Chapter 7 Active recall missed

During embryonic thymus development, which epithelial cells are found in the outer cortex and which are found in the inner medulla?

Epithelial cells of the cortex → found in the outer region

Epithelial cells of the medulla → found in the inner region

what is the rudimentary embryonic thymus called, and what colonizes it?

Thymic anlage; it is colonized by bone marrow-derived cells including:

Progenitor cells → become thymocytes and dendritic cells

Macrophages → populate the medulla (also found in the cortex)

what path do thymocytes take as they mature in the thymus?

Thymocyte Maturation Pathway:

1. Enter the cortex from the blood

2. Migrate inward through the cortex

3. Reach the medulla

4. Undergo positive and negative selection

5. Mature into single-positive CD4 or CD8 T cells

6. Exit into the bloodstream

Where are epithelial cells of the thymus located during embryonic development?

Outer cortex and inner medulla

• Cortical epithelial cells → outer region

• Medullary epithelial cells → inner region

Thymic Anlage

Rudimentary embryonic thymus

What does the macrophage populate?

Populates medulla and cortex

What describes the thymic anlage?

Embryonic structure colonized by bone marrow-derived progenitors, dendritic cells and macrophages

What is the path of thymocyte development?

Blood → cortex → inward migration → Medulla → bloodstream

where do developing thymocytes reside, and what surrounds them?

Developing thymocytes occupy the interstices (spaces) of extensive network of epithelial cells in both the cortex and medulla of the thymus

Which cell types are predominantly found in the thymic cortex?

• Immature thymocytes

• Cortical epithelial cells (thymic origin)

• Macrophages (bone marrow origin)

Which cells types are predominantly found in the thymic medulla?

• Mature thymocytes

• Medullary epithelial cells (thymic origin)

• Dendritic cells (bone marrow origin)

• Macrophages (bone marrow origin)

Cortex

Immature thymocytes, cortical epithelial cells, macrophages

Medulla

Mature thymocytes, dendritic cells, medullary epithelial cells

Where are the cortical epithelial cells and immature thymocytes located?

cortex

What determines whether a thymocyte becomes a CD4+ helper T cell or a CD8+ cytotoxic T cell?

The MHC class its TCR recognizes during positive selection

Which of the following occurs in the thymic medulla?

Final maturation of T cells into single-positive CD4+ or CD8+

Positive Selection →

Negative Selection →

Final Maturation to Single-Positive →

Entry of Thymocytes from Blood →

• cortex

• medulla

• medulla

• cortex

True or False the thymus shrinks with age and is largely nonfunctional in adulthood

True

The thymic cortex is the site of

positive selection

The thymic medulla is the site of

negative selection

What describes the sequence of thymocyte development and selection in the thymus?

Entry → Cortex → Positive selection → Medulla → Negative selection

What is a Hassall’s corpuscle, and where is it found?

Hassall’s corpuscles are:

• A distinct histological feature of the thymic medulla

• made of concentric layers of epithelial cells

• Possibly involved in T cell death or regulatory T cell development

• May serve as sites of cell destruction

Cortical epithelial cells

site of positive selection

Medullary epithelial cells

Present self-antigens to eliminate self-reactive T cells

What is the term for the gradual shrinking and functional decline of the thymus with age?

Involution

What marks the maturation of thymocytes into mature T cells?

the three features that mark T cell maturation:

1. Changes in the TCR gene status (rearrangement and expression)

2. Expression of the TCR protein itself

3. Production of T-cell surface glycoproteins like:

   • CD4

   • CD8

   • CD3 complex

What is the role of Notch1 signaling in early T cell development?

• Notch1 signaling keeps progenitor cells on the T cell developmental path

• It prevents the cell from becoming a B cell

• Specifically, it inhibits BCR gene rearrangement, ensuring TCR genes are activated instead

• It's active through all stages of thymocyte maturation

What is the significance of IL-7 and its receptor in T cell development?

