFN -gamma initiates exchange of beta subunits —improves generation of peptides that bind to MHC class I
immunoproteasome - (PA28 cap + new subunits)
exchange of beta subunits
improves generation of peptides that bind MHC class I
Different caps - speeds up export of peptides
complement induces
inflammation -> initiates adaptive immune response
inflammation awakens adaptive immunity through DC
DC are the sentinels if the immune system, communicating between the innate and adaptive immune system
DC at the sites of infection become triggered to “mature” in response to PAMPs or inflammation
DC migrate through lymphatics to draining lymph nodes interacting with many T and B cells
Maturation of DC greatly enhances their ability to stimulate B and T cells
Immature dendritic cells
Tissue resident, resting
Highly endocytic, phagocytic
low level expression of costimulatory molecules
poor stimulators of T cells
Mature dendritic cells
homes to lymph node
endocytosis shut down
high level expression of costimulatory molecules and cytokines
highly stimulatory for T cells
Mature dendritic cells
homes to lymph node
endocytosis shut down
high level expression of costimulatory molecules and cytokines
highly stimulatory for T cells
local infection, innate immunity ->
dendritic cells take infection to lymph node and stimulates adaptive immune system (T cells)
The lymph node
T and B cells have different regions in the lymph nodes
B cell zone
lympohoid follicle
germinal center - clonal expansion
primary lymphoid organs
bone marrow
thymus
sites where B or T cells undergo selection and development
secondary lymphoid organs
spleen
lymph nodes
sites where B or T cells undergo activation
primary lymphoid organs
Bone marrow and thymus
sites where leukocytes undergo hematopoiesis (development and differentiation). Houses naive leukocytes
Secondary lymphoid organs
all other lymphoid organs, spleen and lymph nodes. sites at which naive, activated and memory cells are housed.
B cell receptor
expressed on surface (named B cell receptor) or secreted (antibody)
2 identical heavy chains + 2 identical light chains
2 antigen binding sites. -> each consists of a variable light chain region + variable heavy chain region.
B cell receptor
expressed on surface (named B cell receptor) or secreted (antibody)
2 antigen binding sites (most variable).
Life stages of B and T cells
- Generating a receptor
- Selection
- Activation
- Differentiation
generating a receptor
rearrangement
selection
making sure the receptor does not react with self
activation
providing all of the signals needed to cause clonal expansion
Differentation
signals received during activation dictate the differentiation of the cells and its specific function
T and B cell receptors are the products of
gene rearrangement (somatic)
every cell line has same germ-line configuration, except for B and T cells
after rearrangement T and B cells
no longer have the germline configuration. however, they did before rearrangement
memory cause
long term changes in repertoire
Each lymphocyte bears
a single type of receptor with a unique specificity
interaction between a foreign molecule and
a lymphocyte receptor capable of binding that molecule with high affinity leads ro lymphocyte activation
the differentiated effector cells derived from an activated lymphocyte will bear receptors of
identical specificity to those of the parental cell from which that lymphocyte was derived
lymphocytes bearing receptors specific for ubiquitous self molecules
are deleted at an early stage in lymphoid cell development and are therefore absent from the repertoire of mature lymphocytes
B cells “see”
native antigen through BCR
T cells “see”
antigenic peptides (digested or processed pieces of antigens) presented by MHC molecules
specific antibody are good for
neutralization and opsonization
B cells can both be
expressed on the cell surface, but after activation, B cells may be secreted
antibody function is critical for viral or bacterial infection
first line of defense at mucosal sites; critical in limiting the dissemination of pathogen in secondary infection (and following vaccination)
autoimmunity
anti-DNA antibodies have been detected in patients with autoimmune syndromes
research
critical for assays such as western blots, ELISAs, flow cytometry and immunohistochemistry
clinically
cancer therapy (non-Hodgkin’s lymphoma) and autoimmunity (rheumatoid arthritits)
immunoglobin
Secreted form of B cell receptor
composed of two heavy chains (50KDa) and two light chains ( 25KDa)
the two heavy chains are identical as are the two light chains
there are multiple disulfide bonds, both intra and interchain
contains discrete antigen binding regions in the N terminus from both the heavy and light chains. Binding region consists of one region from the heavy chain and one region from the light chain
