T Cell Maturation, Differentiation, and Activation

1. Overview and Thymic Origin

• Cortex: Thymic cortical epithelial cells form a meshwork of long cytoplasmic processes, expressing class I and II MHC molecules - the substrate for positive selection.
• Corticomedullary junction: Bone marrow-derived dendritic cells (DCs) reside here.
• Medulla: Medullary thymic epithelial cells (mTECs) and macrophages, critical for negative selection.

2. Stages of Thymocyte Maturation

2.1 Double-Negative (DN) Stage

• DN1/Pro-T cell: CD44+CD25−; not yet T-lineage committed; initiates migration to the mid-cortex.
• DN2/Pro-T: CD44+CD25+; committed to T lineage; RAG1 and RAG2 proteins are first expressed here, initiating TCR gene rearrangement. Dβ-to-Jβ rearrangements at the TCR β locus occur first.
• DN3/Pre-T: CD44−CD25+; Vβ-to-DJβ rearrangement completes. If a productive TCR β rearrangement occurs, the β chain pairs with the invariant pre-Tα chain to form the pre-TCR complex.
• DN4: The pre-TCR signals β-selection - confirming a productive rearrangement - and drives rapid proliferative expansion (a single precursor yields many progeny). It also suppresses further β chain rearrangements (allelic exclusion) and silences the γδ TCR program.

About 90% of DN cells productively rearrange TCR β first and are directed toward the αβ lineage. The remaining ~10% productively rearrange TCR γ and δ before TCR β and commit to the γδ lineage.

2.2 Double-Positive (DP) Stage

• RAG1/RAG2 are re-expressed and TCR α chain rearrangement occurs (Vα-to-Jα joining).
• The complete αβ TCR complex assembles and is expressed on the surface with CD3 and ζ chains.
• Rearrangement of TCR α permanently deletes the TCR δ locus (embedded within TCR α), irreversibly committing the cell to the αβ lineage.
• DP thymocytes then undergo the critical selection processes.

3. Thymic Selection

3.1 Positive Selection

• MHC class I recognition → CD8+ single-positive T cell: TCR-class I interaction activates RUNX3, which maintains CD8 expression and silences CD4.
• MHC class II recognition → CD4+ single-positive T cell: Stronger signaling through CD4-LCK activates GATA3, which commits to the CD4 fate and induces ThPoK (a repressor of CD8 lineage genes).

3.2 Negative Selection

4. T Cell Activation: The Three-Signal Model

Fig. 9.21 from Janeway's Immunobiology 10e: Resting T cells use oxidative phosphorylation and are arrested in G0. Upon activation, blast transformation occurs, metabolism shifts to aerobic glycolysis, and the cell enters active cycling.

Signal 1 - TCR/Antigen Recognition

1. LCK phosphorylates ITAMs (immunoreceptor tyrosine-based activation motifs) on CD3 ζ, γ, δ, ε chains.
2. ZAP-70 (ζ-chain-associated protein kinase 70) is recruited and activated, phosphorylating the adaptor LAT (linker for activation of T cells).
3. LAT recruits a signaling complex including PLCγ1 (phospholipase C gamma 1) and Grb2/SOS.
4. PLCγ1 cleaves PIP2 into DAG (diacylglycerol) and IP3 (inositol triphosphate):
   • IP3 → releases Ca²⁺ from ER → activates calcineurin → dephosphorylates and activates NFAT (Nuclear Factor of Activated T cells) → transcription of IL-2 and other effector genes.
   • DAG → activates PKC-θ → activates IKK → releases NF-κB from IκB inhibition → NF-κB enters nucleus.
5. Grb2/SOS activates Ras → MAPK/ERK cascade → activates AP-1 (Fos/Jun heterodimer) transcription factor.

Signal 2 - Co-stimulation

• CD28 signaling activates PI3K → Akt → mTOR, augmenting glucose uptake (Glut1 upregulation), promoting aerobic glycolysis (the Warburg-like metabolic shift), and driving cell cycle entry from G0 to G1.
• CD28 also stabilizes IL-2 mRNA and amplifies NFAT and NF-κB signals.
• Additional co-stimulatory pairs include ICOS/ICOS-L, OX40/OX40L, and 4-1BB/4-1BBL that reinforce or sustain activation.
• Negative co-stimulatory receptors CTLA-4 (CD152) and PD-1 (CD279) compete with or inhibit these signals to prevent uncontrolled activation.

