Immunological Synapse

1
Immunological Synapse

• Description What is it?
• Mechanism How does it form?
• Function Why does it form?

2
Initial encounter between T cell and an APC or
target cell
3
Early studies of T cell/B cell conjugates

• Observed
• Polarization of actin and microtubule
  cytoskeletons towards B cell
• Accumulation of certain cell surface molecules at
  the interface
• Polarized secretion of cytokines
• By analogy to neuronal synapses (which show
  polarized exocytosis) this led William Paul to
  term this an Immunological synapse

4
Advances in imaging led to better space and time
resolution

• Higher resolution images possible using
• Confocal microscopy or
• Image deconvolution
• both methods decrease out-of-focus light
• Analysis of molecular movement in live cells
  possible using
• Green (Red/Blue/Cyan) fluorescent protein tags
  (poor spatial resolution)
• Use of artificial planar lipid bilayers into
  which ligands that have been tagged with
  fluorescent probes are presented (excellent
  spatial resolution but highly artificial)

5
Segregation of molecules within the T cell and
APC interface
(Avi Kupfer et al 1998)
6
Supramolecular activation clusters (SMACs)

d-SMAC
7
Figure 8-30
8
Planar bilayer system allows real-time analysis
ligand distribution
(Mike Dustin et al from 1996)









9
Segregation within the immunological synapse
changes over time
from Grakoui et al 1999
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Mature Immunological synapse
T cell
APC
excluded (d-SMAC)
CD43 and CD45
LFA-1
central zone (c-SMAC)
TCR/CD3 CD28 CD2
1 mM
peripheral zone (p-SMAC)
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Immunological synapse (IS) definition
There are varying definitions ranging from the
very broad Any stable contact zone between an
immune cell and another cell at which molecules
accumulate ..to the very narrow classic
IS The above but with cytoskeletal
polarization, segregation of molecules within
zone, and polarised exocytosis In T cells the
segregation pattern changes over time from
immature to mature IS
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IS formation by other lymphocytes

• Initially classic IS formation was observed in
  CD4 T cells
• Now also observed in
• CD8 cells
• NK cells
• Partial IS formation has been observed in
• thymocytes (segregation is atypical and unstable,
  no polarized secretion observed)
• B cells (no segregation or polarised secretion
  observed)

13
Polarized secretion of secretory lysosomes by
cytotoxic T cells
14
Patterns of molecular segregation observed at
lymphocyte interfaces
15
Role of APCs in IS formation

• Initially IS formation was observed using B
  cells.
• Different results obtained when DCs were studied
• T cells showed some response (?Ca2, adhesion,
  IS) even when no Ag presented
• DCs seems to play a more active role
• Polarised movement of MHC II and B7 to IS
• Role for DC cytoskeleton
• Interactions with DCs in vitro seems to be much
  more short-lived (10 min vs hours)
• Still not clear whether the classic IS forms when
  Dendritic cells used as APCs ( more difficult to
  image DCs)
• A recent study of T cell/DC interactions in vivo
  has shown that dynamics of the interaction change
  over time.

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Three phases of naïve T-cell priming by dendritic
cells in peripheral lymph nodes
T.R. Mempel, S.E. Henrickson and U.H. von
Andrian, Nature 427 (2004), p1549.
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Immunological Synapse

• Description What is it?
• Mechanism How does it form?
• Function Why does it form?

18
Mechanisms of redistribution/segregation of
molecules at the cell surface

• Passive (binding and steric factors)
• Active
• lateral movement on the surface
• polarised exocytosis of vesicular stores

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Molecules at the T cell surface vary dramatically
in size
(Tim Springer, 1990)
CD148
CD45
10 nm
LFA-1
ICAM-1
KIR CD80
CD4lck
CD58
MHC II
CD43
CTLA-4 CD28
CD154
CD2
TCR/CD3
tyrosine phosphatase domains
20
CD4 and CD8 co-receptors
CD8??
CD4
21
Comparison of size of TCR and CD2 ligand complexes
Predicted 134 Å Measured 1323 Å
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Effect of lengthening CD2/CD48 complex
Lengthening CD2/CD48 complex inhibits TCR
triggering
TCR triggering
spacer
TCR triggering
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Requirement for segregation according to
ectodomain size
Molecules presumably segregate according to size
at the T cell/APC contact area
ICAM-1
Antigen presenting cell
10 nm
MHC
CD40
CD45
B7
Class II
CD48
CD43
ss
CD4
CD40L
LFA-1
CD28
TCR
tyrosine
CD2
phosphatase
T lymphocyte
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Are passive mechanisms sufficient?

