Lecture 9 - T Cell Development 2 Flashcards
what is central tolerance vs peripheral tolerance?
central = in thymus
5 possible fates for highly self-reactive thymocytes?
- “escape” from thymus
- Tregs
- negative selection / clonal deletion
- anergy / hyporesponsive cells
- diverted to other T cell lineages
what happens to highly self-reactive thymocytes if they “escape” from the thymus?
peripheral tolerance mechanisms will catch them
what ultimately determines thymocyte fate?
TCR affinity for self-peptide:MHC
describe how TCR transgenic mice are made
- clone a TCR that you know is specific to an antigen
- express in mice so all TCRs will have that alpha and beta chain and be specific for a known peptide in the context of MHC
why can TCR transgenic mice maintain the beta chain throughout all TCRs?
allelic exclusion for the beta chain is so strong that once the mouse has a functional beta chain, there will be no more endogenous rearrangement
why can TCR transgenic mice maintain the alpha chain throughout all TCRs?
allelic exclusion for the alpha chain is weaker so there may be some rearrangement –> make the mice Rag deficient to prevent this rearrangement
describe how H-Y TCR transgenic mice are produced
- female mice are injected with male cells
- female makes TCR for male HY antigen in the context of MHC I
where is the male HY antigen encoded?
on the Y chromosome
in mice expressing HY transgenic TCR that recognizes the male-specific antigen in the context of MHC I, what T cells will develop in FEMALE mice and why?
CD8+ T cells
since the females were originally able to recognize the male antigen, the T cells must have been selected on another self-antigen with low affinity as CD8+ SP cells to be able to mount T cell response against the non-thymus antigen
since females could respond to the HY antigen, what does this indicate in general about T cells responding to antigens?
the self-antigen that a T cell gets selected on in the thymus does not have to be the same antigen that it will bind once it is mature
in mice expressing HY transgenic TCR that recognizes the male-specific antigen in the context of MHC I, what T cells will develop in MALE mice and why?
the male TCR will bind too strongly to its own protein and will have much less positive selection
describe the experiment to determine the affinity of self-peptide that allows negative vs positive selection
MICE: use OT-I transgenic TCR which recognizes OVA peptide in context of MHC I and has beta2m KO
- add beta2m back with OVA with small aa changes
- can see which ones require higher or lower concentrations to stimulate TCR –> tells you the affinity
why do we use beta2m KO? why do we add it back?
beta2m is a subunit of MHC I that stabilizes it at the surface –> KO means no peptide can be presented and T cell stays at the DP stage
when we add it back with the peptide, we can control when stimulation can occur
what happens if the TCR doesn’t respond to a peptide in the affinity experiment?
the cells remain as DP
what happens if the TCR binds a peptide with high affinity in the affinity experiment?
high affinity = negative selection
what happens if the TCR binds a peptide with low affinity in the affinity experiment?
low affinity = positive selection
describe T4 in the affinity experiment
at low [ ] allowed positive selection
at high [ ] allowed negative selection
describe the threshold btwn positive and negative selection
very narrow! –> a small change in affinity can determine whether DP is positively or negatively selected
describe the range of affinities that allow for positive selection
very wide –> big range of affinities that can allow DP cell to be positively selected
describe the 5 steps of TCR signaling
- TCR recognizes peptide:MHC and co-receptor
- LCK phosphorylates ITAMs of CD3
- ZAP70 is recruited to phosphorylate tyrosine residues of LAT
- many components of signaling pathways are recruited, including MAPK
- allows changes in transcription to determine cell fate
if TCR signaling goes thru the same pathways with negative and positive selection, how does the cell determine which selection to undergo? (5)
- difference in TCR affinity for self-antigen
- difference in amount of phosphorylation of intermediates
- difference in activation and subcellular localization of Ras and MAPK signaling intermediates
- difference in Erk activation
- difference in gene expression programs
describe the difference in amount of phosphorylation of intermediates in positive vs negative selection
more phosphorylation of LAT in negative selection
describe the difference in activation and subcellular localization of Ras and MAPK signaling intermediates
in negative selection, there is increased activation and accumulation of MAPK components at the cell surface
in positive selection, components are more localized at the golgi
describe the difference in Erk activation
in negative selection: high immediate Erk activation that quickly goes away
in positive selection: low level of Erk activation that is sustained over time
WHERE does positive vs negative selection occur?
positive: cortex
negative: cortex and medulla
at what stage does positive selection occur?
what cells allow for positive selection?
co-stimulation?
- DP stage
- cTEC
- no co-stimulation
at what stage does negative selection in the CORTEX occur?
what cells allow for negative selection in the cortex?
co-stimulation?
- DP stage
- DCs
- generally there is co-stimulation
at what stage does negative selection in the MEDULLA occur?
what cells allow for negative selection in the medulla?
co-stimulation?
- SP stage
- mTECs / DCs / B cells
- generally there is co-stimulation
why is there co-stimulation in negative selection but not positive selection?
co-stimulation allows for a stronger binding of TCR to peptide:MHC to help promote negative selection
describe the antigens used in negative selection in cortex vs medulla
cortex: negative selection of thymocytes expressing TCRs specific to ubiquitously expressed self-antigen
medulla: specialized microenvironment to support negative selection of thymocytes expressing TCRs specific to TRA
