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resting B cell
naïve- never seen antigen that it recognizes
Where are B cells found?
lymphatic system
Where do B cells arise from?
pluripotent hematopoietic stem cells in bone marrow give rise to common lymphoid progenitor cells
What are antigen binding sites made up of?
variable region of a light chain and the variable region of the heavy chain
isotypes of heavy chains
G, M, A, D, E
isotypes of light chains
kappa, lambda
hypervaraiable regions
areas where the amino acid sequence differs the most between antibodies
epitope
part of an antigen where antibody binds
multivalent antigen
any antigen that contains more than one epitope (can be copies of same epitope)
linear epitope
amino acids are adjacent (denatured protein can still be used)
discontinuous epitope
amino acids that are not adjacent
invitro vaccine
linear epitope is best bet
Monoclonal antibody steps
immunize mouse w/ antigen
screen via ELISA to ID mouse
kill mouse, harvest spleen, make a paste
isolate B cells
fuse B cells w/ myeloma cells —> hybridoma
dilute into 1 cell per well in a 96-well plate
screening (ELISA) to find right hybridoma
take right hybridoma and mass produce
purify antibody
make $$$
light chain V region
V (variable) and J (joining)
heavy chain V region
V (variable), D (diversity), and J (joining)
light chain gene segment
L (leading), V (variable), J (joining), C (constant)
heavy chain gene segment
L (leading), V (variable), D (diversity), J (joining), C (constant)
each V, D, J segment is flanked by _________ __________ ___________ that direct recombination
recombination signal sequences (RSS)
RAG
recombination-activating genes
RAG _____ and ______ your DNA; the DNA left in the middle is eventually degraded
cut; paste
recombination enzymes
RAG binds to RSSs, brings them together forming hairpin loop
RAG cleaves DNA at specific sequences, removing the hairpin loop and adding P nucleotides
terminal deoxynucleotidyl transferase (TdT) randomly adds N nucleotides
addition of P and N nucleotides = junctional diversity
rearrangement of VDJ segments produces a functional ______ chain gene
heavy
random V, D, J explains:
diversity in B cells; can build functional proteins or not
allelic exclusion
even though every B cell has two copies or alleles of the heavy/light chain locus, only one of each are rearranged to produce functional genes
naive B cell circulates until it comes into contact with the _______ (hopefully on a pathogen) that it recognizes
antigen
immunoglobulins are 1st made as _______________ on B cell surface; after exposure to antigen, Ig are produced in _________ form (antibodies)
membrane-bound; secreted
somatic hypermutation
random point mutations introduced at a high rate in V domain
point of mutations and diversifications of antibodies after antigen exposure
affinity maturation
isotype switching
produces Ig with different C regions but identical antigen specificities; happens via more recombination at switch sequences
IgM function
really good at activating complement through the classical pathway
IgM unique properties
1st expressed as BCR; always 1st antibody to be produced
IgD function
sensitization of basophils during resp bacterial infections
IgD unique property
relatively rare
IgG function
opsonization, neutralization, sensitizing NK cells
IgG unique property
only antibody that can cross placenta
IgA function
neutralization
IgA unique property
found in mucosal secretions
IgE function
activates mast cells
When do B cells develop?
Birth— death
H chain gene development in B cells
(stem cell) germline, (early pro-b cell) D-J rearrangement, (late pro-B cell) V-DJ rearrangement, (large & small pre-B cell, immature B cell) VDJ rearranged
L chain gene development in B cells
(stem, early & late pro-b, large pre-B cells) germline, (small pre-B cell) V-J rearranging, (immature B cell) VJ rearranged
Ig status in B cells
(stem, early & late pro-b cell) none, (large pre-B cell) micro heavy chain is made, (small pre-B cell) micro chain in endoplasmic reticulum, (immature B cell) micro heavy chain, lambda or kappa light chain, IgM on surface
early B cell development dependent on BM ______ cells
stromal
checkpoints in B cell development
late pro B cell stage- formation of functional pre-BCR or not (functional heavy chain)
small pre B cell stage- successful light chain rearrangement
immature B cells tested on _____ ______ for self affinity
bone marrow
if B cells have self affinity for bone marrow then ______
receptor editing (light chain editing only)
B cell receptor editing occurs until:
no self affinity or runs out of DNA
3 fates for self-reactive B cells
survive through receptor editing
die by apoptosis
become anergic (nonresponsive to antigen) (1-5 day lifespan)
developing B cells leave the BM, circulate between blood, ________ lymphoid tissues, and lymph
secondary
final stage of B-cell maturation occurs in _______ _______
lymph nodes
encounter with antigen leads to ________/________ B cells
activated, differentiated
Where do T cells develop?