Fc portion - bottom part of the heavy chains
heavy chain consists of
one variable regions
several constant regions - changes between different isotypes
variable region of the heavy chain is
the part that forms the antigen binding site and can vary
light chain consist of
one variable region, and one constant
Fc region
fraction crystalize
Formed by two constant heavy chains
they do not change (no rearrangement)
Fab region
Fragment antigen binding
variable + constant constant of both heavy and light chains (all of light chains)
antibody structure
allows for flexibility and binding to a range of antigens
determines the antibody isotype
Fc region
B cell receptor cytoplasmic tails
very little …
must co-reside with signaling molecules in the membrane known as IgBeta and IgAlpha
IgBeta + IgAlpha
provide the longer cytoplasmic tail needed by B cell receptors to bind with kinases and to initiate signaling cascades and phosphorylation
if there is no IgBeta and IgAlpha - the B cell receptor cannot be expressed on the surface
CDRs ( complementarily determining regions)
“fingers” sticking out of the VH and VL region of the antigen binding site
Each antigen may be contacted by
6 hyper-variable loops, 3 in the light chain, 3 in the heavy chain
most of the diversity between antibodies is in these regions
the most diversity is observed in the complementary determining region
CDR3 (most likely to make contact with the antigen because it is located outside)
The highest number of Ab:Ag contacts are usually with the CDR3 region
conformational determinant
if an antibody is folded in its native form and it is then heated or chemically treated, the antibody will lose its form by denaturation
the epitope would be unwound and not able to bind to that same receptor
linear determinant
Ig binds to determinant in both native and denatured protein - not sensitive
Ig binds to determinant in denatured protein only - not to the native form of the protein
five major types of isotypes of human antibodies - no difference in the antigen binding sites. The Fc portion is what changes between the different isotypes
IgG
IgM
IgD
IgA
IgE
IgM
first expressed isotype + first one to be secreted.
But through the lifetime of a B cell, it may express many of the isotypes
IgM + IgA = can be expressed as a monomer or multimer
when IgM binds to J-chain, it is expressed as a pentameric IgM.
IgA can be expressed as a
dimer, when it binds to the J-Chain
Valency and Avidity of antigen-antibody interactions
Valency of interaction ( monovalent, bivalent, polyvalent - essentially how many binding sites of the antibody is occupied by the antigen)
Avidity - the collective affinity of an interaction.
Ex:
valence of interaction: monovalent -> avidity of interaction:low
Valence of interaction: polyvalent -> avidity of interaction: very high
IgM function
Mostly activation of complement
+IgG
IgG function
Neutralization
Opsonization
Sensation for killing by NK cells
Complement activation
Transport across placenta
Diffuse into extravascular sites
IgA function
Neutralization
Transport across epithelium
IgE function
Sensitization of Mast cells
Sensitization of Basophils
IgA Isotype function
transport across mucosa, neutralizatio n
IgD isotype function
antigen receptor on naive B cells, Sensitizes Basophils
IgE Isotype function
immediate hypersensitivity, sensitizes Mast Cells
IgG Isotype function
Neutralization, opsonization, complement activation, neonatal immunity (crosses placenta)
IgM isotype function
antigen receptor on naive B cells, complement activation
How to generate monoclonal antibodies
Isolate spleen cells from mouse immunized with antigen X
Mixture of spleen cells including some producing anti-X-antibody (B cells) fusing with myeloma cells (tumor cells)
HAT medium - allows for killing of myeloma cells and B cells that had not fused
Selective group of hybridomas (fused version of B and myeloma cells)
Grow the cell that has the highest affinity -> hybridomas producing monoclonal anti-X-antibody
Flow cytometry
good for measuring antibody labeled cells
monoclonal cells originally was developed from
mouse cells
Four types of therapeutic monoclonal antibody
mouse
chimeric
humanized
human
antibodies as drugs in Rheumatoid Arthritis
Anti-TNF monoclonal antibodies
TNF receptors
combinatorial diversity
multiple germ line gene segments
Heavy chain locus
V, D, J regions
Light chain locus
V, J regions
Heavy chain event (first)
Germline DNA
- somatic recombination
DJ joined DNA
- somatic recombination
VDJ-joined rearranged DNA - these attach to a constant region
Light chain event
Germline DNA
- somatic recombination
VJ joined rearranged DNA - then attaches to a constant region
