1. 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

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

3. Crosslinking and receptor tyrosine kinases

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

5. 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

6. 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

7. 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

8. 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

9. 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

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

11. Sequential tyrosine kinase activation in B cells

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

13. Cytoplasmic adaptors of lymphocytes

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

15. Transmembrane lymphocyte adaptors
LAB/NTAL B cells

16. 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

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

18. Small G protein activation is aided by Guanine nucleotide Exchange Factors

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

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

21. Three major transcription factors downstream of the TCR
PIP2

22. 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

23. 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

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

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

26. 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 ?

27. 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

28. MIRR = multi-chain immune recognition receptor

29. 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

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

31. 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

32. 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)

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

34. Some Toll-Like receptors and their ligands

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

36. Cytokine Receptor Signaling
that
bind

37. TNFr family - death inducers

38. TNFr family - co-stimulators
ligands
co-stimulators

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

40. TGF- Receptor Signlaing