T Cell Activation: Immunity & Signal Transduction

• T cell activation culminates humoral and cell-mediated immunity via activation and clonal expansion.
• Initiation of T cell activation requires the interaction between the TCR, CD3 complex, and processed peptide antigens.
• A key element of T cell activation involves TCR recognition of MHC antigen complexes on APCs.
• This recognition triggers events starting at the membrane surface and concluding in the nucleus, facilitating signal transduction.
• T cell induction has two phases: initiation and signal generation.

Initial Recognition Structure

• Recall the initial recognition structure: MHC antigen complex binding to TCR, CD3 complex, with associated CD4 or CD8 molecules.
• The associated protein activation and cellular effects should also be noted.
• The downstream signal transduction outcome involves NF-κB activation and migration to the nucleus, which results in cytokine expression.

Second Signal Requirement

• Recall structures involved in recognition
• Note the supported adhesion molecules expressed at the plasma membrane level.
• The second signal involves the interaction between CD28 on the T cell surface and CD80/86 on the APC surface.
• CD80/86 may also be referred to as B7.

Superantigens

• Superantigens are viral or bacterial proteins which activate T cells
• They bind to the TCR and class II MHC molecule simultaneously, causing T cell activation and proliferation.
• Endogenous and exogenous superantigens include viral and bacterial antigens, respectively.
• They bind to the variable beta domain of the TCR and the alpha chain of the class II MHC molecule.
• Superantigens produce a unique activating signal, unlike the traditional two-step T cell activation.
• Superantigens activate a large number of T cells.

Endogenous Superantigen T Cell Activation

• Endogenous superantigens cross-link the variable beta region on the TCR with the alpha region of the class II MHC molecule.
• Exogenous superantigens are secreted by bacteria, e.g., staphylococcal enterotoxins.
• Endogenous superantigens are cell membrane proteins encoded by certain viruses, e.g., mouse mammary tumor virus.
• Superantigens can cause overproduction of cytokines, leading to systemic toxicity.

CD3 Peptide Chains

• The T cell sends signals to the nucleus using the intracellular component of CD3 peptide chains after receiving signal one and signal two
• These chains include two epsilon, two zeta, and one each of alpha, beta, gamma, and delta chains.
• All chains except alpha and beta have at least one immune receptor tyrosine-based activation motif, or ITAM.
• ITAMs include two tyrosine amino acids spaced perfectly apart.

ITAM Regions

• Each gamma and delta chain has one ITAM region.
• The two epsilon chains each have one ITAM region.
• The two zeta chains each have three ITAM regions for a total of 10 ITAM regions.
• Binding of CD4 or CD8 to the MHC causes the kinase LCK, bound to CD4 or CD8, to phosphorylate all ITAMs in the CD3 complex.
• Another tyrosine kinase called ZAP70 binds to the phosphorylated ITAMs on the zeta chains of the CD3 complex, and becomes activated.
• Activated ZAP70 then phosphorylates the linker of activated T cells, or LAT, a transmembrane protein.
• ZAP70 also phosphorylates SLP76, and LAT, which together trigger a chain of events that activates NF-κB and NFAT.
• These transcription factors increase the expression of cytokines like IL-1, IL-2, IL-6, and TNF-alpha.
• They also upregulate cell surface markers like BCL-2, providing a strong survival signal to the T cell, leading to proliferation and differentiation.

T Cell Activation Steps

• Step 1: Antigen Recognition
  • T cell activation starts when T cells recognize antigenic peptides presented by major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APCs).
  • CD8+ T cells recognize antigens presented by MHC class I.
  • CD4+ T cells recognize antigens presented by MHC class II.
• Step 2: T Cell Receptor (TCR) Engagement
  • The TCR on the surface of T cells interacts with the peptide-MHC complex on the APC.
  • The CD4 or CD8 co-receptor helps stabilize the interaction between the TCR and the MHC-peptide complex.
• Step 3: Co-stimulation
  • Co-stimulatory signals are provided by interactions between co-receptors on T cells and ligands on APCs.
  • CD28 on T cells interacts with CD80 (B7-1) or CD86 (B7-2) on APCs, providing a crucial co-stimulatory signal for T cell activation.
• Step 4: Formation of Immunological Synapse
  • Antigen recognition leads to the formation of an immunological synapse: a specialized region of close contact between the T cell and APC.
  • The immunological synapse facilitates efficient signaling and the exchange of molecular signals.
• Step 5: Signal Transduction
  • Phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in the CD3 complex of the TCR occurs upon antigen recognition.
  • Lck (Lymphocyte-specific protein tyrosine kinase) and ZAP-70 (Zeta chain-associated protein kinase 70) are activated and play key roles in signal transduction.
• Step 6: Calcium Signaling
  • TCR engagement triggers an increase in intracellular calcium levels, a critical signaling event.
  • Calcium signaling is essential for the activation of downstream pathways and the initiation of T cell responses.
• Step 7: Activation of MAP Kinase Pathway
  • The mitogen-activated protein kinase (MAPK) pathway is activated, leading to the phosphorylation of ERK (extracellular signal-regulated kinase).
  • MAPK signaling contributes to T cell activation and proliferation.
• Step 8: Activation of Transcription Factors
  • Transcription factors, including NFAT (nuclear factor of activated T cells), AP-1 (activator protein 1), and NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), are activated.
  • These transcription factors translocate to the nucleus and initiate the transcription of genes involved in T cell activation and effector functions.