• While sufficient for small scale segregation they
  probably do not account for the large scale
  segregation seen in the IS
• Evidence for active mechanism
• Modelling studies
• Large scale segregation follows TCR triggering
• Co-capping of molecules
• Interactions with cytoplasmic tails which
  modulate movement/segregation
• This implies that active processes are involved
• Cytoskeletally-driven movement on the cell
  surface
• Polarized exocytosis

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Cytoskeletal changes following TCR triggering

• Changes seen
• Accumulation of talin (actin-binding protein that
  binds to integrins)
• Movement of the MTOC to the site of contact
• Polymerization of actin adjacent to the contact
  area (followed by clearing from in the central
  area)
• Extrusion of processes towards the APC (filopodia
  and lamellopodia)
• Many cell surface proteins couple directly or
  indirectly to the actin cytoskeleton
• Movement of cell surface proteins has been shown
  to be dependent on the actin cytoskeleton or
  interactions with actin cytoskeleton

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How does TCR triggering lead to cytoskeletal
changes ?
TCR
LAT
SLP-76
vav
Fyb/SLAP
cdc42
Ena/VASP
WASP
Wiskott-Aldrich Syndrome X-linked. Infection,
Bleeding and Eczema
Arp2/3
Actin branching and polymerization
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Polarized exocyosis

• Several cell surface molecules are transported
  from intracellular vesicular compartments to the
  IS (e.g. CTLA-4, and FasL)

28
Immunological Synapse

• Description What is it?
• Mechanism How does it form?
• Function Why does it form?

29
Functions of Immunological Synapse

• Clearly the IS is a site of bidirectional
  signalling between T cells and APC
• To T cell
• TCR (signal 1)
• Costimulatory receptors (signal 2)
• To APC/target cell
• Soluble or cell surface molecules
• Provide help to APCs (CD4 cells) or kill targets
  (CD8 cells)
• But what is the reason for the molecular
  segregation?

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Function of the IS

• TCR signalling
• Secondary signalling events
• Polarised exocytosis
• TCR down-modulation

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Observations that led to the kinetic-segregation
model of TCR triggering

• Molecules with large ectodomains will tend be
  excluded from the immediate area where TCR
  engages pep-MHC
• Several membrane tyrosine phosphatases that
  inhibit TCR triggering (e.g. CD45 and CD148) have
  very large ectodomains.
• Tyrosine phosphatase inhibitors induce dramatic
  tyrosine phosphorylation and T cell activation,
  indicating that
• Tyrosine kinases (e.g. lck) are constitutively
  active, and continuously phosphorylate their
  normal substrates (e.g. CD3).
• The steady-state level of phosphorylation is low
  largely because tyrosine phosphatases are even
  more active.
• There is a delicate tyrosine kinase/phosphatase
  balance.
• This balance is easily disturbed

32
Kinetic-segregation model of T cell receptor
triggering (I)
Kinetic-segregation model of TCR
triggering (Davis and van der Merwe, 1996)
Resting T cell
CD45
CD4
TCR
CD3
CD2
tyrosine
phosphatase
antigen presenting cell
Antigen
presentation
ITAM Phosphorylation by lck
ZAP-70 recruitment
10 nm
Activation of ZAP-70 by phosphorylation
Phosphorylation of substrates
33
Dual role of CD45 in TCR triggering
Activatory Maintains lck in an primed state in a
resting T cell by dephosphorylating inhibitory
site
TCR
CD3
P
P

• Inhibitory
• Dephosphorylates lck substrates
• Dephosphorylates lck at activatory site

P
P
lck active
lck inactive
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Does segregation in the IS enhance TCR signalling?

• There is exclusion of CD45 from the mature IS and
• a correlation between the mature IS pattern and T
  cell activation
• This suggested that formation of the mature IS
  might TCR triggering
• BUT, analysis of the sequence of events cast
  doubt on this.

