• M. Carrie Miceli• May 4, 2005

• TCR Signal Transduction and Membrane Dynamics

• Reading • Huppa, J. B., Davis, M. M. T-cell-antigen

recognition and the immunological synapse. Nat Rev Immunol 3: 973-83 (2003)

• Behind the scenes of anergy: a tale of 3 E3s, Davis and Ben-Neriah, Nature Immunogy March 2004 5:238

• "In the context of immune responses, the most critical feature of the cell is its surface. The surface of a cell is not smooth and flat, like a ball bearing; it is more like a microscopic garden tended in darkness, bathed by warm salty fluid, a rounded and shaggy convex landscape with cellular vegetation waving like seaweed above the cell membrane. This vegetation represents the aboveground tendrils of proteins and other molecules, which are anchored in the membrane. These are the eyes, the ears, the taste buds, the nerve endings of the cell. At any given time, the surface of a typical cell may be studded with thousands of different tendrils. But perhaps the hardest concept to bear in mind, sometimes even for scientists, is that this moist, shaggy landscape is dynamic. It is constantly changing. New vegetation shoots up and old vegetation collapses as in in a time-lapse film. In real time, these proteins bloom and shrivel, unfurl and fold up, in the course of hours, sometimes minutes, depending on what's going on in the immediate neighborhood, because cells are exquisitely sensitive, and constantly reacting, to their local ecology. Certain receptors, like perennial flowers, grow on the surface, so permanent and unchanging a fixture of the cellular landscape that they can in essence serve as a reliable molecular landmark, or fingerprint, that reveals the identity of the cell itself. Indeed, they are known as the clusters of differentiation...."

• From "A Commotion in the Blood: Life, Death and the Immune System" by Stephen S. Hall. Henry Holt, 1997 ISBN 0-8050-5841-9

Allen group and Germain group both publish in cell

Agonists processively phosphorylate zeta to the pp21 (pp23) isoform; ZAP 70 is associated and phosphorylated (activated)

Antagonists/partial agonists induce pp18 (21) isoform and then stall; ZAP-70 associates but doesn’t get phosphorylated (activated)

Anti-pY western total cell lysates big small

Hb agonist

Gln, ser 79 pa/antag

ppZAP70

pp

TH1 T cells clones

Is the TCR must be dimerized/multimerized and undergo a conformational change to signal?

Alam…..Travers PJ. "Qualitative and quantitative differences in T cell receptor binding of agonist and antagonist ligands. Immunity, 1999 Feb, 10(2):227-37."

• Boniface JJ……..Davis MM. Initiation of signal transduction through the T cell receptor requires the peptide multivalent engagement of MHC ligands. Immunity, 1998 Oct, 9(4):459-66.– Using recombinant MHC/peptides, monovalent or multimerized

demonstrate that MHC/peptide monomers are not sufficient• Single peptide MHC is enough on the surface of an APC (davis,april

NI 2004)• Role for partial agonists/antagonists helping? • Role for costimulators/adhesion molecules• Contraints imposed by mimbrane micoenvironment

Kinetic proofreading models of T cell activation; it takes time

The engagement of peptide MHC does not immediately lead to TCR triggering because a series of phosphorylation steps followed by recruitment and

activation of ZAP70 need to be performed, a process that requires time. If the duration of the interaction is sufficient, the phosphorylation and docking events will proceed until the fully active complex is assembled and can

transmit a signal to downstream pathways. In contrast a premature dissociation before the process has been completed will lead to the formation of inactive intermediates that by sequestering substrates inhibit activating by agonists

resulting in TCR antagonism. Alternatively, intermediates generated might deliver an intermediate or negative signal , thus explaining partial agonism and

antagonism.

Increases Fidelity

Allows for discrimination of small differences in off rates/affinity.

p18 pp21 pp23

TCR Chain Undergoes a Series of OrderedPhosphorylation Events Upon TCR Ligation

Antagonists Agonists

ZAP-70 recruitment, But no activationT cell inactivation

ZAP-70 recruitment,Complete activationT cell activation

Lat functions as an adapter to link TCR to multiple downstream pathways (Annual review of Immunology 2002, Samelson)

LAT is multiply phosphorylated, different tyrosines adapt to different signaling pathways…is this a site of control? Could degree of processive phoshorylation control functional outcome?

2) Naïve T cell activation requires sustained and continual receptor engagement: 6-12 hours of APC:T cell contact.