Signal 3 - Cytokine/Polarizing Signals

5. Effector T Cell Differentiation

5.1 CD4+ T Helper Cell Subsets

5.2 CD8+ Cytotoxic T Lymphocytes (CTLs)

1. Perforin/granzyme pathway: Polarized exocytosis of cytotoxic granules; perforin creates pores in the target cell membrane, and granzymes B (key in humans) and A enter and activate caspases → apoptosis.
2. Fas/FasL (CD95/CD95L) pathway: CTL surface FasL engages Fas on the target cell → DISC formation → caspase-8 activation → apoptosis.
3. Cytokine secretion: IFN-γ and TNF-α produced by CTLs directly kill infected cells and activate macrophages.

6. Memory T Cells

• Central Memory (Tcm): CCR7+, CD62L+; reside in lymph nodes; rapidly proliferate on reencounter with antigen.
• Effector Memory (Tem): CCR7−, CD62L−; reside in peripheral tissues and blood; immediate effector function.
• Tissue-Resident Memory (Trm): CD69+, CD103+; permanently reside in tissues (lung, gut, skin); first-line responders.
• Memory Stem Cells (Tscm): CD45RA+, CCR7+; long-lived, highly self-renewing; the apex of memory hierarchy.

7. Downstream Activation of Other Immune Cells by T Cells

7.1 B Cell Activation and Antibody Production

• CD40L (CD154) on Tfh binding CD40 on B cells - the essential contact signal for B cell class switching and survival.
• Cytokines: IL-21 drives B cell proliferation and differentiation into plasma cells; IL-4 induces IgE class switching; IFN-γ drives IgG subclass switching.
• The GC reaction produces high-affinity antibody-secreting plasma cells and long-lived memory B cells.

7.2 Macrophage Activation

• Upregulates MHC II, CD80/86, and iNOS.
• Induces the "classically activated" M1 macrophage phenotype, producing TNF-α, IL-12, IL-1β, and reactive oxygen/nitrogen species.
• Enhances intracellular killing (critical against Mycobacterium tuberculosis, Leishmania).

7.3 Neutrophil Recruitment

• Induce G-CSF and CXCL8 (IL-8) production → neutrophil mobilization and recruitment.
• Drive production of antimicrobial peptides (defensins, calprotectin) at mucosal barriers.

7.4 Eosinophil Activation and IgE-Mediated Immunity

7.5 NK Cell and Innate Cell Cross-talk

• IL-2 secreted by activated CD4+ T cells drives NK cell proliferation and enhances cytotoxicity.
• IFN-γ from T cells primes NK cells and macrophages.
• Conversely, NK cell-derived IFN-γ feeds back to reinforce Th1 differentiation.

7.6 Dendritic Cell Licensing

8. Epigenetics and Metabolic Checkpoints

• Pioneer transcription factors displace nucleosomes at gene enhancers (creating open chromatin before the transcription machinery arrives).
• Histone modifications: H3K4me3 (active) marks Th1-associated IFN-γ locus in Th1 cells; H3K27me3 methylation represses the same locus in Th2 cells.
• DNA methylation at CpG islands silences lineage-inappropriate cytokine genes.
• mTORC1 (activated by Akt downstream of CD28) drives aerobic glycolysis and is required for Th1 and Th17 differentiation; mTORC2 promotes Th2. Treg development preferentially uses fatty acid oxidation and OXPHOS rather than aerobic glycolysis - explaining why glucose deprivation (as in tumors) enriches for Tregs.
• HIF-1α, induced in low-oxygen environments, reinforces Th17 fate via direct activation of RORγt, while simultaneously targeting FoxP3 for proteasomal degradation - tilting the Th17/Treg balance toward inflammation.

Summary Table: Key T Cell Maturation Checkpoints

• Abbas AK, Lichtman AH, Pillai S. Cellular and Molecular Immunology, 10th ed. - Chapters 8 (T cell development), 9 (T cell activation), 10 (effector T cells)
• Murphy K, Weaver C. Janeway's Immunobiology, 10th ed. - Chapters 8, 9 (T cell signaling and activation)
• Roitt I, Delves P, Martin S, Burton D. Roitt's Essential Immunology, 13th ed. - Chapters 7-8 (T cell activation, metabolic reprogramming, Th polarization)
• Yamada T et al. Yamada's Textbook of Gastroenterology, 7th ed. - T cell lineages in mucosal immunity
• Fishman AP et al. Fishman's Pulmonary Diseases and Disorders, 5th ed. - Adaptive T cell responses in lung immunity