Thymocyte Development and Fibroblasts

Thymic Fibroblasts & Thymocyte Development

• Thymic fibroblasts secrete extracellular matrix (ECM) components, such as collagen.
• Collagen aids in concentrating cytokines necessary for thymocyte development.
• Collagen also controls thymocyte adhesion to stromal cells.

DN Stages of T Cell Development

• DN1 subset:
  • TCR genes are in germline configuration.
  • DN1 cells reside in the thymic cortex.
  • cTECs provide stem cell factor (SCF), which binds to c-kit on DN1 cells.
  • SCF delivers a survival signal to DN1 cells.
  • Transcription factor GATA-3 is crucial for DN1 generation.
• DN2 subset:
  • Also known as Pro-T cells.
  • Primarily located in the outer cortex.
  • TCR genes remain in germline configuration.
  • Expression of CD3 chains begins.
  • DN2 thymocytes proliferate rapidly under IL-7 and SCF influence.
• DN3 subset:
  • DN3 cells stop proliferating and stay in the outer cortex.
  • Five key events occur during DN3 stage:
    • Commitment to T cell lineage, generating alpha/beta and gamma/delta T cells.
    • V(D)J recombination commences at TCRG, TCRD, and TCRB loci.
    • Upregulation of RAG and TdT occurs.
    • DN3 cells destined to become alpha/beta T cells express a functional 'Pre-TCR complex'.
    • The complex determines if a functional TCR-beta chain has been produced.
  • Successful TCR-beta locus rearrangement halts further rearrangement at TCRG and TCRD loci.
  • DN3 thymocytes become early 'pre-T cells' committed to the alpha/beta T cell lineage.
  • DN3 cells express a diverse repertoire of TCR-beta chains.
  • The TCR-beta locus is the first to undergo VDJ recombination in alpha/beta T cells.
  • 'Beta-selection': DN3 thymocytes with successfully arranged TCR-beta chains undergo this process.
  • Cells surviving beta-selection pass the pre-TCR checkpoint, leading to DN3 cell proliferation and differentiation.
  • Pre-TCR complex formation: A glycoprotein called the 'pre-T-alpha chain' on DN3 cells acts as a surrogate for the real TCR-alpha chain.
  • The pre-T-alpha chain assembles with a successfully arranged and translated beta chain, along with CD3 complex proteins.
  • The pre-TCR acts as a sensor, initiating signal transduction.

Naive T Cell Activation

• Naive T cells enter lymph nodes through HEVs.
• They inspect pMHCs displayed by mature DCs near HEVs.
• T cells 'crawl' slowly over DC surfaces aided by adhesion molecules, facilitating pMHC screening.
• TCRs with sufficient affinity/avidity for pMHCs can activate the T cell.

Signal 1 for T Cell Activation

• Signal 1 is delivered when specific pMHCs on DCs bind to multiple TCRs on a naive Th or Tc cell surface.
• TCR engagement induces a conformational change in CD3 chains.
• This allows phosphorylation of CD3 ITAMs by Lck kinase associated with CD4 and CD8.
• Intracellular signaling enzymes are recruited to cytoplasmic tails of CD4, CD8, and CD3 chains.
• These enzymes mediate a cascade leading to activation of other enzymes.
• Multiple TCR activations result in signal 1.
• A single pMHC engagement with TCR is insufficient for complete activation of a naive T cell due to low affinity.
• Transient interaction between pMHC-TCR pairs is also insufficient.
• Sustained interaction between naive T cell and DC for many hours is needed for optimal T cell activation.
• The formation of an immunological synapse between T cell and DC interface is crucial for sustained signaling.
• Actin cytoskeletons and polarization occur in both cells, leading to three concentric rings in the immunological synapse:
  • Inner ring (cSMAC): Contains aggregated TCRs and costimulatory molecules.
  • Middle ring (pSMAC): Contains signaling adaptor talin, integrins, and adhesion molecules.
  • Outer ring (dSMAC): Contains actin-based cytoskeletal structures and large proteins.
• Some Th subsets promote effector functions against extracellular pathogens.

Th Effector Cell Activation

• Most resting Th effectors migrate back to the site of inflammation or tissue containing the antigen after differentiation.
• The same antigen that initially activated naive T cells, when presented by an APC, activates Th effectors.
• Th effectors then secrete subset-specific panels of cytokines, mediating effector functions.
• Th effector cell differentiation pathways are not fixed and can change based on circumstances and transcription program shifts.

