Cellular interactionsin the immune system.

Dr. C. PiccirilloCanada Research Chair

Department of Microbiology & ImmunologyMcGill University

MIMM-414ALecture 4- Nov. 17, 2006

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CC

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p56 lck

peptide

CD3CD28

CD40

LFA-3

LFA-1

ICAM-1MHC IIB7

CD40L

CD4

CD45

TCR

CD2

APC

Molecular Interactions between T Cells and APC

B7

CTLA-4

VLA-1

Collagen

CD80/CD86

T cell

T cell homing to secondary lymphoid tissues

Active recirculation between blood, lymphoid andNon-lymphoid tissues

HEV of T cell areas of LNCD62L dependent

Recirculate approx. 2-3/day.

Under inflammed conditions, T cells are retained forApprox. 2 days.

Stop signal for T cell migration:

Ca+ dependent, extent of polarity of T cells for APCDependent on specific molecular patterns at the interface

Between T/APCICAM-1 dependent

Immunological Synapse (I.S)

Concept of Immunological synapse

Concept philosophically derives from neurobiology

Initially described in the context of cytokine secretion and Th differentiation

• Cellular proximity and [cytokines].

Major research effort to track individual molecules and image immune response in real-time.

Visualization of immune synapses

• Fixed T-DC samples– Antibody stain and confocal microscopy

• Live cell imaging– In vitro: GFP-tagged protein expressed– In vivo: confocal or 2 photon laser

scanning microscopy (TPLSM)

Different types of Immunological synapses

Functions may vary:1. Directing secretion2. Integrating positive and negative signals3. Timing, duration, and strength of a response.

Distinct stages with distinctMembrane and cytoskeletal requirements

• Assembly of the IS can occur in different circumstances for a variety of functions:– T cell motility– Antigen recognition: TCR threshholds

• Establishing checkpoints for T cell activation• Balancing signaling

– Antigen sensing: specificity versus affinity– T cell activation and suppression

• Enhancing signaling• Termination signaling and effector functions

– Th cell differentiation : IFNR vs IL-4R– Directed secretion: eg; granules in CTL.

Functions of the Immunological synapse

Supra-Molecular Activation Cluster (SMAC)

ImmaturePeripheral SMAC

pSMAC

MatureCentral SMAC

cSMAC

5 m

Cross-sectional view of Immune synapses.Many players

Pre-contactInitial TCRTriggering(clusters)

Cytoskeleton-directedRe-organization

cSMAC

Forest of giants and rigidity followed by fluid membrane dynamics.

Mechanisms and structures of basis immune synapses.

Role for the cytoskeleton in directing the TCR in cSMAC

• TCR accumulation in cSMAC is dependent on actin cytoskeletal processes, likely controlled by the ezrin-radixin-moesin (ERM) family of cytoskeletal proteins.

• The ERM cytoskeletal proteins are regulate T cell “shape” and T cell activation.

• ERM proteins act as crosslinkers between the actin filament network and the plasma membrane, operating through their binding to the cytoplasmic regions of integral membrane proteins like CD43 in T cells.

• Ezrin and moesin stably anchor CD43 outside of the synapse at the mature stage (about 30 min after the initial T cell-APC contact in the presence of antigen). The crosslinking function of ERM is regulated through conformational changes controlled by threonine phosphorylation on a motif conserved in all 3 members of the ERM family.

• ERM molecules are highly phosphorylated in resting T cells but are inactivated with respect to their membrane protein-actin cytoskeletal crosslinking capacity by dephosphorylation within the first minute of TCR triggering. Alteration in ERM function after early TCR signaling contributes to T cell shape change during I.S. formation by reducing tethering of the lipid bilayer to cytoskeleton, allowing the T cell to more readily accommodate its surface membrane shape to that of the APC.

• Transient interaction of TCR with pMHC in pSMAC - Ag sensing

• Sustained interaction of TCR with pMHC in cSMAC.- Ag recognition and activation

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TCR/pMHC in cSMACreal-time imaging

Development of immunological synapses in naïve T cells.

Immune synapses, downstream mediators and T cell activation.

Seconds

Minutes

MatureIS

Many minutes

T cell signals precede IS formation

Lck and ZAP-70 are activated at the periphery of the immunological synapse.

A mature immunological synapse is not required to initiate T cellactivation, but appears to form as early as signaling is waning.

Ca+2 responses in activated T cells

T cells loaded with a Ca++ sensitive intracellular dye are added to the fibroblasts, on which the peptide-loaded MHC molecules are shown in blue.

When initial antigen recognition occurs, the T cells change color from yellow to green as the intracellular Ca++ concentration suddenly increases.

Following antigen recognition, MHC molecules accumulate in the contact region between the T cell and the APC, forming an immunological synapse

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Stages in IS

formation

IS always necessary for T cell function?

• CTL cytotoxicity can be induced at [Ag] too low to trigger other responses (IFN and CD3 internalization).

• At these [Ag], killing precedes IS formation• Thus, at least some aspects of mature IS are

necessary for T cell function.

Time course of phosphotyrosine activation in T cell-APC conjugates.

Two hours of conjugate formation is sufficient for T cell activation.

Naïve T cells were loaded with CFSE and then conjugated with antigen-loaded APCs. Cell conjugates were disrupted. T cells were reisolated and recultured with unloaded APCs.

After 72 hours, T cell proliferation was determined by FACS analysis CFSE staining of T cells. Panel 1, disrupted after 1 hour.

Panel 2, disrupted after 1 hour, 30 min.

Panel 3, disrupted after 2 hours.

Panel 4, disrupted after 2 hours 30 min.

Panel 5, disrupted after 4 hours.

Panel 6, disrupted after 6 hours.

Synapses in T cell activation and function.

Mechanism of CD4+CD25+ regulatory T cell function ?Requirement for cognate cell interactions?

• Requires TCR engagement

• Cytokine independent

• Cell-cell contact dependent• Co-stimulation/APC independent• T-T suppressor synapse

• Antigen non-specific suppression of effector functions

• Suppress IL-2 mRNA in T cells • Proliferation• Inflammatory cytokines• Differentiation

• Effector molecules are unknown.

T cellEffector

CD4+

CD25+

Pathways to T cell suppression

Antigen Presenting CellCo-stimulation

Adhesion

XA

B

• CD4+CD25+ cells modulate co-stimulation?• MHC class I/II, B7.1/2, CD40 expression is unaffected.• Suppression is still operative with fixed LPS blasts. • Not overcome with numbers of APCs.

CD4+CD25+ mediated suppression is contact-dependent.

Requirement for APC?

Piccirillo et al. J.Immunology 167:1137-1140.

In vitro suppression is APC-independent?

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Proliferation CytokineCo-stimulation independent

Responder Tg CD8+ T cells

CD4+CD25+ Regulatory T cells

Class I TetramersH-2Kb/

OVA257-264

nTregTeff cell

Antigen Presenting Cell

CD4+CD25+

CD4+ Teff

APC

CD4+

CD25+

CD4+ nTreg cell cognate interactions in suppressor function.

Suppressor Synapse

X

Imaging of Treg “suppressor synapses”?

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T-T suppressor synapse model