MHC Class II Molecules (CD4+ T Cells)

• Formation of the MHC Class II Complex:
  • Endocytosis: Extracellular antigens (e.g., bacterial or fungal proteins) are internalized by APCs.
  • Proteolytic Processing: The antigens are degraded into peptides within the acidic environment of endosomes/lysosomes.
  • MHC Assembly in ER: In the ER, MHC class II molecules are synthesized with an invariant chain (Ii) that prevents premature peptide binding.
• Transportation and Loading of MHC Class II Molecules:
  • Invariant Chain Removal: The MHC class II-invariant chain complex is transported to endosomes/lysosomes, where the invariant chain is degraded, leaving a small fragment (CLIP) in the peptide-binding groove.
  • Peptide Loading: HLA-DM facilitates the removal of CLIP and the loading of antigenic peptides onto MHC class II molecules.
• Presentation on Specific Cell Types:
  • Cell Types: MHC class II molecules are expressed on professional antigen-presenting cells (APCs), including dendritic cells, macrophages, and B cells.
  • Presentation: The MHC class II-peptide complex is transported to the cell surface for recognition by CD4+ helper T cells.
• Binding with the TCR:
  • The TCR on CD4+ T cells binds specifically to the peptide-MHC class II complex.
  • The CD4 co-receptor stabilizes the interaction between the TCR and MHC class II.
• Signal Transduction Pathways:
  • First Signal:
    • TCR binds to the MHC class II-peptide complex.
    • CD4 stabilizes the interaction.
    • ITAMs on CD3 and ζ-chains are phosphorylated by Lck, a tyrosine kinase associated with CD4.
    • Phosphorylated ITAMs recruit ZAP-70, which gets activated and propagates the signal through downstream molecules (e.g., LAT, SLP-76).
  • Second Signal:
    • Co-stimulatory interaction between CD28 on the T cell and B7-1/B7-2 (CD80/CD86) on APCs.
    • This interaction enhances the signal, leading to activation of transcription factors, driving the production of cytokines and T cell differentiation into effector subsets.

Detailed Signal Transduction Pathway Following TCR-MHC Binding (MHC Class I - CD8+ T Cells)