• Premature distruption of an agonist signal-> aborted signal. Lanzavecchia

• Whereas the earliest TCR signals ( chain phosphorylation), ZAP-70 activation happens within seconds to minutes of receptor engagegment , sustained engagement is required to for T cell activation.

• 3) The TCR is serially engaged. Also Lanzavecchia The finding that TCR engagement results in its internalization in an antigen dose and time dependent fashion provided investigators with a readout as to the number of TCR that had been engaged. Using TCR internalization as a readout of TCR engagegment and by accurately labeling and counting the number of receptors that were internalized it was determined that few (100 per APC) agonistic peptide-MHC complexes engage and trigger a much larger number of TCRs (2000-18,000).

• Andrey S. Shaw and Michael L. Dustin . Making the T Cell Receptor Go the Distance: A Topological View of T Cell Activation Immunity 1997 6: 361-369

• It needs a space

Topologic model of T cell activation; it needs a space• Emphasizes the requirements for the rearrangement of membrane

proteins at the area of contact between the T cell and APC in addition to TCR engagement for successful T cell activation.

• Liganded engagement of short similarly sized molecules are proposed to concentrate at the activation cap while taller heavily glycosylated are proposed to relocate outside the area of contact

• The actin cytoskeleton is proposed to play a role in affecting this molecular reorganization.

• Such interactions are proposed to increase the local 2D affinity by concentrating T cell activation molecules/transducers within the contact cap and increasing local membrane rigidity and signal procession.

• Coincident with recruitment of T cell accessory molecule and the exclusion of CD45 is the recruitment of associated intracelluar tyrosine kinase activity and the exclusion of phosphatase activity.

The T Cell:APC contact site is organized into the cSMAC and pSMAC (Monks CR et al, Nature 1998 395:82-86)

The immune synapse partitions into cSMACs and pSMACS

• Supramolecular activation complexes• Central cSMAC

– TCR/CD3– Pep/MHC– CD28/CD80– PKC theta– Lck/Fyn– Lipid rafts???

• Peripheral SMAC– LFA-1 (ICAM-1)– Talin

• Even further out CD45 -D-SMAC (distal) and CD43

The Immunological Synapse: A Molecular Machine Controlling T Cell Activation Science 1999 July 9; 285: 221-227. Arash Grakoui, 1 Shannon K. Bromley, 1 Cenk Sumen, 2 Mark M. Davis, 2 Andrey S. Shaw, 1 Paul M.

Allen, 1 Michael L. Dustin 1*

The specialized junction between a T lymphocyte and an antigen-presenting cell, the immunological synapse, consists of a central cluster of T cell receptors surrounded by a ring of adhesion molecules. Immunological synapse formation is now shown to be an active and dynamic mechanism that allows T cells to distinguish potential antigenic ligands. Initially, T cell receptor ligands were engaged in an outermost ring of the nascent synapse. Transport of these complexes into the central cluster was dependent on T cell receptor-ligand interaction kinetics. Finally, formation of a stable central cluster at the heart of

the synapse was a determinative event for T cell proliferation.

Quicktime movie= synapse live

TCR transgenic T cells plated on lipid bilayer reconstituted with fluorescent MHC/peptide and ICAM-1 (LFA-1 ligand)

Figure 2. Immunological synapse formation and MHC-peptide dose. 2B4 T cells on Oregon green Ek-GPI loaded with different peptides and Cy5 ICAM-1-GPI at 200 molecules per square micrometer. Images show accumulated MHC-peptide (green) and ICAM-1 (red). (A) to (E) Ek(MCC88-103) agonist at 80 molecules per square micrometer. (A) Bright-field image; (B) IRM image; (C) Ek only (green); (D) ICAM-1 only (red); (E) Ek and ICAM-1 overlay. (F) Ek(MCC88-103) agonist at 0.6 molecules per square micrometer Ek plus ICAM-1 overlay. (G) Ek(MCC88-103 K99A) null at 80 molecules per square micrometer Ek plus ICAM-1 overlay. (H) Dose-response for Ek(MCC88-103) agonist for T cell proliferation on bead-supported bilayers (41, 42). (I) Dose-response for Ek(MCC88-103) agonist for cluster formation at 30 min. The asterisk indicates no immunological synapse formation. Original image elements = 0.018 µm2. Data are representative of two

experiments.

Agonist peptide induces SMACs

Antagonist peptide doesn’t induce SMACs

30 min post antigen

proliferationdose response

Wulfing and Davis Dec 1998 Science

• Peripheral T cells from TCR transgenic loaded with Ca+ sensitive dye.

