Molecular events in T cell activation

• Describe signals 1, 2 and 3 in T cell activation

  Signal 1: antigen recognition by CD4+ T cell receptor, presented by MHCII

  Signal 2: co-stimulatory molecule interaction, e.g., CD28-CD80/86. This interaction is strengthened by adhesion receptors, such as LFA1-ICAM1 interaction

  Signal 3: cytokine signal to promote differentiation of T cells
• Describe the T cell receptor complex

  T cell receptor complex = T cell receptor + CD3 molecules

  • alpha and beta chains on the TCR recognise antigen but do no have signalling ability
  • CD3 stabilises the receptor via electrostatic interactions between negative residues on the CD3 molecules and positive charges on the TCR chains
• Describe the structure of the CD3 complex

  CD3 complex is composed of:

  • Heterodimer of epsilon and delta chains
  • Heterodimer of epsilon and gamma chains
  • Homodimer of zeta chains

  There are a total of 10 ITAM motifs on the cytoplasmic domains (3 on each zeta chain), which are required for signalling.
• Describe ITAMs

  Immunoreceptor tyrosine-based activation motifs that are present in several different immunoreceptors, such as B cell receptors, NK cell receptors and Fc receptors. These motifs contain a consensus sequence: YXXL/I - YXXL/I. Phosphorylation of tyrosines in this motif, creates a binding site of additional signalling proteins, which is important for signal transmission across the plasma membrane.
• Describe how ITAMs are involved in signal transmission

  CD4 molecule recruits Lck, which is a tyrosine kinase that phosphorylates ITAMs on the CD3 complex. This produces adapter binding sites for proteins that contain the SH2 domain, such as kinase ZAP-70.
• Describe ZAP-70 action

  ZAP-70 is a kinase that is recruited to the phosphorylated ITAMs on CD3 complex. ZAP-70 is activated via phosphorylated by Lck. ZAP-70 then phosphorylates multiple target proteins to trigger signalling, which is enhanced by TCR complex clustering. ZAP-70 phosphorylates the scaffold proteins LAT (linker of activated T cells) and SLP-76.
• Describe the immunological synapse

  This describes the membrane interaction between the APC and the T cell, to form a cluster of signalling molecules, known as the SMAC complex:

  • Central SMAC: TCR-CD3 complex interacting with MHC-bound peptide, CD28 – CD80/CD86
  • Peripheral SMAC: CD4 with Lck, LFA1-ICAM1
  • Distal SMAC: CD44 and CD45, which is a phosphatase that regulates Lck activity
• Describe the action of the scaffold proteins LAT and SLP-76

  LAT and SLP-76 can form a complex with the adaptor protein Gads. This complex can recruit phospholipase C gamma, which is initially recruited to the plasma membrane by PIP3. PLC-gamma is activated via phosphorylation by ITK.

  
• Describe the action of PLC-gamma

  PLC-gamma is recruited by the Gads : LAT : SLP-76 complex and cleaves phospholipid phosphatidylinositol bisphosphate (PIP2) to form inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 triggers an increase in intracellular calcium ion concentration by activation nuclear calcium channels. DAG recruits signalling proteins to the plasma membrane, such as PKC and RasGRP.
• Describe the role of CD28

  CD28 binds to CD80/86, which induces CD28 cytoplasmic tail phosphorylation by LKC. PI3K is recruited to and activated by CD28 and produces PIP3, which is the binding molecule for the PH domain for PLC-gamma.
• How does PIP3 reinforce signals from the TCR?

  Recruits and activates PLC-gamma, as well as recruiting the kinases PDK and Akt. Akt is important in pro-survival signalling and metabolism, therefore promoting the survival of and metabolically preparing the T cell for activation.
• Describe the Calcineurin pathway

  This pathway is stimulated by an influx of calcium ions:

  1. Calcium ions act on calmodulin, which stimulates it to open up and interact with calcineurin. 2. Calcineurin can then de-phosphorylate NFAT and prevent it from being sequestered and translocated to the nucleus 3. Active NFAT is a transcription factor that modulates gene expression
• Describe the NF-kB pathway

  This pathway is stimulated by DAG-mediated PKC activation:

  1. CARMA1 phosphorylated by PKC-theta, which is activated by DAG
  2. CARMA1 multimerises and recruits MALT1 and BCL10, which recruit TRAF-6.
  3. TRAF-6 is an E3 ubiquitin ligase that ubiquitylates itself and other proteins to produce long chains, which recruit kinases via adaptor proteins TAB1/2
  4. TAK1 regulates kinase complex IKK, which regulates IkB kinase complex, which degrades IkB and activates NF-kB
• Describe the IKK complex

  This is the IkB kinase complex and consists of:

  • IKK-alpha
  • IKK-beta
  • NEMO subunits, which phosphorylate and degrade IkB

  This results in activation of NF-kB
• Describe how the IL-2 gene is targeted by different TFs

  IL-2 is a major mitogen for T cells and allows rapid clonal expansion to occur. Expression is regulated by the requirement to occupy multiple regulatory sites in both its enhancer and promoter regions:

  • NFAT
  • NF-kB