• TCR Binding and CD8 Stabilization:
  • The TCR specifically binds to the MHC class I-peptide complex on the target cell.
  • The CD8 co-receptor binds to the α3 domain of the MHC class I molecule, stabilizing the interaction and bringing Lck into proximity with the CD3 and ζ-chains of the TCR complex.
• Phosphorylation of ITAMs:
  • CD3 (γ, δ, ε subunits) and ζ-chains each contain immunoreceptor tyrosine-based activation motifs (ITAMs).
  • Lck phosphorylates the tyrosine residues in the ITAMs of the CD3 and ζ-chains upon MHC-TCR binding.
• Recruitment and Activation of ZAP-70:
  • Phosphorylated ITAMs serve as docking sites for ZAP-70, a cytoplasmic tyrosine kinase that bind to the phosphorylated ITAMs.
  • Once recruited, ZAP-70 is activated by phosphorylation at specific tyrosine residues by Lck.
• Activation of LAT and SLP-76:
  • Activated ZAP-70 phosphorylates downstream scaffold proteins
    • LAT (Linker for Activation of T cells): A transmembrane adaptor protein.
    • SLP-76: A cytosolic adaptor protein.
  • Phosphorylated LAT recruits multiple signaling molecules through its phosphorylated tyrosine residues.
• Formation of the Signalosome:
  • Phosphorylated LAT forms a complex with SLP-76, Grb2, Gads, and PLCγ1, assembling a large multi-protein signaling complex (signalosome).
• Activation of PLCγ1:
  • ZAP-70 phosphorylates and activates PLCγ1, which catalyzes the hydrolysis of PIP2 into two key second messengers:
    • DAG (diacylglycerol): Activates protein kinase C (PKC) and facilitates activation of the Ras pathway.
    • IP3 (inositol 1,4,5-trisphosphate): Binds to IP3 receptors causing release of intracellular calcium stores.
• Calcium Signaling:
  • Increased intracellular calcium binds to calmodulin, activating calcineurin
  • Calcineurin dephosphorylates NFAT allowing NFAT to translocate to the nucleus and initiate transcription of genes, including IL-2.
• Activation of DAG-Dependent Pathways:
  • DAG recruits and activates Protein Kinase C-θ (PKCθ), which in turn activates the NF-κB pathway:
    • PKCθ phosphorylates CARMA1, leading to the activation of the IκB kinase (IKK) complex.
    • The IKK complex phosphorylates IκB, allowing NF-κB to translocate to the nucleus.
  • DAG also activates RasGRP, which activates the Ras-MAPK pathway:
    • Ras activates Raf, which phosphorylates MEK.
    • MEK phosphorylates ERK.
    • ERK activates transcription factors like AP-1.
• Transcriptional Activation:
  • The combined activity of NFAT, NF-κB, and AP-1 leads to:
    • Expression of cytokines
    • Upregulation of survival and effector molecules
• Integration with the Second Signal (CD28-B7 Interaction):
  • CD28 binds to B7-1/B7-2 on antigen-presenting cells, amplifying the first signal.
  • CD28 signaling activates PI3K, leading to the production of PIP3.
  • PIP3 recruits and activates Akt, which promotes cell survival and metabolism.
  • CD28 co-stimulation also synergizes with the TCR signal to fully activate NF-κB, NFAT, and AP-1.
• Outcomes of Signal Transduction:
  • Clonal Expansion: IL-2 secretion and IL-2 receptor (CD25) upregulation enable rapid proliferation of CD8+ T cells.
  • Differentiation: CD8+ T cells differentiate into cytotoxic T lymphocytes (CTLs).
  • Effector Functions: CTLs kill infected cells via perforin and granzyme release or Fas-FasL interactions.

Detailed Signal Transduction Pathway for the Second Signal (CD28-B7 Interaction)

• Overview:
  • The second signal is critical to amplify and sustain T cell activation.
  • The interaction between CD28 (on the T cell) and B7-1/B7-2 (CD80/CD86 on APCs) triggers a cascade of signaling events that synergize with the TCR-MHC pathway.
• Step-by-Step Signal Transduction for the Second Signal:
  • CD28 Engagement:
    • CD28 on the T cell surface binds to B7-1 or B7-2 expressed on professional APCs.
    • This interaction recruits PI3K to the cytoplasmic tail of CD28.
  • Activation of PI3K and Generation of PIP3:
    • PI3K phosphorylates PIP2, generating PIP3.
    • PIP3 serves as a docking site for proteins with PH domains.
  • Activation of Akt Pathway:
    • PIP3 recruits Akt to the plasma membrane.
    • PDK1 phosphorylates and activates Akt.
    • Activated Akt enhances cell survival and metabolism.
  • Enhancement of NF-κB Activation:
    • CD28 signaling amplifies PKCθ activation, which was already initiated by TCR signaling.
    • PKCθ activates the CBM complex
      • The CBM complex recruits and activates the IκB kinase (IKK) complex.
      • IKK phosphorylates IκB, targeting it for degradation.
      • This releases NF-κB, allowing it to translocate to the nucleus.
  • Amplification of MAPK Pathway:
    • The CD28 pathway enhances the activation of the Ras-MAPK cascade
      • Ras activates the Raf-MEK-ERK pathway, culminating in the activation of AP-1.
  • Synergistic Activation of NFAT:
    • Calcium signaling, initiated by TCR engagement, leads to the activation of calcineurin.
    • Calcineurin dephosphorylates NFAT, enabling its nuclear translocation.
    • CD28 signaling sustains the calcium flux, ensuring prolonged NFAT activation.
  • Integration of Signals:
    • The combined activity of NF-κB, NFAT, and AP-1 results in transcription of genes essential for T cell activation and function.
• Key Outcomes of Second Signal:
  • Cytokine Production: Enhanced secretion of IL-2, IFN-γ, and other cytokines critical for T cell expansion and effector function.
  • Proliferation: Upregulation of the high-affinity IL-2 receptor (CD25) enables autocrine and paracrine stimulation by IL-2.
  • Survival: Activation of pro-survival pathways through Akt ensures T cell persistence during the immune response.
  • Differentiation: Activation of pathways like mTOR promotes differentiation into cytotoxic T cells or helper T cells.
• Integration with First Signal: The second signal amplifies and sustains the initial TCR-mediated activation which ensures the T cell response is robust, specific, and regulated.