• APC pulsed with agonist antigen• Beads coated with strepavidin (T cells are biotinylated)• Within 4 minutes of CA+ flux get repolarization of

membrane toward the T cell:APC interface.• Beads are dragged the interface as a result of reversal of

polarity.• Requires presence of B7 and/or ICAM on APCs

(ligands for CD28 and LFA respectively).• The sequel: requires actin:myosin motor

• Synapse Live Quicktime move•TCRs intially engaged at edgesof contact site and then cluster centrally

• How is a synapse constructed?•Wulfing movie•Reversal of polarity •Synaptice membrane recruitment

•Does it function in signal transduction?

Figure 3. CD28-induced raft redistribution to the site of TCR engagement. (A to C) Resting T cells were stained with FITC-CTx and analyzed by confocal microscopy. (A) Unstimulated resting T cells. (B) T cells stimulated for 20 min with beads coated with anti-CD3 alone (10 µg/ml) and (C) stimulated for 20 min with beads coated with anti-CD3 (10 µg/ml) plus anti-CD28. Representative data are from one of six experiments. (D and E) Redistribution of rafts induced by costimulation. (D) Surface staining by FITC-CTx at various times after stimulation of T cells with beads coated with anti-CD3 alone () or anti-CD3 plus anti-CD28 (). () Unstimulated control cells. (E) Surface (filled bars) and total (open bars) FITC-CTx staining in live and permeabilized cells, respectively.

T Lymphocyte Costimulation Mediated by Reorganization of Membrane Microdomains Antonella Viola, Susanne Schroeder, Yoichi Sakakibara, and Antonio Lanzavecchia Science 1999 January 29; 283: 680-682.

Lipid Rafts/Membrane Microdomains

Enriched in GPI-linked proteins (the long and saturated acyl chains of their carboxyterminal GPI-lipid modification exhibit typical features of lipid raft components).

Enriched in doubly acylated Src family members with fully saturated fatty acids including Lck and Fyn.

Enriched in other signal transduction molecules including Ras, heterotrimeric G proteins, nitric oxide synthase, LAT, phosphatidylinositol-(4,5)-biphosphate and sphingomyelin.

Enriched in actin, actin binding proteins and Rho, Rac, Cdc42 implicated in cytoskeletal communication.

Specifically exclude CD43 and CD45 and contribute to the partitioning of phosphatase and kinase activities within T cell membranes.

“Lipid Rafts" are composed primarily of sphingolipids (including the glycosphingolipid GM1) and cholesterol and float as laterally associated units within in an otherwise glycerophospholipid-rich plasma membrane.

LAT

Lck

CD48

CD48

APC Surface

lck

CD2Class II

MHC

T Cell SurfaceGPI

TCR

CD48 is Expressed on the Surface of T cells and is a Ligand for CD2

CD48/TCR Costimulation(Moran and Miceli, Immunity 1998)

• CD48/TCR coengagement enhances TCR tyrosine phosphorylation and cytoskeletal association– requires intact lipid rafts – requires the actin cytoskeleton

• CD48/TCR coengagement induces F-actin redistribution and early morphological changes

• CD48/TCR costimulation enhances TCR induced IL-2 production

Therefore:– Costimulators can enhance TCR signals by recruiting

lipid rafts to the contact site.

– Lipid rafts function to integrate TCR engagement and cytoskeletal reorganization at the contact cap.

TCR/CD48stimulation

CD48 CD48CD48

CD45

CD43

Lck Lck

Fyn

TCR

F-actinF-actin

G-actin

CD43

CD45

Fyn

CD48

Lck Lck

TCR

Modified topological model which included contributions of lipid raftsCostimulators (CD48 and CD28) function to organize the contact cap through

reorganization of membrane microdomains.

Lipid Raft Fractionation

200,000gcentrifugationLysis:

non-ionic detergent

(1% Brij-58)Cell lysate in 40% sucrose

Sucrose

5%

30%

Lipid Rafts/DIGS

Non-Rafts

Detergent insoluble glycoclipid membranes (DIGs) are a biochemical approximation of lipid rafts

Membrane Compartmentation Is Required for Efficient T Cell

Activation, Xavier….Seed; Immunity, June 1998

A) western blots demonstrating membrane microdomain localization of various proteins. B) Anti-phosphotyrosine analysis of proteins in the cytoplasmic membrane and DIG (raft) fractions of Jurkat cells before and after treatment with anti-TCR antibody