• IL-7 is secreted by thymic stromal cells

• The IL-7 receptor on thymocytes is essential for binding IL-7

• This interaction helps guide the next steps in T cell maturation — it's critical during early thymocyte development, especially the double-negative stage

What is the primary function of Notch1 in thymocyte development?

Ensures T cell lineage commitment by inhibiting B cell development

Which new markers appear when a progenitor cell commits to the T cell lineage as a DN thymocyte?

• CD2 – adhesion and signaling

• CD5 – adhesion and signaling

• CD127 – IL-7 receptor (cytokine receptor)

• CD1A – MHC class I–like molecule

• RAG complex – initiates TCR gene rearrangement

What happens when Notch1 binds its ligand on thymic epithelium?

• Notch1 on the thymocyte binds to a Notch ligand on the thymic epithelial cell

• This causes cleavage of the Notch1 receptor

• The intracellular domain of Notch1 is released

• It enters the nucleus and:

  • Displaces co-repressors

  • Recruits co-activators

  • Turns on transcription of genes that commit the cell to the T cell lineage

Notch1 →

Notch ligand →

Intracellular Notch domain →

Thymic epithelial cell →

T cell–specific transcription →

Membrane-bound receptor on thymocyte

Ligand that binds Notch receptor

Turns on T cell development genes

Cell presenting the Notch ligand

Final result of Notch1 signaling

When does T cell lineage commitment occur in relation to TCR rearrangement?

T cell lineage commitment does not occur before TCR rearrangement — instead, it’s a race to obtain a productive rearrangement.

So, rather than choosing a lineage first (αβ or γδ), the thymocyte starts rearranging TCR genes, and whichever receptor successfully rearranges first determines the lineage.

Which chain more frequently rearranges productively first?

The β chain more frequently rearranges productively first, which favors commitment to the αβ T cell lineage.

What is pTα, and what is its role after β chain rearrangement?

pTα is a surrogate (placeholder) α chain. It’s not rearranged, just expressed as-is.

After a productive β chain rearrangement, the β chain pairs with pTα to form the pre-T cell receptor (pre-TCR).

The pre-TCR signals the cell to:

• Stop rearranging β (allelic exclusion)

• Start proliferating

• Begin rearranging the α chain

• Move forward in development

What forms the pre-TCR complex?

β chain and pTα

What happens to the α-chain locus after pre-TCR signaling?

After pre-TCR signaling, the cell becomes permissive for TCR α-chain locus rearrangement.

What effect does pre-TCR signaling have on TCR β-chain rearrangement?

it stops further rearrangement through allelic exclusion.

What other receptors begin to be expressed after pre-TCR signaling?

After pre-TCR signaling, the cell stimulates expression of CD4 and CD8 co-receptors.

These are essential for becoming a double-positive (CD4⁺CD8⁺) thymocyte — the next developmental stage.

How does pre-TCR signaling affect cell numbers?

Pre-TCR signaling induces proliferation, meaning the thymocyte undergoes multiple rounds of cell division.

This expansion increases the number of thymocytes that have a functional β chain, ensuring more cells can proceed to α-chain rearrangement.

What is the role of pTα in T cell development?

Pairs with β chain to form pre-TCR

What happens to recombination machinery after pre-TCR is expressed?

After expression of the pre-TCR, the recombination machinery is reactivated and redirected to rearrange the α-chain locus — and still has access to the γ and δ loci.

Where is the δ chain locus located, and what happens to it during α-chain rearrangement?

The δ chain locus is located within the α-chain locus on the chromosome.

So... When the α-chain rearranges, it deletes the entire δ chain locus.
This makes it impossible for the cell to become a γδ T cell afterward.

Roughly what percentage of thymocytes die in the thymus due to failed rearrangement?

About 98% of thymocytes fail to make a productive rearrangement and undergo apoptosis.

What happens after the β-chain forms the pre-TCR complex regarding CD4 and CD8?

After the β chain pairs with pTα (pre-Tα) to form the pre-TCR complex, the thymocyte then:

• Starts rearranging the α-chain locus

• Expresses both CD4 and CD8 co-receptors, becoming a double-positive thymocyte

What components make up the pre-T cell receptor (pre-TCR)?

