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MIMM 314 > Lecture 10 - Visualizing T Cell Development > Flashcards

Lecture 10 - Visualizing T Cell Development Flashcards

1
Q

describe the motility of T cell thymocytes

A

highly motile to search for self-peptides and must move from cortex to medulla

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2
Q

relationship btwn motility and TCR signaling

A

motility affects TCR signaling and vice versa

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3
Q

describe density of thymic cortex vs medulla

A

cortex is more cell dense than medulla

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4
Q

what are chemokines

A

small, secreted proteins that signal thru cell surface G protein-coupled chemokine receptors

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5
Q

what is the role of chemokines?

A

chemokine gradients stimulate migration of cells

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6
Q

describe the chemokine and chemokine receptor system in the cortex

A

DP cells have CXCR4 receptors which bind CXCL2

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7
Q

describe the chemokine and chemokine receptor system in the medulla

A

SP cells have CCR7 receptors which bind CCL19/21

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8
Q

describe how chemokines allow thymocytes to migrate from cortex and medulla

A

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

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9
Q

what happens if there’s no CCL19/21 or CCR7?

A

cell won’t migrate to medulla and individual will develop autoimmune condition

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10
Q

describe the differences in speed throughout T cell development

A

DN and DP in cortex have slower speed

CD4 and CD8 in medulla have wider range of speeds but have higher speeds

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11
Q

why might DN and DP be slow?

A
  • 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
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12
Q

some DP cells are a bit faster, why?

A

maybe speed increases as they mature so those cells are just more mature

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13
Q

why is there such a large range of SP speeds?

A

again, speed could change depending on maturation level

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14
Q

describe Ca2+ influx downstream of TCR signaling (3)

A
  1. intracellular Ca2+ levels increase
  2. NFAT re-localizes from cytoplasm to nucleus
  3. targets gene expression
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15
Q

what 2 things do Ca2+ levels affect?

A
  1. how much NFAT can go to nucleus
  2. the gene transcription that is affected
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16
Q

what was done in this experiment?

which curve shows negative selecting peptide? positive selecting peptide?

A

OT-1 thymocytes were stimulated with altered peptide ligands and intracellular Ca2+ was measured

orange = negative selecting peptide
blue = positive selecting peptide

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17
Q

describe the level of intracellular calcium with a negative selecting peptide

A

strong burst of Ca2+ that quickly decreases

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18
Q

describe the level of intracellular calcium with a positive selecting peptide

A

slow increase of Ca2+ that is maintained for longer time

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19
Q

what does the difference in intracellular Ca2+ for positive vs negative selection correlate to?

A

level of Ca2+ correlates with compartmentalization and activation of MAPK signaling intermediates for positive vs negative

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20
Q

describe how we can use THYMIC SLICES to analyze positive and negative selection

A

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

21
Q

reminder: what are OT-1 thymocytes?

A

every TCR is specific for OVA presented by MHC I

22
Q

what happens if a MHCnull thymic slice receives DP OT-1 transgenic thymocytes?

A

no MHC = no antigen presentation = TCR cannot be activated

cells remain as DP

23
Q

what happens if a WT thymic slice receives DP OT-1 transgenic thymocytes?

A

positive selection –> cells become SP CD8

24
Q

what happens if a WT thymic slice receives DP OT-1 transgenic thymocytes with OVA?

A

binding is too strong = negative selection –> cells get deleted

25
Q

what is CD69?

A

early activation marker of TCR stimulation

26
Q

what happens to the amount of CD69 in thymic slices that are:

MHCnull
WT
WT + OVA

A

MHCnull –> no CD69

WT –> increased bc positive selection

WT + OVA –> big increase bc strong activation of TCR

27
Q

what happens to the amount of DP in thymic slices that are:

MHCnull
WT
WT + OVA

at 3h vs 24h vs 72h

A

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

28
Q

what happens to the amount of SP in thymic slices that are:

MHCnull
WT
WT + OVA

at 3h vs 24h vs 72h

A

only WT have SP –> small increase at 24h then big increase at 72h as they are being positively selected

29
Q

describe Ca2+ levels in MHCnull vs WT vs WT+OVA thymic slices when using fluorescently dyed thymocytes

A

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

30
Q

3 types of Ca2+ signaling patterns in thymocytes

A
  1. lo
  2. lo <> hi –> temporary spike in Ca2+, then decreases
  3. hi
31
Q

describe the type of Ca2+ signaling pattern in positive selection

A

mostly the lo <> hi signaling, i.e. transient Ca2+ levels

32
Q

describe the type of Ca2+ signaling pattern in negative selection

A

high levels maintained!

33
Q

describe the movement of WT thymic slices and the Ca2+ signaling pattern

A

moving then stopping –> transient Ca2+ signal

(positive selection)

34
Q

describe the movement of WT+OVA thymic slices and the Ca2+ signaling pattern

A

stops migrating, locked into a cell with strong TCR-peptide interaction –> high Ca2+ signal

(negative selection)

35
Q

what occurs during a TCR signaling event?

A

increased Ca2+ levels (as TCR is activating signaling pathways) and decreased thymocyte speed

36
Q

again, what type of signaling occurs during positive selection?

A

infrequent and transient signaling

37
Q

again, what type of signaling occurs during negative selection?

A

high signaling and low motility

38
Q

on avg, how many signaling events occur per hour? how long do they last?

A

1-2 events

last 3-5 min

39
Q

do we observe negative selection thymocytes transitioning from signaling to non-signaling events?

A

no

40
Q

how can such infrequent and transient TCR signaling events lead to long-lasting positive selection?

A

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

41
Q

what is 3-MB-PP1?

A

3-MB-PP1 is a competitive inhibitor of ATP binding on kinases

42
Q

why do we use 3-MB-PP1?

A

TCR signaling events often involve kinases but kinase inhibitors are often not specific

43
Q

how do we use 3-MB-PP1?

A

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

44
Q

describe the experimental setup for using thymic slices with 3-MB-PP1

A
  1. use WT or MHCnull thymic slices
  2. add DP OT-1 transgenic thymocytes with the mutant Zap70
  3. add 3-MB-PP1
45
Q

describe the levels of CD8 for these thymic slices:

MHCnull
WT + DMSO
WT + 3-MB-PP1

what can we conclude from this?

A

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

46
Q

describe the TCR signals required by positive selection

A

many hours of uninterrupted TCR signals

47
Q

how do we know positive selection requires many hours of TCR signals?

A

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

48
Q

how do we know positive selection requires many hours of UNINTERRUPTED TCR signals?

A

if the drug is added after some signaling and then removed (i.e. the drug interrupts the signal), there will be less CD8

49
Q

what happens if TCR signaling is interrupted?

A

Ca2+ levels will decrease and then thymocyte must start accumulating Ca2+ all over again

MIMM 314 (23 decks)

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