Immunological Synapse Description What is it?

Immunological Synapse

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

• Function Why does it form?

Initial encounter between T cell and an APC or target cell

No Ag

Keep moving to another cell

Ag

(<1 min)ArrestScanning

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’

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)

Segregation of molecules within the T cell and APC interface

TCR/CD3 LFA-1

overlay

CD45

(Avi Kupfer et al 1998)

Supramolecular activation clusters (SMACs)

• d-SMAC

Figure 8-30

* * * * * * * * * ** ***

* * * * * * * *

T cell TCR

fluorescently-labelled pep-MHC (lipid-anchored, mobile)

Planar lipid membrane

Planar bilayer system allows real-time analysis ligand distribution(Mike Dustin et al from 1996)

pep-MHC

ICAM-1

Segregation within the immunological synapse changes over time

from Grakoui et al 1999

peripheral zone (p-SMAC)

T cell

APC

CD43 and CD45

LFA-1

TCR/CD3CD28CD2 1 M

central zone (c-SMAC)

Mature Immunological synapse

excluded (d-SMAC)

Immunological synapse (IS) definitionThere 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

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)

Polarized secretion of secretory lysosomes by cytotoxic T cells

Patterns of molecular segregation observed at lymphocyte interfaces

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.

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), p154–9.

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

KIRCD80

CTLA-4CD28

CD58

CD2

MHC IICD4lck

TCR/CD3

LFA-1

ICAM-1

CD45

CD43

CD148

CD154

10 nm

tyrosine phosphatasedomains

Molecules at the T cell surface vary dramatically in size(Tim Springer, 1990)

CD4 and CD8 co-receptors

CD4

CD8

Comparison of size of TCR and CD2 ligand complexes

Predicted 134 Å Measured 132±3 Å

Effect of lengthening CD2/CD48 complexCD2

TcR

CD2 CD2

TcR

CD2

MHC class IICD48 CD48

MHC class IICD48-CD22 CD48-CD22

T CELL

APC / TARGET CELL

1

2

1

2

1

2

11

2

1

1 1

22

22

1 1

2 2

3 3

‘spacer’

TCR triggering

TCR triggering

Lengthening CD2/CD48 complex inhibits TCR triggering

Requirement for segregation according to ectodomain sizeAntigen presenting cell

T lymphocyte

LFA-1

ICAM-1

CD43

CD28

B7

ss

CD2

MHCClass II

CD4

TCR

CD48

CD40L

CD40

10 nm

CD45

tyrosine phosphatase

Molecules presumably segregate according to size at the T cell/APC contact area

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

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

How does TCR triggering lead to cytoskeletal changes ?

vav

cdc42

WASP*

Arp2/3

Actin branching and polymerization

TCR

SLP-76

LAT

Fyb/SLAP

Ena/VASP

*Wiskott-Aldrich Syndrome X-linked. Infection, Bleeding and Eczema

Polarized exocyosis• Several cell surface molecules are transported from intracellular

vesicular compartments to the IS (e.g. CTLA-4, and FasL)

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?

Function of the IS

• TCR signalling

• Secondary signalling events

• Polarised exocytosis

• TCR down-modulation

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

Kinetic-segregation model of T cell receptor triggering (I)

Resting T cell

10 nm

antigen presenting cell

ITAM Phosphorylation by lck

ZAP-70 recruitment

Antigen presentation

Activation of ZAP-70 by phosphorylation

Phosphorylation of substrates

tyrosine phosphatase

CD45

CD2

CD4TCR

CD3

lck tyrosine kinase

Kinetic-segregation model of TCR triggering(Davis and van der Merwe, 1996)

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….

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

•

http://www.sciencemag.org/content/vol295/issue5559/images/large/se0620209003.jpeg


TCR signalling is detected well before central clustering of the TCR (2)


(van der Merwe et al., 2000)

Immunological synapse

• T cell:APC contact formation• passive small-scale segregation• formation of multiple close-contact zones

TCR

LFA-1

• resting T-cell surface

Immunological synapse formation

• active large-scale segregation• formation of immunological synapse

• full activation• commitment

TCR triggering

hrs

tyrosine phosphatases maintain a low level of phosphorylation

Kinetic-segregation model of TCR triggering

Resting T-cell surface

APC

T cell

phosphorylation of TCR/CD3 trapped by pep-MHC bindingin close-contact zone

TCR triggering

T cell:APC close-contact zone

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 (>2 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

Possible functions of the immunological synapse

Other possible functions of the immunological synapse

Other possible functions of Immunological Synapse




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)

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

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)

Figure 9-6

Genetic defects in secretion of secretory lysosomes

[Stinchcombe 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 byImmune deficiency

±Albinism

Cytotoxic lymphocytes (CTL) killing target cells Secretory lysosomes (green) are transported along microtubules to the

microtubule organizing system (MTOC, red)

m mitochondriaG golgi

Gene defects lead to abnormalities in transport and release ofsecretory lysosomes

Chediak-Higashi

Griscelli 2SYNDROMES Hermansky-Podlack 2

TCR down-modulation and the IS[Lee et al (2003) Science 302:1218]

• 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

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.

Immunological Synapse Description What is it?

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