The pre-TCR consists of:

• A functional β chain

• A surrogate α chain called pTα

• CD3 proteins

• The ζ chain

What percentage of T cells successfully rearrange the β-chain gene?

About 80% of T cells successfully rearrange the β-chain gene, which is higher than the success rate for B cell heavy chain rearrangement.

What does a productive rearrangement enable at the mRNA level?

A productive rearrangement allows the transcription of functional β-chain mRNA, which can then be translated into the β protein chain.

What happens to RAG-1 and RAG-2 gene expression during the proliferation phase after pre-TCR signaling?

During proliferation initiated by pre-TCR signaling, RAG-1 and RAG-2 gene expression is repressed.

This repression means the recombination machinery is temporarily turned off, which halts further β-chain gene rearrangement (this is part of allelic exclusion).

Can the TCR α-chain undergo multiple rearrangements?

Yes, the TCR α-chain can undergo several successive gene rearrangements during T cell development.

This increases the chance that the cell will generate a functional α chain.

What gene segments allow for many successive rearrangements in the α-chain locus?

The presence of many Vα (variable alpha) and over 60 Jα (joining alpha) gene segments in the α-chain locus allows for multiple successive rearrangements.

This diversity and abundance enable the cell to try many combinations.

What is the likelihood that a developing T cell will make a productive α-chain rearrangement?

Almost every developing T cell will make a productive α-chain rearrangement, meaning the success rate is very high, close to 100%.

How many successive rearrangements can the TCR α-chain undergo?

The TCR α-chain can undergo several successive gene rearrangements, allowing multiple attempts to generate a functional receptor.

What happens during nonproductive α-chain rearrangements?

it can try multiple successive rearrangements at the α-chain locus until a productive one is made or all options are exhausted.

What results from a productive α-chain rearrangement at the mRNA level?

A productive α-chain rearrangement results in the transcription of functional α-chain mRNA, which then leads to the synthesis of the T-cell receptor α chain protein.

Where are the δ-chain genes located relative to the α-chain locus?

The δ-chain genes are located within the α-chain locus on the chromosome.

At what developmental stages do these checkpoints occur?

• The first checkpoint (β chain + pTα) occurs at the double-negative (DN) stage, specifically before the thymocyte expresses CD4 or CD8.

• The second checkpoint (α and β chain pairing to form TCR) occurs at the double-positive (DP) stage, when the thymocyte expresses both CD4 and CD8.

During which stage(s) of development does Vβ–Dβ/Jβ rearrangement occur?

Vβ–Dβ/Jβ rearrangement takes place during the double-negative (DN) stage, specifically in the DN2–DN3 phases before pre-TCR expression and proliferation.

When are RAG-1 and RAG-2 expressed?

RAG-1 and RAG-2 are expressed during the double-negative stage (DN2–DN3) to drive β-chain rearrangement, then are repressed during pre-TCR–induced proliferation, and are re-expressed in the double-positive stage to enable α-chain rearrangement.

At what stage is pTα (pre-Tα) most highly expressed?

The pTα surrogate chain peaks in expression right after the first checkpoint, during the transition from DN3 to DN4, fueling pre-TCR signaling and the subsequent proliferation.

Which surface co-receptors appear at the double-positive stage?

The CD4 and CD8 co-receptors are both upregulated together, marking the double-positive (DP) stage.

Which transcription factors are upregulated in single-positive thymocytes?

In single-positive (SP) cells, lineage-specific transcription factors like Th-POK (for CD4⁺) and RUNX3 (for CD8⁺), along with GATA-3, become dominant to cement the helper vs cytotoxic fate.

Which CDRs recognize MHC vs. peptide?

• CDR1 and CDR2 primarily contact and recognize the MHC molecule.

• CDR3 is the most variable loop and recognizes the bound peptide.

How is the initial repertoire of T-cell receptors generated in developing thymocytes?

