Lecture 10 - Visualizing T Cell Development Flashcards
describe the motility of T cell thymocytes
highly motile to search for self-peptides and must move from cortex to medulla
relationship btwn motility and TCR signaling
motility affects TCR signaling and vice versa
describe density of thymic cortex vs medulla
cortex is more cell dense than medulla
what are chemokines
small, secreted proteins that signal thru cell surface G protein-coupled chemokine receptors
what is the role of chemokines?
chemokine gradients stimulate migration of cells
describe the chemokine and chemokine receptor system in the cortex
DP cells have CXCR4 receptors which bind CXCL2
describe the chemokine and chemokine receptor system in the medulla
SP cells have CCR7 receptors which bind CCL19/21
describe how chemokines allow thymocytes to migrate from cortex and medulla
IN CORTEX:
- high [CXCL12] in cortex maintains the DP cells in the cortex with the CXCR4 receptor
when the DP is selected with self-peptide, gene expression of CXCR4 decreases and CCR7 increases –> cell follows decreasing gradient of CXCL12 and increasing gradient of CCL19/21
IN MEDULLA:
- high [CCL19/21] in medulla maintains the SP cells in the medulla with the CCR7 receptor
what happens if there’s no CCL19/21 or CCR7?
cell won’t migrate to medulla and individual will develop autoimmune condition
describe the differences in speed throughout T cell development
DN and DP in cortex have slower speed
CD4 and CD8 in medulla have wider range of speeds but have higher speeds
why might DN and DP be slow?
- take time to sample the self-antigens and allow for interaction with TCR and antigen to occur
- data looks at average speed so may include some cells that have been deleted which reduces the average
- mTECs only express some TRAs so takes time to search for the rare TRAs
some DP cells are a bit faster, why?
maybe speed increases as they mature so those cells are just more mature
why is there such a large range of SP speeds?
again, speed could change depending on maturation level
describe Ca2+ influx downstream of TCR signaling (3)
- intracellular Ca2+ levels increase
- NFAT re-localizes from cytoplasm to nucleus
- targets gene expression
what 2 things do Ca2+ levels affect?
- how much NFAT can go to nucleus
- the gene transcription that is affected
what was done in this experiment?
which curve shows negative selecting peptide? positive selecting peptide?
OT-1 thymocytes were stimulated with altered peptide ligands and intracellular Ca2+ was measured
orange = negative selecting peptide
blue = positive selecting peptide
describe the level of intracellular calcium with a negative selecting peptide
strong burst of Ca2+ that quickly decreases
describe the level of intracellular calcium with a positive selecting peptide
slow increase of Ca2+ that is maintained for longer time
what does the difference in intracellular Ca2+ for positive vs negative selection correlate to?
level of Ca2+ correlates with compartmentalization and activation of MAPK signaling intermediates for positive vs negative
describe how we can use THYMIC SLICES to analyze positive and negative selection
thymus is sliced and labeled thymocytes are added on top
thymic microenvironment (chemokine levels), cortex and medulla are maintained
then analyze with flow cytometry or 2-photon time-lapse microscopy
reminder: what are OT-1 thymocytes?
every TCR is specific for OVA presented by MHC I
what happens if a MHCnull thymic slice receives DP OT-1 transgenic thymocytes?
no MHC = no antigen presentation = TCR cannot be activated
cells remain as DP
what happens if a WT thymic slice receives DP OT-1 transgenic thymocytes?
positive selection –> cells become SP CD8
what happens if a WT thymic slice receives DP OT-1 transgenic thymocytes with OVA?
binding is too strong = negative selection –> cells get deleted
what is CD69?
early activation marker of TCR stimulation
what happens to the amount of CD69 in thymic slices that are:
MHCnull
WT
WT + OVA
MHCnull –> no CD69
WT –> increased bc positive selection
WT + OVA –> big increase bc strong activation of TCR
what happens to the amount of DP in thymic slices that are:
MHCnull
WT
WT + OVA
at 3h vs 24h vs 72h
3h:
no change in any
24h:
WT + OVA begin to decrease bc being removed by negative selection
72h:
- WT decreases as DP become SP
- WT + OVA is non-detectable
what happens to the amount of SP in thymic slices that are:
MHCnull
WT
WT + OVA
at 3h vs 24h vs 72h
only WT have SP –> small increase at 24h then big increase at 72h as they are being positively selected
describe Ca2+ levels in MHCnull vs WT vs WT+OVA thymic slices when using fluorescently dyed thymocytes
MHCnull have low Ca2+
WT have more Ca2+ –> migrate, pause, increase Ca2+, then migrate again
WT+OVA have the most Ca2+ and cells are moving less
3 types of Ca2+ signaling patterns in thymocytes
- lo
- lo <> hi –> temporary spike in Ca2+, then decreases
- hi
describe the type of Ca2+ signaling pattern in positive selection
mostly the lo <> hi signaling, i.e. transient Ca2+ levels
describe the type of Ca2+ signaling pattern in negative selection
high levels maintained!
describe the movement of WT thymic slices and the Ca2+ signaling pattern
moving then stopping –> transient Ca2+ signal
(positive selection)
describe the movement of WT+OVA thymic slices and the Ca2+ signaling pattern
stops migrating, locked into a cell with strong TCR-peptide interaction –> high Ca2+ signal
(negative selection)
what occurs during a TCR signaling event?
increased Ca2+ levels (as TCR is activating signaling pathways) and decreased thymocyte speed
again, what type of signaling occurs during positive selection?
infrequent and transient signaling
again, what type of signaling occurs during negative selection?
high signaling and low motility
on avg, how many signaling events occur per hour? how long do they last?
1-2 events
last 3-5 min
do we observe negative selection thymocytes transitioning from signaling to non-signaling events?
no
how can such infrequent and transient TCR signaling events lead to long-lasting positive selection?
cell “remembers” these transient interactions and builds up Ca2+ with each positive selection signal to be able to drive gene expression pathways for positive selection
what is 3-MB-PP1?
3-MB-PP1 is a competitive inhibitor of ATP binding on kinases
why do we use 3-MB-PP1?
TCR signaling events often involve kinases but kinase inhibitors are often not specific
how do we use 3-MB-PP1?
make mutant Zap70 (kinase in TCR signaling) which has ATP pocket of kinase domain more open so 3-MB-PP1 can inhibit it
3-MB-PP1 inhibition is reversible
describe the experimental setup for using thymic slices with 3-MB-PP1
- use WT or MHCnull thymic slices
- add DP OT-1 transgenic thymocytes with the mutant Zap70
- add 3-MB-PP1
describe the levels of CD8 for these thymic slices:
MHCnull
WT + DMSO
WT + 3-MB-PP1
what can we conclude from this?
MHCnull - no CD8 bc no selection
WT + DMSO –> gradual increase in CD8, therefore positive selection
WT + 3-MB-PP1 –> no CD8, therefore negative selection
therefore, Zap70 kinase domain is necessary for positive selection
describe the TCR signals required by positive selection
many hours of uninterrupted TCR signals
how do we know positive selection requires many hours of TCR signals?
allow thymocytes to receive signal for 24h, then add 3-MB-PP1 –> no CD8 cells
36h –> start to make CD8 cells
48h –> making more CD8
therefore, need signals for >36h to have positive selection
how do we know positive selection requires many hours of UNINTERRUPTED TCR signals?
if the drug is added after some signaling and then removed (i.e. the drug interrupts the signal), there will be less CD8
what happens if TCR signaling is interrupted?
Ca2+ levels will decrease and then thymocyte must start accumulating Ca2+ all over again
