1. Cytotoxic T Lymphocyte Antigen-4 Accumulation in the Immunological Synapse is Regulated by Signal Strength Jackson G. Egen and James P. Allison Howard Hughes Medical Institute Department of Molecular and Cell Biology Cancer Research Laboratory University of California, Berkeley

2. Co-Stimulatory Molecule Function Signal 1: TCR + Co-receptor with MHC+peptide  Ligation of T cell receptor and co-receptor does not stimulate naïve T cells to proliferate and differentiate on its own  REQUIRE COSTIMULATORY SIGNALS  Signal 2: Co-stimulatory signal from antigen presenting cell Activating co-stimulatory signals promotes synthesis of IL-2  Required for T cell proliferation and differentiation into effector cells Antigen recognition in absence of co-stimulation results in anergy (inactivation of naïve T cells) Co-stimulatory molecules can also counteract signals provided by the T-cell receptor (inhibitory) Janeway et al., 2001

3. Co-Stimulatory Molecules: Antigen Presenting Cell T Cell Current Opinion in Immunology 2004, 16:321–327

4. B7 Molecules: B7.1 (CD80) and B7.2 (CD86) Homodimeric members of the Immunoglobulin superfamily Principal costimulatory molecules found on professional antigen- presenting cells Bind to CD28 on T cell surface  CD28 constitutively expressed on both naïve and activated T cells Upon ligation with CD28, it leads to:  Enhancement of T cell proliferation  Increased production and stability of IL-2 mRNA  Upregulation of anti-apoptotic genes (ie. Bcl-X L ) Janeway et al., 2001

5. Alternative receptor of B7 molecules: CTLA-4 Cytotoxic T Lymphocyte Antigen-4 (CD152) Similar to CD28 in amino acid sequence Binds B7 molecules with higher avidity than CD28 (by 2500 fold) Protein not detectable in naïve T cells, but upregulated upon T cell activation  Targeted to the endosomal compartment through association of clathrin coated pit adaptor protein (AP-2) and intracellular tail of CTLA-4 (tyr.-based localization motif) Delivers an inhibitory signal to stimulation by APC  Reduces production of IL-2, cyclin D3, cyclin-dependent kinase 4, 5.: Limits T cell expansion Janeway et al., 2001

6. Balance between activating signals from CD28 and inhibitory signals from CTLA-4 determine outcome of a T cell’s interaction with an APC Temporal and spatial localization of proteins are ways of regulating this balance. Therefore, protein trafficking is thought to be important:  Although CD28 and CTLA-4 are homologues, they have different expression patterns and localization  Trafficking of CD28 and CTLA-4 occurs by cytoskeletal rearrangements that occur upon T cell stimulation  Upon T cell stimulation, the microtubule organizing centre reorient facing the contact site and protein accumulates at contact site (formation of imm. synapse)  PURPOSE OF EXPERIMENT:To investigate the trafficking characteristics of CD28 and CTLA-4 to gain insight about how and when these molecules exert their regulatory function on T cell activation

7. Figure 1: Localization of CD28 and CTLA-4 in Migrating T Cells T cells were stimulated for 5 days with irradiated syngenic splenocytes pulsed with residues 88- 103 of moth cytochrome c (agonist peptide). Cells were stained with antibodies against tubulin and A) CD28 or B) CTLA-4. The color overlay images shows tubulin in green, CD28 or CTLA-4 in red and a nuclear stain in blue. Question: What are the localization patterns of CTLA-4 and CD28 in activated T cells?

8. Results: CD28 was evenly distributed throughout the plasma membrane of T cell CTLA-4 primarily found around the MTOC and in the uropod CD28 found at the leading edge of the cell CTLA-4 appeared to be sequestered in intracellular compartment facing away from leading edge Significance: Localization of proteins may function in determining the kinetics of signaling  Allow CD28 to quickly interact with B7 molecules  Delayed CTLA-4 localization due to dependence on TCR-mediated cytoskeletal reorganization MTOC Leading edge Uropod

9. Figure 2: CTLA-4 Has a Rapid Rate of Protein Turnover Activated 5C.C7 TCR transgenic T cells were treated with cycloheximide and/or ammonium chloride. At the indicated time points, cells were permeabilized and stained with antibodies specific for A) transferrin receptor, B) CD28 or C) CTLA-4 Question: What is the turnover rate of CD28 and CTLA-4? Transferrin and CD28 expression levels remained relatively constant

10. Figure 2: CTLA-4 levels ↓ in the absence of new protein translation, accumulation of CTLA-4 when lysosomal degradation is inhibited. Significance: Rapid turnover of CTLA-4 ensures that the level of protein expression is linked to the rate of gene transcription/translation

11. Antigen-Induced Localization of CD28 and CTLA-4 to the T Cell-APC Interface Approach: Mix activated TCR transgenic T cells with B cell pulsed with HB (null) or MCC (agonist) Mix activated TCR transgenic T cells with B cell pulsed with HB (null) or MCC (agonist) Immunofluorescence staining Immunofluorescence stainingConclusion: Upon agonist stimulation, MTOC reorganizes MTOC reorganizes A population of Intracellular CTLA-4 polarizes to a site facing contact siteA population of Intracellular CTLA-4 polarizes to a site facing contact site PKCθ, CD28 and a population of CTLA-4 accumulate at T cell-APC interface PKCθ, CD28 and a population of CTLA-4 accumulate at T cell-APC interface

