L8
what does flow cytometry look like if theres no functional RAG protein on a CD4 and CD8 plot?
its DN, so on the left corner.
RAG proteins are essential for V(D)J recombination of the T cell receptor genes. Without functional RAG, developing T cells cannot rearrange their TCR, and therefore cannot pass the β-selection checkpoint at the DN3 stage. They never progress to the double positive (CD4+CD8+) or single positive stages.
–> essentially no cells anywhere on the plot, which is consistent with a complete block in T cell development — the cells simply can’t mature past the DN stage without a functional TCR, so you get no DP or SP populations at all.
L8
if MHCII expression is restricted to dendritic cells only, which of the following T cells will develop in
the thymus?
- both CD4 and CD8
- only CD4
- only CD8
- none
3
* bone marrow chimera experiments: positive selection requires MHC expression on cTECs, not on hematopoietic cells like DCs.
* If MHC II is restricted to DCs only, then cTECs lack MHC II entirely. Since cTECs need MHC II to positively select CD4+ T cells, CD4 T cell development fails = no positive selecting signal for MHC class II-restricted thymocytes in the cortex, they will die
* However, cTECs still express MHC I normally, so CD8+ T cells can still be positively selected as usual.
* Dendritic cells do play a role in the thymus, but primarily in negative selection in the medulla — not positive selection. So restricting MHC II to dendritic cells won’t rescue CD4 positive selection.
L9
In mice in which the T cells are engineered to express only (RAG-/-) the H-Y-specific transgenic TCR that recognizes a male-specific antigen in the context of MHC I, what T cells will develop in female and male mice?
- female: No H-Y antigen present = no negative selection. The TCR can still weakly recognize self-peptide/MHC I on cTECs, so positive selection proceeds normally → CD8+ T cells develop.
- male: H-Y antigen present in body & thymus. The TCR binds too strongly to H-Y/MHC I → negative selection deletes the cells → no T cells develop. (self-reactive)
L9
You have a TCR transgenic mouse (OT-Il) that is specific for a peptide from the chicken ovalbumin (OVA) protein presented in the context of
MHC II.
You decide to cross this to a mouse expressing chicken ovalbumin (OVA) driven by the Aire-dependent insulin 2 promoter.
- What T cells do you expect will develop?
When developing OT-II thymocytes migrate to the medulla, they encounter OVA peptide presented on MHC II by mTECs and dendritic cells. Because they bind strongly and specifically to OVA/MHC II, they undergo negative selection and are deleted.
Little to no CD4+ T cell development. The self-reactive cells are eliminated in the thymus before they can leave.
L10
Why do you think DN and DP thymocytes may migrate slowly while more mature CD4+ and CD8+ SP thymocytes are “fast”?
DN/DP cells: slow= don’t need to cover much ground, cortical TECs present similar peptides throughout
DP cells don’t need to travel far: cTECs all present similar self-peptides, so positively selecting signals are found nearby
L10
There are some DP cells that are relatively faster than the average. What might these be?
possibly just received positive selection signal, transitioning to SP
L10
Among SP thymocytes, there is extensive heterogeneity in cell speeds. Why might this be?
- SP cells: fastest cells in the body — need to scan entire medulla to sample all tissue-restricted antigens (each mTEC expresses different ones)
- Slow SP cells: possibly undergoing strong TCR engagement → negative selection or anergy
L10
How can such infrequent (~1-2/hour), transient (~3-5 minutes) TCR signaling events lead to the long-lasting dramatic change that is positive selection?
- positive selection is like repeatedly filling a bucket one drop at a time — each 3–5 min signal adds a drop of calcium, and only once the bucket is full (after ~48hrs of serial events) does the transcriptional switch flip. Interrupt it long enough and the bucket empties.
- Each transient TCR signal adds a small calcium spike → calcium accumulates over ~48hrs of serial events → enough calcium to translocate NFAT to nucleus → positive selection transcriptional program. Interrupt it → calcium resets → start over.
L10
The data presented described the TCR signals associated with positive (and negative) selection of MHC I-restricted OT-I TCR transgenic cells.
Based on what you learned about the TCR signals associated with positive selection and differentiation into the CD4 vs CD8 T cell lineages, how might the signals be similar or different for MHC II-restricted thymocytes?
