Lecture 10 - Visualizing T Cell Development

Question 1

describe the motility of T cell thymocytes

Answer 1

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

Question 2

relationship btwn motility and TCR signaling

Answer 2

motility affects TCR signaling and vice versa

Question 3

describe density of thymic cortex vs medulla

Answer 3

cortex is more cell dense than medulla

Question 4

what are chemokines

Answer 4

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

Question 5

what is the role of chemokines?

Answer 5

chemokine gradients stimulate migration of cells

Question 6

describe the chemokine and chemokine receptor system in the cortex

Answer 6

DP cells have CXCR4 receptors which bind CXCL2

Question 7

describe the chemokine and chemokine receptor system in the medulla

Answer 7

SP cells have CCR7 receptors which bind CCL19/21

Question 8

describe how chemokines allow thymocytes to migrate from cortex and medulla

Answer 8

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

Question 9

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

Answer 9

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

Question 10

describe the differences in speed throughout T cell development

Answer 10

DN and DP in cortex have slower speed

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

Question 11

why might DN and DP be slow?

Answer 11

• 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

Question 12

some DP cells are a bit faster, why?

Answer 12

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

Question 13

why is there such a large range of SP speeds?

Answer 13

again, speed could change depending on maturation level

Question 14

describe Ca2+ influx downstream of TCR signaling (3)

Answer 14

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

Question 15

what 2 things do Ca2+ levels affect?

Answer 15

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

Question 16

what was done in this experiment?

which curve shows negative selecting peptide? positive selecting peptide?

Answer 16

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

orange = negative selecting peptide
blue = positive selecting peptide

Question 17

describe the level of intracellular calcium with a negative selecting peptide

Answer 17

strong burst of Ca2+ that quickly decreases

Question 18

describe the level of intracellular calcium with a positive selecting peptide

Answer 18

slow increase of Ca2+ that is maintained for longer time

Question 19

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

Answer 19

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

Question 20

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

Answer 20

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

Question 21

reminder: what are OT-1 thymocytes?

Answer 21

every TCR is specific for OVA presented by MHC I

Question 22

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

Answer 22

no MHC = no antigen presentation = TCR cannot be activated

cells remain as DP

Question 23

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

Answer 23

positive selection –> cells become SP CD8

Question 24

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

Answer 24

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

Question 25

what is CD69?

Answer 25

early activation marker of TCR stimulation

Question 26

what happens to the amount of CD69 in thymic slices that are: MHCnull WT WT + OVA

Answer 26

MHCnull --> no CD69 WT --> increased bc positive selection WT + OVA --> big increase bc strong activation of TCR

Question 27

what happens to the amount of DP in thymic slices that are: MHCnull WT WT + OVA at 3h vs 24h vs 72h

Answer 27

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

Question 28

what happens to the amount of SP in thymic slices that are: MHCnull WT WT + OVA at 3h vs 24h vs 72h

Answer 28

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

Question 29

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

Answer 29

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

Question 30

3 types of Ca2+ signaling patterns in thymocytes

Answer 30

1. lo 2. lo <> hi --> temporary spike in Ca2+, then decreases 3. hi

Question 31

describe the type of Ca2+ signaling pattern in positive selection

Answer 31

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

Question 32

describe the type of Ca2+ signaling pattern in negative selection

Answer 32

high levels maintained!

Question 33

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

Answer 33

moving then stopping --> transient Ca2+ signal (positive selection)

Question 34

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

Answer 34

stops migrating, locked into a cell with strong TCR-peptide interaction --> high Ca2+ signal (negative selection)

Question 35

what occurs during a TCR signaling event?

Answer 35

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

Question 36

again, what type of signaling occurs during positive selection?

Answer 36

infrequent and transient signaling

Question 37

again, what type of signaling occurs during negative selection?

Answer 37

high signaling and low motility

Question 38

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

Answer 38

1-2 events last 3-5 min

Question 39

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

Answer 39

no

Question 40

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

Answer 40

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

Question 41

what is 3-MB-PP1?

Answer 41

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

Question 42

why do we use 3-MB-PP1?

Answer 42

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

Question 43

how do we use 3-MB-PP1?

Answer 43

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

Question 44

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

Answer 44

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

Question 45

describe the levels of CD8 for these thymic slices: MHCnull WT + DMSO WT + 3-MB-PP1 what can we conclude from this?

Answer 45

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

Question 46

describe the TCR signals required by positive selection

Answer 46

many hours of uninterrupted TCR signals

Question 47

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

Answer 47

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

Question 48

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

Answer 48

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

Question 49

what happens if TCR signaling is interrupted?

Answer 49

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