Overview Events controlled by signaling Development, Activation, Homing, Death Antigens, cytokines, chemokines, etc. Antigen receptors: Associated chains, ITAM’s, tyrosine kinases, adaptors, downstream effectors (more ubiquitous) Spatial organization: lipid rafts, immunol. synapse (SMAC) Other receptors: TNFr’s, chemokines, TLR’s

Signal Transduction Pathways Relay Information from the Cell Surface to the Nucleus

Crosslinking and receptor tyrosine kinases

Surface Ig Crosslinking --> B Cell Activation Similarly, TCR crosslinking activates T cells

TCR/CD3/ Complex and Surface Ig with Ig and Ig These are relatively stable complexes, and can isolated intact by immuno-precipitation, when mild detergents are used Assembly of the entire complex is necessary for efficient expression at the cell surface

Irving and Weiss expt. demonstrating that z crosslinking is sufficient for T cell activation CD8a ecto + t.m. transfect into CD8 neg. T cell line X-link with anti-CD8 Ab z intracellular IL-2 Similar results seen by others with chimeras containing intracellular domain of CD3 or Ig or Ig

How do TCR and surface Ig access intracellular signaling pathways? Unlike RTK’s, no intrinsic catalytic activity Conserved motif with 2 tyrosines in , CD3 chains and Ig-a and Ig-b proteins First recognized by Michael Reth Now called an ITAM Immuno-receptor Tyrosine-based Activation Motif Also found in receptors on NK and monocytic cells

TCR- and Surface Ig-Associated Proteins Immuno-receptor tyrosine-based activation motif (ITAM) D/E X X Y X X L X7 Y X X L

Src vs. Syk Family Tyrosine Kinases SH2 catalytic Src Yes Fgr Hck Lyn Blk Fyn Lck Syk - B cells, early T cell development, NK cells, platelets, monocyte lineage ZAP-70 - Throughout T cell development, NK cells B cells T cells

Early events in signaling through the TCR - Sequential involvement of src and Syk family src family tyrosine kinases Syk/ZAP-70 family tyrosine kinase

Sequential tyrosine kinase activation in B cells

Multi-domain adaptors nucleate signaling complexes SH2 (src-homology 2) :: phospho-tyrosine SH3 :: proline-rich sequences Different SH2 and SH3 domains have different specificities

Cytoplasmic adaptors of lymphocytes

Transmembrane adaptors are also intermediates in non-receptor tyrosine kinase systems

Transmembrane lymphocyte adaptors LAB/NTAL B cells

Nucleation of signaling complex at LAT Formation of this complex results in stable membrane association of PLC-1, activation of which is critical for generation of second messengers

Second messengers relay signals from receptors at the plasma membrane DAG and IP3 are second messengers

Small G protein activation is aided by Guanine nucleotide Exchange Factors

Ras activation is also aided by a guanine nucleotide releasing protein (GRP) GEF GRP

Small G proteins activate MAPK Cascades MAPK = mitogen-activated protein kinase

Three major transcription factors downstream of the TCR PIP2

1.Tyrosine phosphorylation 2. “Second messengers” 3. Downstream kinases 4. Transcription factors 1 3 2 IP3 second messengers AP-1 activation transcription factors NF-kB activation

Ways to study the contributions of signaling proteins to T cell development and activation Generate mouse knockout Germ-line; tissue-specific; inducible Generate mutant cell line Chemical or radiation mutagenesis RNAi - oligos or plasmid-based - variable Each approach has advantages and drawbacks

Effect of SLP-76 deficiency on T cell development LAT k.o. looks identical

Signaling defects in SLP-76-deficient Jurkat T cells Ras/MAPK IP3/Ca2+

Some Remaining Big-Picture Questions Exactly how does crosslinking initiate the whole signaling cascade? Is crosslinking/dimerization actually sufficient? Any role for conformational change? Maybe a little of both… Changes in local concentration of ITAMs, sub-cellular localization (i.e. access to kinases and/or lack of access to phosphatases ?

Lipid rafts and Ig/TCR signaling Distribution of lipids in p.m. not uniform High concentration of sphingolipids and cholesterol in mobile ‘rafts’ Singer/Nicholson fluid mosaic model… Proteins with certain lipid modifications partition preferentially to lipid rafts including some signaling molecules (lck, LAT) Chemical disruption of rafts prevents activation May be important for initiation of signaling

MIRR = multi-chain immune recognition receptor

SMAC or Immunologic Synapse SMAC = supra-molecular activation cluster Kupfer - deconvolution IF microscopy of T cell/APC interactions - --> organized, bulls-eye type structure c-SMAC p-SMAC LFA-1/Talin, CD45 c-SMAC TCR/CD3, CD28, PKC q, lck - Non-activating, altered, peptides do not support the formation of these structures - Precise functional role still controversial

SMAC or Immunologic Synapse LFA-1 TCR APC T cell LFA-1: an integrin; binds ICAM on APC

Negative regulation and down-regulation of TCR and BCR signaling Internalization; trafficking to endosomes/lysosomes role for the immune synapse in this process? Ubiquitination --> proteasomal degradation of receptor Cbl and other adaptors Phosphatases - remove tyrosines, etc. SHP-1 and others

Other types of receptors in lymphocyte biology G-protein coupled receptors (GPCR) e.g. chemokine receptors Jak/Stat signaling cytokine receptors Toll-like receptors (DC activation, etc.) TNF receptor family TNFr, CD40, Fas (co-stimulation; death) TGF- Receptor (Treg/Th17 development/function)

GPCR Signaling DAG PLCb PI4,5P2 ATP Ca2+ (CREB, etc.)

Some Toll-Like receptors and their ligands

Major Signaling Pathways Activated by TLR’s and IL-1R MyD88-Independent Pathway MAPKs

Cytokine Receptor Signaling that bind

TNFr family - death inducers

TNFr family - co-stimulators ligands co-stimulators

TNFr family - proximal signaling Downstream pathways: MAP Kinases + AP-1 IKK’s + NF-κB anti-apoptotic Caspases (apoptosis)

TGF- Receptor Signlaing