Lecture 8 - T Cell Development 1 Flashcards
what do immune cells arise from?
multi-potent hematopoietic stem cells
describe the progression from mutli-potent hematopoietic stem cells to specific immune cells
series of differentiation steps with increasingly restricted differentiation potential to make specific cells
what happened when adult mice had their thymus removed?
nothing
what happened when neonatal mice had their thymus removed?
in sterile environment –> nothing
in non-sterile environment –> died from infection
what did they know about the thymus and lymphocytes before the discovery of thymus?
knew that lymphocytes existed but didn’t realize there were T and B cells
initially thought thymus was “graveyard” for lymphocytes
describe the difference of T cells in mice and humans in terms of when mature T cells can be detected
mature T cells can be found at birth in mice
mature T cells can be found at 14-15 weeks of pregnancy in humans
what would happen if you remove the thymus from a human at birth?
they would be fine –> since mature T cells are there by 14-15 weeks of pregnancy, the baby would have T cells
what are the intermediate cells in T cell development?
DN1 –> DN2 –> DN3 –> DN4 –> DP –> SP
describe a flow cytometry plot of CD4 vs CD8 for T cell precursors
looks like a bird
how can we gate the DN population of cells to look at DN1 vs DN2 vs DN3 vs DN4
Use CD44 vs CD25 to look at specific DN populations
what are the 6 general steps for T cell maturation?
- Progenitor seeding of the thymus
- T cell lineage commitment
- B-selection
- Positive selection
- CD4 vs CD8 cell fate choice
- Impact of basal self-reactivity on T cell function
what cells seed the thymus? where do they come from in fetal vs adult mice?
multi-potent HSCs seed the thymus
- in fetal mice from liver
- in adult mice from BM
describe T cells in fetus vs adult mouse
fetus: invariant with less diverse Ag receptor
adult: more specific and very diverse T cell population
why are fetal T cells more invariant and less specific?
they don’t have fully functioning immune system so fast-acting, non-specific cells (more immune-like) are better to get rid of infection
what allows for the different types of T cells in fetal vs adult mice?
Lin28 and let-7 interaction
where is Lin28 expressed?
Lin28 is expressed in HSC progenitors in fetal liver
what is the role of Lin28?
Lin28 is an RNA-binding protein that inhibits the maturation of the let-7 miRNA
Let-7 levels in fetal vs adult mice
fetus has Lin28 so low let-7 levels
adult has no Lin28 so high let-7 levels
what happens when there’s forced expression of Lin28 in adult bone marrow?
it is sufficient to cause differentiation of SOME fetal T cell subtypes
what happens when fetal progenitors are transferred into an adult thymus?
can support SOME fetal T cell subtypes
what’s the next step once progenitors have been seeded in the thymus?
commitment to the T cell lineage
what is known as the “master regulator” of T cell fate?
Notch
4 Notch receptors and 5 Notch ligands
RECEPTORS:
- Notch1
- Notch2
- Notch3
- Notch4
LIGANDS:
- Jagged 1
- Jagged 2
- Delta 1
- Delta 4
- Delta 3
how does Notch signaling work? (3 steps)
- Thymic epithelial cell has notch ligand which binds notch receptor on thymocyte
- Interaction forces intracellular domain of notch to be released
- drives T cell fate
4 main functions of Notch signaling
- cell fate choice
- cell survival
- proliferation
- stem cell maintenance
Notch is _______ and ________ to determine T cell fate
Notch is NECESSARY and SUFFICIENT to determine T cell fate
what happens with GAIN OF FUNCTION NOTCH in lymphoid progenitor cell?
lymphoid progenitor cell makes T cells but NOT B cells
what happens with INACTIVATION OF NOTCH in lymphoid progenitor cell? why?
lymphoid progenitor cell makes B cells but NOT T cells because notch is necessary
what is the difference between the notch ligands?
most are redundant with the same roles
what is the main notch ligand in the thymus? what cells is it expressed on?
delta 4
found on cortical thymic epithelial cells
what happens to multipotent progenitor cells that become bone marrow cells without notch ligand?