The initial TCR repertoire is generated by somatic V(D)J recombination in developing thymocytes:

• TCR β chain: Random recombination of V, D, and J gene segments

• TCR α chain: Random recombination of V and J gene segments

• This process is mediated by the RAG1/2 enzymes and diversified further by TdT adding (and deleting) nucleotides at the junctions

Which specialized cells present self-peptide–MHC complexes to thymocytes during negative selection in the medulla?

medullary thymic epithelial cells (mTECs)

What transcription factor in mTECs drives the expression of tissue-restricted antigens during negative selection?

The transcription factor is AIRE (Autoimmune Regulator), which drives expression of tissue-restricted antigens in medullary thymic epithelial cells to delete strongly self-reactive T cells.

Which enzyme complex is directly responsible for initiating the V(D)J recombination that generates TCR diversity in thymocytes?

RAG1/2 (Recombination-activating genes 1 and 2)

At the stage when positive selection occurs, thymocytes express which of the following phenotypes?

Double positive (CD4⁺ CD8⁺)

In your own words, describe the role of cortical epithelial cells in the positive selection of double-positive thymocytes. What do these cells do, and how do they interact with the developing T cells?

Cortical epithelial cells in the thymic cortex:

• Extend a lattice of cell processes that physically envelop the CD4⁺CD8⁺ thymocytes.

• Present self-MHC class I and II molecules loaded with self-peptides on their surface.

• Test each thymocyte’s αβ TCR by seeing if it can bind one of these peptide–MHC complexes.

• If binding occurs within about 3–4 days, they deliver a positive (survival) signal; if not, the thymocyte undergoes apoptosis and is cleared by macrophages.

What types of MHC molecules do cortical epithelial cells present during positive selection?

Class one and two

Where do the self-peptides presented by cortical epithelial cells during positive selection actually come from? Describe their origin in your own words.

They’re simply fragments of the cell’s own proteins produced right there in the thymus (by cortical epithelial cells and nearby thymic cells).

If someone is heterozygous at the six major HLA loci (three class I and three class II, two alleles each), about how many total self-peptides could be presented during positive selection? (Hint: multiply the per-MHC estimate by the number of distinct MHC molecules.)

if you’re heterozygous at all six HLA loci, you have 18 distinct MHC molecules (six loci × two alleles each) × ~10,000 peptides each, which is about 180,000 self-peptides presented during positive selection.

During positive selection, how does engagement of the TCR with MHC class I vs. class II influence whether the thymocyte becomes a CD8⁺ or CD4⁺ T cell? Describe which co-receptor is recruited and which is excluded in each case.

• If the TCR binds a self-peptide:MHC class I complex, the CD8 co-receptor is recruited into the signaling complex and CD4 is excluded. The cell downregulates CD4 and becomes a CD8⁺ T cell.

• If the TCR binds a self-peptide:MHC class II complex, the CD4 co-receptor is recruited and CD8 is excluded. The cell downregulates CD8 and becomes a CD4⁺ T cell.

Why does ongoing α-chain rearrangement matter for the thymocyte’s TCR specificity?

ongoing α-chain rearrangement during those 3–4 days is all about rescuing thymocytes that didn’t initially bind self-MHC. Each new V–J recombination attempt generates a different α chain, pairing with the same β chain to try again. That way, cells that would otherwise die get multiple chances to produce a TCR capable of positive selection.

After a thymocyte finally produces a TCR that binds self-MHC and receives the survival signal, what happens to RAG1/2 expression and further TCR gene rearrangement? Explain why.

Once a thymocyte’s TCR successfully binds self-MHC and it receives its survival signal, RAG1/2 expression is shut off. This halts further V(D)J recombination to “lock in” that functional TCR and maintain allelic exclusion.

What is allelic exclusion in the context of TCR gene rearrangement, and why is it crucial for T-cell specificity?

allelic exclusion ensures that once a productive β-chain rearrangement occurs on one allele, the other allele is prevented from rearranging, so each T cell expresses a single, unique β chain (and thus a single TCR specificity).

Which thymic cell types are the most important mediators of negative selection?

the most important mediators of negative selection are the bone marrow–derived dendritic cells and macrophages in the thymic medulla.