- OT1 (MHC I → CD8): individual signals ~3–5 min
- MHC II-restricted TCR: individual signals ~15 min (stronger, longer interactions)
- total time to positive selection was shorter for MHC II despite longer individual signals — possibly because longer/stronger signals accumulate calcium faster, needing fewer events
- CD4 lineage commitment requires sustained signalling → longer individual events compensate
Kinetic signaling model of CD4 versus CD8 T cell lineage choice
* Previous models
Instructive: TCR signal quality/quantity dictates cell fate choice
Stochastic: Random downregulation of a co-receptor, second selection step to ensure TCR specificity and co-receptor are matched
* Current model
* Kinetic signaling: the persistence of TCR signaling influences cell fate choice
The CD4 (MHC II) and CD8 (MHC I) coreceptors stabilize the interaction with MHC upon antigen recognition (and participate in signaling – CD4 has a stronger association with Lck)
L11
You have a mouse strain with a spontaneous mutation that leads to an absence of CD4+ SP thymocytes. How could you determine if the defect is hematopoietic cell-intrinsic or -extrinsic (impacts the thymic stroma)?
- transfer bone marrow from a wild type mouse into a lethally irradiated mutant mouse strain and determine if CD4 SP thymocytes develop
- transfer bone marrow from a mutant mouse strain into a lethally irradiated wild type mouse and determine if CD4 SP thymocytes develop
- transfer mature CD4 T cells into the mutant mouse strain
- determine if MC I is expressed on thymic epithelial cells from the mutant mouse strain
- transplant a thymus from a wild type mouse into the mutant mouse and determine if CD4 SP thymocytes develop
1,2,5
Which of the following statements are true:
1. dendritic cells express genes encoding tissue restricted antigens
2. medullary thymic epithelial cells (mTEC) express genes encoding tissue restricted antigens
3. mTEC present tissue restricted antigens to developing T cells in the thymus
4. dendritic cells present tissue restricted antigens to developing T cells in the thymus
5. cortical thymic epithelial cells present tissue restricted antigens to developing T cells in the thymus
2,3,4
DP thymocytes in CCR7-defiiceint mice will:
1. mis-localize to the cortex
2. mis-localize to the medulla
3. not udnergo positive selection
4. none
4
There is a second lineage of T cells that express a TCR composed of gamma and delta chains (rather than the conventional alpha/beta heterodimer). What evidence would suggest that Notch signals are NECESSARY for gamma delta T cell development?
1. gamma delta T cells are present in the thymus of mice constitutively expressing active Notch in hematopoietic cells
2. Culture of thymic progenitor cells on OP cells does not support gamma delta cell development but culture and OP9-DLL1 cells does
3. Notch-deficient hematopoietic cells do not give rise to gamma delta T cells
4. DIl4, a Notch ligand, is expressed on cortical thymic epithelial cells
2,3
L11
You harvest bone marrow from an MHC I-restricted OT-I TCR transgenic mouse that is deficient in Psmb11, the gene that encodes the ß5t subunit of the thymoprotesome, and you transfer the bone marrow into lethally irradiated wild-type mice. What cells will develop?
1. CD4+ SP thymocytes
2. CD8+ SP thymocytes
3. Neither CD4+ or CD&+ SP thymocytes
4. Both CD4+ and CD8+ SP thymocytes
2
- What thymocyte subset?
- DN1 → DN2 → DN3 → DN4 → DP → SP (CD4 / CD8).
Key checkpoints: - β-selection at DN3 (require successful TCR beta chain rearrangement)
- positive/negative selection at the DP stage. Lineage commitment to CD4 or CD8 occurs as
- What cells are they interacting with (and is there something special about these cells)?
In cortex: developing thymocytes interact with cTECs (responsible for positive selection)
* unique antigen processing: thymoproteasome (unique β5T subunit) for MHC I peptide presentation
* Cathepsin-L and TSSP for MHC II peptide presentation (generate unique peptide repertoire)
In the medulla: thymocytes interact with mTECs and DCs: critical for negative selection.
- What are the ‘master regulators’ that influence T cell development in the thymus?
Notch signaling drives commitment to the T cell lineage over other lymphoid fates at the DN stage.
ThPOK: master regulator of CD4 lineage commitment. its expression, driven by sustained TCR signaling, locks in the CD4 fate, and without it CD4 T cells fail to develop.
* absence of ThPOK favors CD8 lineage
