MIMG 185a Exam Questions and Correct
Answers
what do T cell receptors recognize?
processed antigens presented by MHCs on cell surface
do TCRs recognize soluble or particulate antigens?
no
what part of the MHC-peptide complex does the TCR recognize?
BOTH the peptide and the MHC itself
describe the structure of the TCR
two subunits, alpha and beta, disulfide bond in constant regions
each chain with immunoglobulin domains -constant and variable region
variable regions with three CDRs that form peptide binding site
constant regions with TM regions and short intracellular domains
what are the two types of TCRs?
1. alpha-beta (ab) - more common + higher specificity
2. gamma-delta (yd) - generally recognize polysaccharides and lipids (non traditional APCs)
what do ab TCRs recognize?
peptides presented by MHC
what does the varaible region do?
binds to MHC peptide complex
what does the constant region do?
,lifts antigen binding domain away from the plasma membrane
what are the three CDRs for?
CDR1 and CDR2 bind to MHC
CDR3 binds to peptide
what are the coreceptors of TCRab?
CD4
CD8
CD3
why does TCRab use coreceptors for?
increase binding affinity to the MHC-peptide complex (binding very weak alone)
what is CD3 for?
coreceptor, signal transduction complex
upon antigen binding, ITAM motifs in their intracellular domain are phosphorylated and
initiate the TCR signaling cascade
how does TCR-CD3 assembly work?
assembled intracellularly
without CD3, TCR does not translocate to cell surface
why is the costimulatory signal from CD28 and CD80/86 necessary?
to avoid recognition of self antigens
what experiment demonstrated the TCR-peptide-MHC interaction is restricted by the
HLA haplotype?
,sinkernagel and doherty (1975) - demonstrated CD8 cells are restricted to recognizing
peptides in the context of self-MHC
1. immunized H2k mice with LCMV
2. isolated spleen cells (contained Tc cells)
3. added to H2k uninfected cells --> no 51Cr release (no lysis)
4. added to H2k LCMV infected cells --> lysis
5. added to H2b LCMV infected cells --> no lysis
how were TCR genes discovered?
1980, Kappler + Marrack
1. immunized mice with Ova
2. purified T cells from LN and incubated with OVA to induce proliferation
3. fuse with cancer cells to create OVA specific hybridomas
4. seed into single clones (dilution)
5. incubate with cells expressing OVA and select clones secreting IL-2 because those are
OVA specific
6. immunize mice with T cell hybridoma
7. select B cells from spleen
8. make hybridomas and clone to get one specific to TCR (monoclonals)
9. take DCpresenting OVA and stimulate with T cell hybridoma to force proliferation
10. add Ab to T cells then add to DC --> NO proliferation because TCR blocked
How did Davis and MAk clone TCRb in 1984?
1. used T cell hybidomas and isolated mRNA bound to rER
2. made cDNA because more stable
, 3. hybridize with B cell RNA to create duplexes to remove all genes present in BOTH
4. rest of cDNA must be only in one
5. used cDNA as probe in sourthern blot against T cells, B cells, and liver cell DNA
6. TCR genes were same size in B and liver cell but different size in T cell due to
recombination
what assumptions did Davis and Mak make when cloning TCRb?
1. TCRs are only expressed in T cells and not B cells
2. TCRs are membrane bound so TCR specific mRNA should be isolatable from ER bound
ribosomes (and not cytosolic ribosomes)
3. TCR genes are rearranged to generate diversity
4. TCRs have constant and variable regions
yd T cells
appear early in thymic embryonic development
represent 0.5% of mature thymocytes in periphery
recognize unusualantigens (lipids + glycolipids_ associated with unconventional MHCs
reside in mucosal and skin tissue with innate cells
pricipal role = protecting barrier tissues from outside infections
how does TCR recombination compare to VDJ?