Th1 Cells

• Intracellular pathogens (viruses and intracellular bacteria) trigger IFN-gamma, IL-12, and IL-27 production by macrophages and DCs.
• These cytokines activate STAT4 in Th0 cells, leading to Th1 commitment.
• At the site of inflammation or in tissues, Th1 cells are stimulated by antigen, activating transcription factor T-bet.
• T-bet drives IFN-gamma production.
• Th1 cells oppose Th2 cell differentiation.

Th2 Cells

• Extracellular pathogens do not induce IL-12 production by macrophages and DCs.
• They stimulate an unknown cell type (possibly mast cells or NKT cells) to secrete IL-4.
• IL-4 activates STAT6 in Th0 cells, driving differentiation to Th2 effectors.
• At the site of inflammation or in tissues, Th2 cells are stimulated by antigen, activating transcription factor GATA-3.
• GATA-3 drives the production of IL-4, IL-5, and IL-13, which are signature Th2 cytokines.
• Th2 cells oppose Th1 cell differentiation.

Cross-Regulation of T Helper Subsets

• Helper T cell subsets cross-regulate each other.
• Their secreted cytokines enhance their own differentiation and expansion while inhibiting other helper T cell lineages.
• This is called cross-regulation.
• Prominent cross-regulation occurs in the Th1/Th2 and Th17/iTreg pairs.
• IL-4 and IL-5 produced by Th2 cells suppress Th1 expansion.
• IFN-gamma produced by Th1 cells inhibits Th2 expansion.

Cytotoxic T Lymphocyte (CTL) Effector Functions

• Apoptosis induction:
  • CTLs kill target cells by expressing FAS ligand (FasL).
  • FasL binding to Fas on the target cell induces apoptosis.
• Cytotoxic cytokines:
  • CTLs produce TNF and LT, which induce apoptosis by binding to TNF receptor 1 on the target cell surface.
• IFN-gamma production:
  • IFN-gamma produced by CTLs indirectly aids B cells in antibody production, increasing antibody-dependent cell-mediated cytotoxicity (ADCC) and upregulating MHC class I expression.

CTL Dissociation and Re-arming

• After delivering a lethal hit, CTLs detach from the damaged target cell within 5-10 minutes due to a low affinity conformation of adhesion molecules.
• The target cell undergoes apoptosis within 3 hours of dissociation.
• The CTL resumes synthesis of new cytotoxic granules and inspects other host cells.
• A single CTL can sequentially attack multiple host cells, delivering lethal hits.
• The mechanism preventing CTL self-destruction by its granules remains unknown.

Termination of Effector T Cell Responses

• Th effector cells and CTLs are maintained by inflammatory cytokines (IL-12) and transcription factors (ID2 and BCL-3).
• After threat removal, effector cells are no longer needed.
• Continued exposure to inflammatory environment without antigen causes downregulation of IL-7R and IL-15R, reducing their ability to receive survival signals.
• Three mechanisms induce effector cell death:
  • Activation-induced cell death (AICD)
  • Cytokine withdrawal
  • T cell clonal exhaustion

Memory T Cells

• Approximately 5-10% of antigen-specific T cell progeny generated in a primary response survive AICD or IL-2 withdrawal.
• These cells differentiate into long-lived memory T cells, which are the basis of vaccination.
• Memory T cells recognize the same pMHC as naive and effector T cells but have intermediate properties.
• Memory T cells are usually found in a resting state but can undergo self-renewal for long-term survival.
• Upon reinfection by the same pathogen, memory T cells mount a secondary response faster and stronger than the primary response.
• Continuous exposure to an antigen can lead to rapid effector cell proliferation and burnout without memory T cell generation.

Peripheral Tolerance Mechanisms in B Cells

• Autoreactive B cells that escape central tolerance are controlled by unique peripheral tolerance mechanisms.
• If an autoreactive B cell encounters self-antigen in the periphery, it receives signal 1 but still requires an antigen-specific Th effector cell for signal 2 and 3.
• Without the required Th cell (due to central or peripheral tolerance), B cells cannot be activated, even in the presence of DAMPs/PAMPs.
• The B cell becomes anergic and undergoes apoptosis within 3-4 days.
• This dependence on Th cells for B cell activation minimizes autoreactive responses from Ig gene somatic hypermutation.
• Even with an autoreactive Th cell in the periphery, DCs are more likely to delete or anergize the Th cell than activate it in the absence of DAMPS/PAMPs.
• An anergic autoreactive Th cell cannot deliver signal 2 to a B cell, causing the B cell to become anergic and undergo apoptosis.

Explore the essential role of thymic fibroblasts in thymocyte development through extracellular matrix components like collagen. Understand the different DN stages of T cell development, including the specifics of the DN1, DN2, and DN3 subsets.