Xavier et al Immunity 1998

TCR antigen receptor complex localizes to DIG/Raft Compart-ment following TCR activation

Anti-CD3 mediated redistribution of Signaling molecules

•Zhang…. Samelson LAT Palmitoylation: Its Essential Role in

Membrane Microdomain Targeting and Tyrosine

Phosphorylation during T Cell Activation Immunity

1998 9: 239

Wt Lat partitions within lipid rafts (GEMS),

C26/29A; C26A and C29A palmitoylation mutants don’t. Mutants can’t be tryosine phosphorylated.Without LAT phosphory-lation, no T cell activation

The Lck SH3 Domain is Required forPolarized Lipid Raft Clustering at the TCR Contact Site

Patel, Moran, Low and Miceli, JI 166(2):754, 2001

neoCD3

F505CD3

neoCD3 + CD48

F505CD3 + CD48

YLDY/F505CD3 + CD48

AF/F505CD3 +CD48

LckF505 second site SH3 mutation

does not affect:

Induction of protein tyrosine phosphorylation

apoptosis does affect:

Sustenance of protein tyrosine phosphorylation

Raft clustering at the TCR contact

IL-2 production

Costimulator shortening of TCR engagement requirements for IL-2

A Two Signal Model for T Cell Activation Reliant on Distinctly Regulated Raft-Mediated Signals One and Two

Synaptic raft clustering requires: Lck SH3 Patel et al JI 2001; MAGUK scaffold Dlgh1 Round et al J Exp Med 2005; WASP Dupre et al Immunity, 2002

Cbl-b ablation lifts the requirement for CD28 in activation and enhances synaptic raft/TCR clustering through WASP Krawczyk…Penninger Immunity 2000

Immunity, Vol 17, 809-822, December 2002Dynamics of p56lck Translocation to the T

Cell Immunological Synapse following Agonist and Antagonist Stimulation

Lauren I. Richie Ehrlich1, Peter J.R. Ebert1, Matthew F. Krummel , Arthur Weiss, and Mark M.

Davis• Lck-GFP Translocates to the Immunological Synapse following

Agonist but Not Antagonist Stimulation in D10 T Cells (from intracellular vesicles)

• To observe lck translocation to the immunological synapse, lck-GFP D10 cells were imaged as they interacted with CA-pulsed CH27 B cells. The T cells were loaded with the calcium indicator dye fura-2 before imaging. Agonist-pulsed CH27 B cells were added to the T cells, and images were acquired for 15 min at 15 s time intervals. At each interval, four images were acquired: first, a differential interference contrast (DIC) image, second and third 340 nm and 380 nm images for calcium analysis, and fourth a 20 µm z stack of GFP images acquired at 1 µm intervals. From this z stack, we generated 3-dimensional (3-D) reconstructions of lck-GFP that were rotated to view the immunological synapse en face. In addition, we followed a central horizontal GFP slice of the cell over time.

Staging and resetting T cell activation in

SMACsBenjamin A.

Freiberg…. Abraham Kupfer Nature

Immunology 3, 911 - 917 2002

CD45 greenLck red

hang together early (3 minutes); by 8 minutes start to

segregate. CD45 outside both SMACs

3 min

8 min

23 min

Green TCR; blue CD45; red talin at 23 minutes

The Immunological Synapse--a Multitasking System P. Anton van der

Merwe and Simon J. Davis Science 2002 February 22; 295: 1479-1480. What is the purpose of the immune synapse?A machine for modulating signals-TCR or-costimulators? A mechanism for polarized secretion

A mechanism for internalization,ligand counting and TCR desensitization

Figure 1. Simplified overview of signals provided by TcR, Cbl-b and CD28. Binding of the TcR to antigen/MHC results in recruitment of Src family kinases such as Lck, followed by recruitment of ZAP-70. ZAP-70 subsequently phosphorylates the membrane-associated adaptor molecule LAT, which forms a complex with the adaptor SLP-76 and the guanine nucleotide-exchange factor Vav1. Calcium mobilization, IL-2 production and SMAC formation depend, in part, on Vav1, which is negatively regulated by the E3 ubiquitin ligase Cbl-b. Costimulation through CD28 represses Cbl-b, permitting activation of Vav1 and the lipid kinase PI3K. This allows Vav1-mediated activation of a molecular complex that induces cytoskeletal changes leading to SMAC formation. Further, Vav1 and PI3K co-operate to localize PKC to the SMAC, where it acts upstream of PLC1 to drive calcium flux and activation of IL-2 transcription factors such as AP-1, NF-B and NF-AT. The Vav1 homologues Vav2 and Vav3 may have overlapping functions with Vav1. Integration of TcR and/or CD28 signals might have several outcomes. Appropriate ones include tolerance to self-Ag and immune response to pathogen; the decision between tolerance and response is influenced by the function of Cbl-b. However, in situations of immune dysregulation, autoimmune attack against self-tissue can proceed. Recent studies suggest that GRAIL and Itch might promote T cell tolerance