12. Approach: Immunoflurescence co-staining with PKCθ and CD28/CTLA-4 Results: A population of CTLA-4 localizes on T cell surface at immunological synapse independently from its intracellular polarized population To better localize CTLA-4 population at the synapse …

13. Kinetics of CTLA-4 and CD28 Localization to T Cell-APC Interface Upon Stimulation Retrovirally infected activated TCR transgenic T cell with CD28-GFP and CTLA-4-GFP Live cell fluorescence microscopy: GFP/brightfield time-lapse

14. Model: Trafficking of CTLA-4 Intracellular polarized CTLA-4 B7 CD28Surface CTLA-4 APC T cell Non-polarized CTLA-4 As APC and T cell interact: CTLA-4 rapidly localize to a site facing contact interface from uropod CTLA-4 rapidly localize to a site facing contact interface from uropod intracellular polarized CTLA-4 translocates to surface synapse intracellular polarized CTLA-4 translocates to surface synapse  Regulatory check point to control CTLA-4 induced inhibitory signal

15. Hypothesis: CTLA-4 and CD28 trafficking to synapse depends on TCR signal strength Step 1: Identify peptides that elicit different T cell response Test MCC and its variants for : IL-2 production T cell-APC conjugates formation Results: Identified 2 agonists and 2 weak agonists for further experiments

16. More on MCC and its variants Conclusion: Both agonist peptides and weak agonist peptides were equally capable of inducing MTOC reorientation Test their ability to reorganize MTOC

17. Step 2: test effect of TCR signal strength on surface CTLA-4 expression Approach: stimulate naïve TCR transgenic T cells with MCC, restimulate with different peptides along with APC stimulate naïve TCR transgenic T cells with MCC, restimulate with different peptides along with APC immunofluorescence staining and flow cytometry immunofluorescence staining and flow cytometryMust: use10uM peptide to ensure constant degree of conjugate formation for all use10uM peptide to ensure constant degree of conjugate formation for all measure total CTLA-4 to ensure constant protein synthesis for all measure total CTLA-4 to ensure constant protein synthesis for allConclusion: CTLA-4 expression on T cell surface is proportional to the TCR signal strength

18. Figure 7. TCR signal strength determines translocation of CTLA-4 to T cell-APC interface

19. Summary of Results CD28CTLA- 4 Localization in activated T cells Evenly distributed throughout the plasma membrane Primarily found near the MTOC and in the uropod Turnover rate in activated T cells Relatively slowRapid Localization upon antigen-specific T cell interaction with APC Detected on entire surface of T cell but most concentrated at center of T cell- APC interface Polarization of intracellular CTLA-4 to contact site + accumulation on the surface of interface Kinetics of localization to T cell- APC interface Almost instantaneousPolarization follows the kinetics of MTOC reorientation Effect of TCR signal strength on trafficking NoneStronger TCR signals induce higher CTLA-4 surface expression

20. Discussion  Intracellular retention of CTLA-4 may serve as a checkpoint to spatially and temporally regulate its function as an inhibitor of T cell activation  Different expression and trafficking patterns between CD28 and CTLA-4 are potentially important for regulating the balance between the activating and inhibitory signals upon T cell-APC interaction

21. Dynamic nature of CTLA-4 expression in activated T cells Intracellular CTLA-4 levels are proportional to its rate of protein synthesis (due to rapid turnover)  arresting its gene transcription/translation may quickly downmodulate CTLA-4-mediated inhibitory signals T cell-APC interaction (both TCR signaling and B7 engagement) could alter the turnover of both CD28 and CTLA-4  another possible regulatory mechanism for controlling protein expression levels

22. Trafficking of CTLA-4 during T cell-APC interactions Translocation of CTLA-4 to the immunological synapse is favored under stronger TCR signals Possible mechanisms of this correlation are: a) stronger signals inducing stronger, more sustained calcium fluxes- calcium ionophores are known to upregulate CTLA-4 surface expression by increasing its rate of export to the surface from intracellular compartments b) TCR-mediated protein tyrosine kinase actvity phosphorylating tyrosine in CTLA-4 ’ s AP2-binidng site, thereby inhibiting its endocytosis and thus increasing expression of functionally relevant receptors on the surface of immunological synapse Modulation of composition of immunological synapse by TCR signal strength would provide a means to selectively activate and/or maintain specific T cell signaling pathways

23. TCR regulation of CTLA-4-mediated inhibition: from the single cell to the population Preferential CTLA-4-mediated inhibition of high TCR signals  attenuation of affinity maturation  greater diversity in the T cell response This could: a) improve immune system to respond to mutated or heterologous antigens derived from pathogens b) allow strength of TCR signals to dictate the functional nature of the Ag-specific response (e.g. cytokine expression profile of a particular T cell) c) elaborate a protective T cell response by preventing oligo or even monoclonal responses to a complex set of Ags derived from a pathogen

24. The Message This study provides the basis for : 1) novel feedback control mechanism where a stimulatory signal regulates the recruitment of an inhibitory receptor to a functionally relevant site on the cell surface 2) previously unrecognized function of CTLA-4 – expanding the diversity of a T cell response by restricting the expansion of T cells receiving stronger stimulation