B cells are produced
what happens to multipotent progenitor cells that become bone marrow cells with notch ligand?
T lineage cells are produced
progenitor cells that seed the thymus are ______
progenitor cells that seed the thymus are MULTIPOTENT
what does it mean for progenitor cells that seed the thymus to be multipotent?
make T or B cells depending on the signal
describe production of B and NK cells from DN1 vs DN2 vs DN3 cells with or without notch
with notch:
- cannot make B cells!
- can make some NK cells but less as progenitor cells are more differentiated
without notch:
- can make B and NK cells
- but lose ability to make either B or NK cells as progenitor cells are more differentiated
describe production of DP T cells from DN1 vs DN2 vs DN3 cells with or without notch
T cells can only be made when notch is present
what is a way that therapeutic T lineage cells can be made? what is the issue with this?
can use a bead with notch ligand on its surface to interact with notch receptor on hematopoietic progenitor
easy to reach the DP stages but hard to reach mature T cells
what happens once the progenitor cells have committed to the T lineage?
Beta selection! make beta chain for TCR
at what intermediate cell does TCRbeta chain rearrangement begin?
DN2
how does TCRbeta chain rearrangement work?
select 1 V, D, J, and C to make unique combination
what gene causes TCR gene rearrangement?
recombination activating genes (RAG) 1 and 2
what allows for the most TCR diversity?
terminal deoxynucleotidyl transferase (TdT) adds non-template nucleotides at junctions of the gene segments
what percent of TCR diversity is due to TdT?
95%
is TCRalpha made at the same time as TCRbeta?
no
what is the preTCR?
preTCR = rearranged TCRbeta chain paired with invariant preTCRalpha chain
3 things that a functional TCRbeta chain leads to:
- transient downregulation of RAG, allelic exclusion
- proliferation
- differentiation
why is RAG downregulated?
so that only 1 beta chain is rearranged
what are the 2 hypotheses for how beta-selection works?
either ligand DEPENDENT or ligand INDEPENDENT
what does it mean for beta selection to be ligand independent?
only requires rearranged beta chain for beta selection
evidence that preTCR signaling/beta-selection is ligand independent (2)
- delete MHC, differentiation from DN to DP is unaffected
- delete extracellular domain of preTCR (where ligand would bind), differentiation from DN to DP is unaffected
evidence that preTCR signaling/beta-selection is ligand dependent (3)
preTCR can bind to a peptide presented by MHC I and an absence of this interaction leads to:
1. decreased beta-selection efficiency
2. decreased TCR repertoire diversity
3. abnormal thymocytes
describe the binding of preTCR to peptide presented by MHC I and what this allows for
binds in diff conformation than mature TCR which means more beta chains can bind less specifically to peptide presented by MHC I
which of the following plots of thymocytes would we get if there was no functional RAG? why?
C
without RAG, there will be no differentiation to DP
what happens after beta selection?
positive selection
what happens during positive selection?
DN4 becomes DP as TCR is sampling environment
when does TCRalpha chain rearrangement occur?
at DP stage
describe TCRalpha chain rearrangement
only V and J are rearranged
does TCRbeta chain rearrangement occur during TCRalpha chain rearrangement?
no!! that has been completed
what are the 3 outcomes for thymocytes with TCRs during positive selection
- death by neglect –> apoptosis
- positive selection –> survival and differentiation
- negative selection –> apoptosis
what causes death by neglect?
TCR receives no signal from any peptides therefore will never be able to bind peptide
what causes positive selection?
TCR binds with low affinity to a SELF PEPTIDE
what causes negative selection?
TCR binds too strongly to self peptide, therefore too much self-reactivity
are T cells self-reactive? why?
yes, all T cells are self-reactive
bc the low affinity interaction with self peptide is what allows for positive selection
how many different TCR heterodimers are possible?