Current Opinion in Pharmacology August 2004, Pages 415-422 Rangachari and Penninger

Anergizing conditions upregulate E3 ligases (some sustained NFAT without AP1) 

Figure 4. Upregulation of E3 ligases in T cells subjected to sustained Ca2+ signaling.(a) Upregulation of Itch, Cbl-b and Tsg101 in anergic T cells. D5 cells were left resting (-) or were stimulated (+) with ionomycin (Iono), CsA or both. Cell extracts were evaluated for Itch, Tsg101, Cbl-b and Nedd4 by

immunoblotting, and relative protein expression was quantified (below lanes). (b) D5 cells were left untreated or were stimulated with ionomycin (Iono) or ionomycin plus CsA for 10 h, and expression of Itch, Cblb, Rnf128 (GRAIL) and Plcg1 mRNA was evaluated by real-time RT-PCR, normalized to amounts of mRNA

encoding the ribosomal protein L32. Data represent the average s.d. of the ratio of mRNA expression in ionomycin-treated or ionomycin and CsA-treated to that in untreated cells..

Restimulation of anergized cells induces targeted proteolysis PLC PKC, RasGAP degradation (ubiquitination and lysosomal targeting) and induces

anergy/unresponsiveness

c) Cells were pretreated for 16 h with ionomycin and restimulated for 1 h with anti-CD3, anti-CD3 plus anti-CD28, ionomycin, or PMA plus ionomycin. A) pretreated wth iono w or w/out CSA and then restimulated

(a,b) Primary TH1 cells from 2B4 TCR transgenic mice were left untreated (top rows; control) or were pretreated with ionomycin (bottom rows), then were incubated for 40 min on planar phospholipid bilayers containing Oregon green-labeled I-EK-agonist MCC peptide complexes and indocarbocyanine-labeled ICAM-1. The distribution of ICAM-1 (red) and I-Ek-MCC (green) molecules in T cell-bilayer contact zones was captured at different times. The gray panels in a are interference reflection microscopy images in which cell-bilayer contacts appear as dark areas. We have obtained similar results in more than four independent experiments. (c) Involvement of PLC-1 in synapse stability. Mature T cell synapses were allowed to form, then weak or strong PLC- inhibitors were added. Right, percentage of cells with mature synapses relative to the same cells before the addition of inhibitors.

Calcineurin imposes T cell unresponsiveness through targeted proteolysis of signaling proteins Heissmeyer… Anjana Rao Nature Immunology 5, 255-265 Figure 5. Ionomycin-anergized T cells show decreased stability of the immunological synapse.

Figure 1. A model for destabilization of the T cell synapse due to anergy-induced ubiquitination: 'Unauthorized' APC engagement of TCR in the absence of costimulation induces NFAT-dependent

transcription of 'anergy genes', including those encoding several E3s (Cbl-b, Itch and GRAIL).After subsequent engagement with an 'authorized' APC, the upregulated E3s are activated, targeting SMAC

components (PLC-1 and PKC-) to lysosomal degradation. This might be achieved in three steps: SMAC component recruitment to the endosome mediated by Cbl-b, CIN85 or endophilin; subsequent ubiquitination of SMAC and

endocytic components by Itch and/or GRAIL in the endosome; and trafficking of the ubiquitinated proteins into the lysosome, where the SMAC proteins could be degraded. Protein degradation will eliminate recycling of PLC-1 and PKC- back to the synapse and thus may shorten the lifespan of the synapse. In addition, Cbl-b may compromise SMAC through inhibition of the WASP-Arp2/3 actin polymerization pathway by negative modification of the WASP

activator PI3K and by elimination of PKC- , a negative regulator of WASP inhibitor WIP.