What transcription factor expressed by medullary thymic epithelial cells drives the presentation of tissue-restricted self-peptides during negative selection?

The transcription factor is AIRE (Autoimmune Regulator).

Negative selection can’t delete T cells specific for self-peptides not present in the thymus. What mechanism helps expose thymocytes to those otherwise tissue-restricted antigens?

AIRE-driven expression by medullary thymic epithelial cells

What happens to any self-reactive T cells that do escape negative selection because their antigen wasn’t presented in the thymus?

Self-reactive T cells that slip through because their specific antigen wasn’t presented in the thymus are dealt with by peripheral tolerance mechanisms, such as:

• Anergy: They become functionally unresponsive when they encounter antigen without proper costimulation.

• Suppression by regulatory T cells (Tregs): Natural Tregs (selected in the thymus) and induced Tregs in the periphery help suppress autoreactive clones.

• Deletion: In some contexts, they can undergo apoptosis upon repeated or aberrant stimulation in the periphery.

What key surface marker and unique transcription factor define thymus-derived regulatory T cells (Tₙₙₙ)?

Regulatory T cells are a subset of CD4⁺ T cells that express high levels of the IL-2 receptor α-chain (CD25) and the lineage-defining transcriptional repressor FoxP3.

How do Tregs suppress autoreactive CD4⁺ T cells? Describe the key requirements and the types of mediators involved.

Regulatory T cells (Tregs) suppress autoreactive CD4⁺ T cells by two key features:

1. Antigen-Specific Contact

   • Both the Treg and the target autoreactive T cell must engage the same antigen-presenting cell (APC) via their TCR/CD4:MHC-II interactions.

   • This close proximity lets the Treg deliver inhibitory signals directly at the immunological synapse.

2. Non-Inflammatory Mediators

   • Cytokines: Tregs secrete high levels of IL-10, TGF-β, and sometimes IL-35, which dampen activation and proliferation of nearby T cells and APCs.

   • CTLA-4 Expression: Tregs express CTLA-4, which can strip costimulatory molecules (CD80/86) off the APC, reducing its ability to activate other T cells.

After surviving thymic selection, where do naïve single-positive T cells go, and what triggers their final differentiation into effector cells?

naïve single-positive T cells recirculate through the secondary lymphoid tissues (lymph nodes, spleen, Peyer’s patches), and when they encounter their specific antigen presented by an APC (with the right costimulatory signals and cytokine milieu), they undergo their final differentiation into effector T cells.

Once activated in secondary lymphoid organs, what effector functions do:

• CD8⁺ T cells adopt?

• CD4⁺ T cells adopt, and what determines whether they become TH1 versus TH2?

• CD8⁺ T cells → become cytotoxic T lymphocytes (CTLs), capable of killing infected or malignant cells via perforin/granzyme and Fas–FasL pathways.

• CD4⁺ T cells → become helper T cells.

  • TH1 differentiation is driven by IL-12 (from dendritic cells/macrophages) and IFN-γ, and TH1 cells secrete IFN-γ to activate macrophages and support cell-mediated immunity.

  • TH2 differentiation is driven by IL-4, and TH2 cells secrete IL-4, IL-5, and IL-13 to support B-cell help and humoral immunity.

Looking at the summary diagram of T-cell development:

1. What are the four major thymic regions/stages (with their CD profiles) that a T cell passes through from entry to exit?

2. Where do mature single-positive T cells exit the thymus?

• Four major regions/stages in the thymus:

  • Subcapsular zone: proliferating double-negative (CD3⁻ CD4⁻ CD8⁻) thymocytes

  • Cortex: double-positive (CD4⁺ CD8⁺) thymocytes undergoing positive selection

  • Cortico-medullary junction / Medulla: still primarily double-positive but undergoing negative selection (interaction with DCs, mTECs, macrophages)

  • Medulla: single-positive (CD4⁺ or CD8⁺) thymocytes completing selection

• Exit point: Mature single-positive T cells leave via blood venules in the medulla and enter the circulation.