1. Cellular interactions in the immune system. Dr. C. Piccirillo Canada Research Chair Department of Microbiology & Immunology McGill University MIMM-414A Lecture 4- Nov. 17, 2006

2.    V  C  C  V    p56 lck peptide CD3 CD28 CD40 LFA-3 LFA-1 ICAM-1 MHC II B7 CD40L CD4 CD45 TCR CD2 APC  Molecular Interactions between T Cells and APC B7 CTLA-4 VLA-1 Collagen CD80/CD86 T cell

3. T cell homing to secondary lymphoid tissues Active recirculation between blood, lymphoid and Non-lymphoid tissues HEV of T cell areas of LN CD62L dependent Recirculate approx. 2-3/day. Under inflammed conditions, T cells are retained for Approx. 2 days. Stop signal for T cell migration: Ca+ dependent, extent of polarity of T cells for APC Dependent on specific molecular patterns at the interface Between T/APC ICAM-1 dependent Immunological Synapse (I.S)

4. 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.

5. 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)

6. Different types of Immunological synapses Functions may vary: 1.Directing secretion 2.Integrating positive and negative signals 3.Timing, duration, and strength of a response. Distinct stages with distinct Membrane and cytoskeletal requirements

7. 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 : IFN  R vs IL-4R –Directed secretion: eg; granules in CTL. Functions of the Immunological synapse

8. Supra-Molecular Activation Cluster (SMAC) Immature Peripheral SMAC pSMAC Mature Central SMAC cSMAC 5  m

9. Cross-sectional view of Immune synapses. Many players Pre-contact Initial TCR Triggering (clusters) Cytoskeleton-directed Re-organization cSMAC Forest of giants and rigidity followed by fluid membrane dynamics.

10. Mechanisms and structures of basis immune synapses.

11. 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

12. TCR/pMHC in cSMAC real-time imaging

13. Development of immunological synapses in naïve T cells.

14. Immune synapses, downstream mediators and T cell activation. Seconds Minutes Mature IS Many minutes

15. T cell signals precede IS formation

16. Lck and ZAP-70 are activated at the periphery of the immunological synapse. A mature immunological synapse is not required to initiate T cell activation, but appears to form as early as signaling is waning.

17. 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

18. Stages in IS formation

19. 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.

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

21. 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.

22. Synapses in T cell activation and function.

23. 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.

24. T cell Effector CD4 + CD25 + Pathways to T cell suppression Antigen Presenting Cell Co-stimulation Adhesion X A 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.

25. In vitro suppression is APC-independent? # CD4 + CD25 - (  ) or CD4 + CD25 + (  ) Tcells CPM Anti-B7.1/2 CTLA-4Ig CPM ProliferationCytokine Co-stimulation independent Responder Tg CD8+ T cells CD4 + CD25 + Regulatory T cells Class I Tetramers H-2K b / OVA 257-264

26. nT reg T eff cell Antigen Presenting Cell CD4 + CD25 + CD4 + T eff APC CD4 + CD25 + CD4 + nTreg cell cognate interactions in suppressor function. Suppressor Synapse X

27. Imaging of Treg “suppressor synapses”? T-T suppressor synapse model