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
what do mTECs express?
mTECs can express almost 90% of the coding genome, including thousands of TRA
why do mTECs express TRAs?
to predict tissue-specific antigens that will be seen once the T cell leaves the thymus
how many TRAs are expressed by 1 mTEC?
100-300
what percent of mTECs express a given TRA?
1-5% of mTECs express a given TRA
What is happening when CD4 and CD8 SP thymocytes are in the medulla?
sampling the TRAs expressed by mTECs
what does Aire stand for?
AutoImmune REgulator
what does Aire do?
promiscuous/non-specific TF that binds other regulatory mechanisms to regulate gene expression of TRAs
5 examples of mechanisms that are influenced by Aire?
- RNA pol II pausing
- epigenetic modifications
- RNA splicing
- superenhancer activity
- chromatin looping
how many genes in mTEC are directly or indirectly regulated by Aire?
~4,000
what happens if there are Aire deficiencies? why?
Aire deficiency causes broad autoimmunity because then DP TCRs are not sampling all the TRAs and T cells cannot undergo sufficient selection
is Aire required for all TRA expression in mTECs?
example
No! there are some Aire-independent TRA
CRP doesn’t require Aire for expression in thymus
what is another regulator of TRA expression?
Fezf2
what are the 2 types of mTECs and their characteristics?
- mTEClo –> low levels of Ag presentation and co-stimulatory molecules
- mTEChi –> high levels of Ag presentation and co-stimulatory molecules AND AIRE
why do mTEChi cells have more Aire?
there is more Ag presentation and processing so more Aire is required to promote more TRAs that can be presented
describe the gene expression profiles of post-Aire mTEClo
very very similar to the peripheral version!
what does the mTEClo population contain?
mTEClo population includes immature mTEC and mature “post-aire” mTEC
what technique did we use to identify that post-Aire mTEClo cells have characteristics of peripheral cells?
single cell RNA sequencing
what is another name for the mTEChi cells that look like peripheral cells?
mimetic cells
how does Aire work in mTEChi cells?
drives key TFs that are important for the diff gene profiles of peripheral-like cells
what is the only cell type that supports negative selection in cortex and medulla?
DCs
where are most DCs found in the thymus? (cortex or medulla) and what does this indicate about negative selection?
medulla –> medulla is specialized in negative selection
how do DCs allow for negative selection?
they are professional APCs that express MHC and co-stimulatory molecules
what type of thymocytes are deleted by DCs?
DCs support deletion of thymocytes with TCRs specific to ubiquitously expressed antigens
describe how migratory DCs help with negative selection
DCs can migrate from peripheral lymphoid organs to thymus and bring peripheral antigens so TCRs can sample these antigens for and be negatively selected
what happens to those migratory DCs during infection?
this process stops so DCs don’t bring real pathogenic antigens to thymus and allow for tolerance
do DCs only present ubiquitously expressed antigen?
no! they can also present TRAs
how do DCs present TRAs?
they don’t express them but can acquire via antigen transfer from mTECs
besides mTECs and DCs, what is the 3rd cell type that supports negative selection in the medulla?
B cells
what percent of total thymic cells are B cells?
0.1-0.3%
where are B cells mostly found in the thymus?
cortico-medullary junction
what do B cells express that allows them to support negative selection? what does this indicate about the type of T cells that it negatively selects for?
high levels of MHC II and co-stimulatory molecules AND AIRE
therefore involved in negative selection of MHC II-restricted CD4 T cells
how do B cells acquire the antigens to be presented for negative selection?
capture from the environment and present via MHC II
do all T cells that bind peptide too strongly undergo negative selection?
no, negative selection is “incomplete”
describe how the negative selection in HY TCR transgenic mice shows that negative selection is incomplete
the males are reacting to self-peptide with specific TCR so all should be negatively selected and die –> we see a reduction but not fully 0
describe how we can track antigen-specific T cells with TETRAMERS
Tetramer = many MHC loaded with peptide are tethered to biotin and streptavidin and conjugated to a fluorochrome and can study the cells with flow cytometry
3 possible reasons why negative selection is imperfect?
- Tregs
- maintain basal level of inflammation
- react to sick cells
- all T cells are somewhat self-reactive so getting rid of all of them = reduced diversity required to find pathogen
2 possibilities for autoreactive thymocytes that survive negative selection
- anergy
- lineage diversion
what does it mean when T cells become anergic?
hyporesponsive –> fail to become activated and won’t proliferate
what does it mean for T cells to undergo lineage diversion?
develop into non-conventional T cells like Treg and TCRaB DN T cells
what is the role of Tregs?
in central tolerance they suppress autorective cells
what are Tregs called in mice?
Foxp3+/CD4+ T cells
Treg TCR vs CD4+ TCR
TCR sequences have minimal overlap
what happens when antigen-specific mature Tregs recirculate back to the thymus?
compete with developing Tregs so you won’t get new Treg for the same antigen
2 types of Treg development
CD4 SP has strong signal thru TCR and doesn’t get negatively selected
- increased CD25, IL2 and IL15 signal for it to become Treg with increased Foxp3
- others have low Foxp3
4 differences btwn Tregs that derive from diff progenitors
- gene expression profiles
- TCR repertoires
- affinity for self-peptide:MHC
- function
what do Tregs derived from CD25+ progenitors prevent?
can prevent autoimmune disease development
what can Tregs from low Foxp3 progenitors prevent in the lab?
can prevent weight loss in a T cell transfer model of colitis
OT-II transgenic mouse that is specific to OVA peptide presented in context of MHC II
cross this to mouse expressing OVA driven by Aire-dependent insulin 2 promoter
what T cells do you expect will develop?
will see reduced CD4+ cells because OVA will be driven by Aire in mTEC to encourage negative selection
will develop OVA-specific Tregs