thymus
When do T cells develop?
birth-involution
involution
thymus shrinks w/ age
What does the thymus become after involution?
adipose tissue
stages of T cell development are characterized by ____ and _____ expression
CD4, CD8
progenitor cells that enter the thymus are not yet committed to the ___ cell lineage
T
upon interaction with thymic stromal cells, the progenitors are signaled to ______ and _______
divide, differentiate
it is a race to rearrange lambda, delta, or beta 1st and once successful:
cells express both CD4 and CD8
T cell development
lymphoid progenitor cells enter thymus
double - T cell progenitor
beta chain rearranged
Pre-TCR checkpoint (Beta chain and surrogate alpha chain)
CD4 and CD8 expression turned on
alpha chain rearranged
TCR checkpoint (still double +)
positive selection (MHC compatibility, single +)
negative selection (self-reactive T cells die)
mature T cells exit thymus
checkpoints in T cell development
assembly of pre-TCR- alpha and beta chains are equal in size
central tolerance maintained by ___ and ___ selection
+, -
T cells that recognize self MHC molecules are ____ selected in the thymus
+
__ selection determines expression of either CD4 or CD8 co-receptor
+
receptor binds self-peptide: self-MHC I =
CD8 T cell
receptor binds self-peptide: self-MHC II =
CD4 T cell
tissue specific proteins are expressed in the thymus and participate in ___ selection
-
transcriptions factor called ____ causes peripheral tissue proteins to be expressed by thymic epithelial cells
AIRE
T cells that strongly recognize self antigen are deleted from the population
negatively selected
T cell receptor structure
resembles a membrane-bound Fab fragment of Ig
TCR diversity is generated by ______ ___________
gene rearrangement
genetic defects in RAG can lead to
severe combined immunodeficiency syndrome (SCID)
expression of TCR on cell surface requires association with additional ______
proteins
MHC I—> CD8 T cells:
intracellular pathogens
MHC II—> CD4 T cells:
extracellular pathogens
MHC I is always expressed by
nucleated cells
when nucleated cells are not infected by a virus they present ____
self
MHC II always expressed by
macrophages and dendritic cells
MHC I structure
shorter peptides, 3 large alpha chains, tiny beta chain, CD8 binds the alpha3 domain
MHC II structure
longer peptides, equal alpha and beta chains, CD4 binds the beta2 domain
MHC has ______ binding specifcity
promiscuous
intracellular pathogens are degraded by
cytosol
peptides generated in the cytosol are transported into the ___ where they bind MHC I
ER
MHC I antigen processing
sources of cytosolic antigens- self or pathogens
proteasome degrades cytosolic proteins
transport of peptides from the cytosol to the ER (TAP)
assembly of peptide- MHC I complexes in ER
surface expression of MHC I peptide complexes
IFNg causes proteasome to favor production of _____ that bind MHC I
peptides
class I heavy chain is stabilized by ______ until B2-microglobulin binds
calnexin
calnexin is released and the heterodimer of class I heavy chain and B2m forms the
peptide-loading complex (calreticulin, tapasin, TAP, ERp57, and PDI)
a peptide delivered by TAP binds to the class I heavy chain, forming
mature MHC class 1 molecule
MHC I dissociates from the peptide-loading complex and is exported from
endoplasmic reticulum
extracellular pathogens are _____ and ______ in the phagolysosome
phagocytosed, degraded
peptides presented by MHC II are generated in ________ intracellular vesicles (__________)
acidified, phagolysosome
certain pathogens (_______) hide out and replicate in these vesicles
mycobacteria
MHC II antigen processing
uptake of extracellular proteins into vesicular compartments of APCs
processing of internalized proteins in endosomal and lysosomal vesicles
biosynthesis and transport of MHC II molecules to endosomes
association of processed peptides with MHC II molecules in vesicles
MHC II molecules are prevented from binding peptides in the ____ by the ______ chain
ER, invariant
vesicle:
MHC II, invariant chain stabilizing, HLA-DM (helper protein)
most of invariant chain degraded with ____ left over
CLIP
phagolysosome and ________ peptides fuses w/ vesicle
extracellular
pH change down activates _______ removes CLIP so peptides can bind
HLA-DM
alpha chain, beta chain, and peptide —>
gets shipped to plasma membrane
MHC II expressed by
professional antigen presenting cells (B cells, macrophages, dendritic cells)