Nature Immunology  5, 238 - 240 (2004) Matti Davis & Yinon Ben-Neriah

Only after restimulation and E3s synaptic recruitment does “defect’ present

THE SYNAPSE AT DIFFERENT STAGES OF T CELL DEVELOPMENT MAY

DIFFER

Lck (and likley raft) distribution differs thoughout development of antigen specific CD8 T cellsBachman…Viola J exp Med

Eric Hailman, W. Richard Burack, Andrey S. Shaw, Michael L. Dustin, and Paul M. Allen Immature CD4+CD8+ Thymocytes Form a Multifocal Immunological Synapse with

Sustained Tyrosine PhosphorylationImmunity 2002 16: 839-848.

The Immunological Synapse of DP Thymocytes

(A) DP thymocytes from 3A9 H-2b RAG-1-/- mice were incubated on lipid bilayers containing fluorescently labeled ICAM-1 (red) and I-Ak-HEL (green).

(B) (B) DP thymocytes from N3.L2 or 3A9, H-2b RAG-1-/- mice were incubated on lipid bilayers containing fluorescently labeled ICAM-1 (red) and I-Ek loaded with agonist peptide (for N3.L2) or I-Ak with covalently linked agonist peptide (I-Ak-HEL, for 3A9). Similar patterning, with multiple areas of exclusion (holes) in the ICAM-1 fluorescence pattern, was seen in >90% of adherent cells in 15 separate experiments.

(C) 3A9 H-2b RAG-1-/- thymocytes were incubated on lipid bilayers containing fluorescently labeled ICAM-1 (red) and I-Ak-HEL (green). The same cell is shown at three time points, with intervals of 2 min between images.

Balamuth, F….Bottomly, K Immunity Nov. 2001Distinct

patterns of membrane microdomain partitioning in Th1 and Th2 cells

• Th1s cluster rafts at synapse, TH2 don’t

• Dsiruption of lipid rafts affects TH1, but not TH2

• Th1 induce SMACs (TCR/LFA-1); TH2 dont

Figure 4. CTL Secretory Lysosomes Insert between the Signaling Molecule Patch and the Adhesion Ring Lck (red) and either CD11a (green) and cathepsin D (blue, [A–

C]), or granzyme A (green) and actin (blue, [D–G]).

• CTL Secretory Lysosomes Insert between the Signaling Molecule Patch and the Adhesion Ring

• CTL-P815 target cell conjugates stained with antibodies against Lck (red) and either CD11a (green) and cathepsin D (blue, [A–C]), or granzyme A (green) and actin (blue, [D–G]). CTL and P815 target cells are shown on the right and left, respectively, in (A), (B), and (E–G). Three separate conjugates are shown (A–C, D–F, and G).

• (A), (E), and (G) are single confocal sections, (B) and (F) are projected confocal sections taken 0.4 É m apart through the sample (shown as 3D image reconstructions in Supplemental Movies S2 and S3), and (C) and (D) are z axis image reconstructions shown in the plane of the contact site. The lytic granules insert to the side of the signaling protein marker (A, B, E, and F) within the adhesion ring (A and B). (C) and (D) clearly show that the signaling and secretory molecules are distributed into two distinct domains within the adhesion ring (green in [C] and shown by the black ring lacking signal in [D]) at the contact site. Note, especially in (B) and (F), that different stages of granule polarization and secretion are happening simultaneously with some granules already inserted between the signaling patch and adhesion ring while others are still polarizing.

• (G) A single confocal section demonstrating granule content protein (green) appearing on the target cell (left) side of the CTL (right) membrane defined by the Lck (red) signal. The scale bars represent 10 É m (A, B, E, and F) and 4 É m (G).

Copyright © 2004 Cell Press.Immunity, Vol 15, 751-761, November 2001

The Immunological Synapse of CTL Contains a Secretory Domain and Membrane Bridges

Jane C. Stinchcombe , Giovanna Bossi , Sarah Booth , and Gillian M. Griffiths

Remaining Questions•What controls alternate synapse assembly/raft clustering in thymocytes vs naïve T cells?•What controls raft clustering and transducer partitioning throughout T cell development

PreTCR constitutively raft associatednaïve T cells have a significant fraction of their rafts contained in intracellular vesicles, whereas as activated effectors release them to the surfaceTH1 are more raft dependent than TH2?

•Can engagement of costimulators/accesory molecules function to modify arrangement within rafts/SMACs?•How does synapse assembly influence costiimulator signaling?•How are effectors specifically routed to the synapse?•How does macromolecular assembly translate into alternate signals (or is this just about receptor internalizationor directed secretion)?•Relationship betwee rats microdaomins and SMACS•More than one flavor of lipid raft microdoamain?Alternate organizers of membrane microdoains (sugar packing)