10^20
what percent of DP thymocytes can pass positive selection?
only 5-10%
describe positive selection and MHC restriction
positive selection ensures that the TCR has dual specificity for peptide + MHC (MHC restriction)
what is MHC restriction?
TCR can only recognize peptide presented by self-MHC and will NOT recognize the same peptide presented by another MHC
describe bone marrow chimera
mouse is irradiated and BM cells are killed –> donor BM cells from another mice are added to the irradiated mouse
what cells are radiation resistant?
what cells are not radiation resistant?
thymic epithelial cells ARE radiation resistant
hematopoietic cells (BM cells) are NOT radiation resistant
what happens to lymphocyte generation when WT mouse is donor for irradiated WT mouse?
positive selection can occur
what happens to lymphocyte generation when MHCnull mouse is donor for irradiated MHCnull mouse?
no MHC = no positive selection = no CD4+/CD8+ DP cells
what happens to lymphocyte generation when WT mouse is donor for irradiated MHCnull mouse?
no positive selection because the thymic epithelial cells don’t have MHC
what does the WT –> MHCnull bone chimera tell us?
positive selection requires MHC expression in thymic epithelial cells
specifically, what type of thymic epithelial cells are required for positive selection?
cortical thymic epithelial cells
what allows cortical thymic epithelial cells to induce positive selection?
THYMOPROTEASOME
what is the thymoproteasome?
beta5t proteasome subunit encoded by Psmb11 gene in cortical thymic epithelial cells allows for peptide fragments to be made for positive selection
specifically, what is the thymoproteasome required for?
required for CD8+ T cell development to present peptides on MHC I
what happens in beta5t KO? significance
reduced CD8+ cells –> therefore required for positive selection
why are the peptides made by the thymoproteasome unique?
they bind to MHC I with lower affinity than normal peptides so they can fall off more easily –> this helps with the low affinity interactions with self-peptides
what happens after positive selection?
CD4 vs CD8 T cell lineage choice
what are the 2 previous models of CD4 vs CD8 T cell lineage choice?
- instructive model
- stochastic model
describe the instructive model
when TCR on DP binds peptide on MHC, the QUALITY and QUANTITY of the signal instructs the DP to downregulate CD4 or CD8
ex. if there’s stronger signal when TCR binds MHC II with CD4, the cell knows to downregulate CD8
describe the stochastic model
when TCR on DP binds peptide on MHC, one of the co-receptors is randomly downregulated, then there’s a second step to ensure the TCR specificity and remaining co-receptor match
what is the current model of CD4 vs CD8 T cell lineage choice?
kinetic model
describe the kinetic model
when TCR on DP binds peptide on MHC, CD8 is downregulated and depending on whether the signal continues or is interrupted will instruct whether it is CD4 or CD8 involved in TCR binding
if signal persists –> CD4
if signal is lost –> CD8
what is the master regulator of CD4 vs CD8 T cell lineage choice?
ThPOK
what happens with ThPOK overexpression in DP cells?
only CD4 cells are produced
what happens with ThPOK KO in DP cells?
only CD8 cells are produced
ThPOK is ________ and ________ to make ___ T cells
ThPOK is NECESSARY and SUFFICIENT to make CD4 T cells
what happens with ThPOK if TCR signal is persistent with CD8 downregulation?
if there is a persistent signal in cells with MHC II when CD8 is downregulated, ThPOK is turned on leading to:
- expression of CD4-specific genes
- inhibition of cytokines for CD8 development
if MHC II expression is restricted to DCs, what type of T cells will be found in the thymus?
Cortical thymic epithelial cells require MHC I and MHC II to make CD4 and CD8 –> so if MHC II is only on DCs, will only make CD8!
what happens once T cells become single positive?
thymic egress
are T cells finished maturing once they leave the thymus?
no, they continue differentiating until they become fully mature T cells
what are T cells called once they leave the thymus but before they become mature T cells?
recent thymic emigrants
where do recent thymic emigrants mature?
in peripheral lymphoid organs