- same machinery as BCR VDJ
- b and d segments have VDJ
- y and a segments only have V and J
- only one constant region each
- a and b have more V exons than y and d
- a has higher variability in J region than b
Answers
what do T cell receptors recognize?
processed antigens presented by MHCs on cell surface
do TCRs recognize soluble or particulate antigens?
no
what part of the MHC-peptide complex does the TCR recognize?
BOTH the peptide and the MHC itself
describe the structure of the TCR
two subunits, alpha and beta, disulfide bond in constant regions
each chain with immunoglobulin domains -constant and variable region
variable regions with three CDRs that form peptide binding site
constant regions with TM regions and short intracellular domains
what are the two types of TCRs?
1. alpha-beta (ab) - more common + higher specificity
2. gamma-delta (yd) - generally recognize polysaccharides and lipids (non traditional APCs)
what do ab TCRs recognize?
peptides presented by MHC
what does the varaible region do?
binds to MHC peptide complex
what does the constant region do?
,lifts antigen binding domain away from the plasma membrane
what are the three CDRs for?
CDR1 and CDR2 bind to MHC
CDR3 binds to peptide
what are the coreceptors of TCRab?
CD4
CD8
CD3
why does TCRab use coreceptors for?
increase binding affinity to the MHC-peptide complex (binding very weak alone)
what is CD3 for?
coreceptor, signal transduction complex
upon antigen binding, ITAM motifs in their intracellular domain are phosphorylated and
initiate the TCR signaling cascade
how does TCR-CD3 assembly work?
assembled intracellularly
without CD3, TCR does not translocate to cell surface
why is the costimulatory signal from CD28 and CD80/86 necessary?
to avoid recognition of self antigens
what experiment demonstrated the TCR-peptide-MHC interaction is restricted by the
HLA haplotype?
,sinkernagel and doherty (1975) - demonstrated CD8 cells are restricted to recognizing
peptides in the context of self-MHC
1. immunized H2k mice with LCMV
2. isolated spleen cells (contained Tc cells)
3. added to H2k uninfected cells --> no 51Cr release (no lysis)
4. added to H2k LCMV infected cells --> lysis
5. added to H2b LCMV infected cells --> no lysis
how were TCR genes discovered?
1980, Kappler + Marrack
1. immunized mice with Ova
2. purified T cells from LN and incubated with OVA to induce proliferation
3. fuse with cancer cells to create OVA specific hybridomas
4. seed into single clones (dilution)
5. incubate with cells expressing OVA and select clones secreting IL-2 because those are
OVA specific
6. immunize mice with T cell hybridoma
7. select B cells from spleen
8. make hybridomas and clone to get one specific to TCR (monoclonals)
9. take DCpresenting OVA and stimulate with T cell hybridoma to force proliferation
10. add Ab to T cells then add to DC --> NO proliferation because TCR blocked
How did Davis and MAk clone TCRb in 1984?
1. used T cell hybidomas and isolated mRNA bound to rER
2. made cDNA because more stable
, 3. hybridize with B cell RNA to create duplexes to remove all genes present in BOTH
4. rest of cDNA must be only in one
5. used cDNA as probe in sourthern blot against T cells, B cells, and liver cell DNA
6. TCR genes were same size in B and liver cell but different size in T cell due to
recombination
what assumptions did Davis and Mak make when cloning TCRb?
1. TCRs are only expressed in T cells and not B cells
2. TCRs are membrane bound so TCR specific mRNA should be isolatable from ER bound
ribosomes (and not cytosolic ribosomes)
3. TCR genes are rearranged to generate diversity
4. TCRs have constant and variable regions
yd T cells
appear early in thymic embryonic development
represent 0.5% of mature thymocytes in periphery
recognize unusualantigens (lipids + glycolipids_ associated with unconventional MHCs
reside in mucosal and skin tissue with innate cells
pricipal role = protecting barrier tissues from outside infections
how does TCR recombination compare to VDJ?
- same machinery as BCR VDJ
- b and d segments have VDJ
- y and a segments only have V and J
- only one constant region each
- a and b have more V exons than y and d
- a has higher variability in J region than b