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TCR signalling is detected well before central
clustering of the TCR (1)

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TCR signalling is detected well before central
clustering of the TCR (2)
37
Immunological synapse
Immunological synapse formation
Kinetic-segregation model of TCR triggering

• resting T-cell surface

Resting T-cell surface

• T cellAPC contact formation
• passive small-scale segregation
• formation of multiple close-contact zones

tyrosine phosphatases maintain a low level of
phosphorylation
T cellAPC close-contact zone
TCR triggering
APC

• active large-scale segregation
• formation of immunological synapse

T cell
TCR
phosphorylation of TCR/CD3 trapped by pep-MHC
binding in close-contact zone
LFA-1
hrs

• full activation
• commitment

TCR triggering
(van der Merwe et al., 2000)
38
TCR triggering and the IS

• TCR triggering precedes and is maximal well
  before formation of the mature IS.
• Thus mature IS formation clearly does not enhance
  early TCR triggering.
• Does it enhance sustained TCR triggering?
• Sustained (gt2 hrs) TCR signalling is required for
  full activation of naïve T cells
• There is direct evidence for low level sustained
  TCR signalling at mature IS
• Blocking TCR/pep-MHC interaction causes cell to
  move apart
• Sustained TCR signalling may be required simply
  maintain IS for other functions

39
Possible functions of the immunological synapse
Other possible functions of the immunological
synapse
40
Other possible functions of Immunological Synapse

• Secondary signalling events
• Polarised exocytosis
• TCR down-modulation

41
Secondary (non-TCR) signalling

• How would IS enhance secondary signalling?
• Better contact interface
• Active clustering or polarised secretion of
  molecules (TCR, CTLA-4, FasL etc)
• Secondary upregulation of molecules
• Signalling would be bidirectional
• To T cell (e.g. CD28 ligation)
• To APC (e.g. B7, CD40, MHC ligation, cytokines)

42
Other possible functions of Immunological Synapse

• Secondary signalling events
• Polarised exocytosis
• TCR down-modulation

43
Polarised exocytosis.

• ..requires polarization of the actin and
  microtubule cytoskeleton, which is a key feature
  of IS formation
• reduces bystander effects by targeting
  cell-surface and secreted molecules to the cells
  presenting appropriate antigen
• is always accompanied by mature IS formation

44
Several types of material undergo polarized
exocytosis

• Proteins stored in secretory lysosomes (perforin,
  granzymes, fasL)
• Newly-synthesized proteins emerging from the
  trans-Golgi (e.g. cytokines)
• Proteins internalized from the surface that are
  being recycled (e.g. CTLA-4)

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Figure 8-31
46
Figure 9-6
47
Genetic defects in secretion of secretory
lysosomesStinchcombe et al (2004) Science 305
55

• Secretory lysosomes are found in haematopoeitc
  cells and melanocytes
• Mutations in genes in this pathway lead to
  syndromes characterised by
• Immune deficiency
• Albinism

48
Cytotoxic lymphocytes (CTL) killing target cells
Secretory lysosomes (green) are transported
along microtubules to the microtubule organizing
system (MTOC, red)
m mitochondria G golgi
49
Gene defects lead to abnormalities in transport
and release of secretory lysosomes
Chediak- Higashi
Hermansky- Podlack 2
Griscelli 2
SYNDROMES
50
Other possible functions of Immunological Synapse

• Secondary signalling events
• Polarised exocytosis
• TCR down-modulation

51
TCR down-modulation and the ISLee et al (2003)
Science 3021218

• TCRs are constitutively internalised and recycled
• If they have been triggered they are degraded
  following internalization leading to
  down-modulation
• Cells deficient in the cytosolic adaptor protein
  CD2AP do not downimodulate their TCRs
• Cells deficient in CD2AP also show abnormal
  segregation in the IS
• This led to proposal that segregation in the IS
  accompanies TCR down-modulation
• but mechanism unclear

52
Conclusion

• Immune receptor signalling leads to formation of
  a stable contact interface termed the IS
  characterised by
• Accumulation and large-scale segregation of
  molecules
• Polarisation of the cytoskeleton
• The mechanisms underlying IS formation are still
  poorly understood but involve cytoskeletal
  activation
• While the IS is clearly a site of bidirectional
  intercellular communication, the reasons for the
  large scale segregation remain controversial.