Cytotoxic T Lymphocyte Antigen-4 Accumulation in the Immunological Synapse is Regulated by Signal Strength
Jackson G. Egen and James P. AllisonHoward Hughes Medical Institute Department of Molecular and Cell BiologyCancer Research LaboratoryUniversity of California, Berkeley
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
Co-Stimulatory Molecules:
Antigen Presenting Cell
T Cell
Current Opinion in Immunology 2004, 16:321–327
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-XL)
Janeway et al., 2001
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
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
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:Question:What are the localization patterns of CTLA-4 and CD28 in activated T cells?
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
Figure 2: CTLA-4 Has a Rapid Rate of Protein TurnoverActivated 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
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
Antigen-Induced Localization of CD28 and CTLA-4 to the T Cell-APC Interface
Approach:Approach:
• Mix activated TCR transgenic T Mix activated TCR transgenic T cells with B cell pulsed with HB (null) cells with B cell pulsed with HB (null) or MCC (agonist)or MCC (agonist)
• Immunofluorescence staining Immunofluorescence staining
Conclusion:Conclusion:
Upon agonist stimulation,Upon agonist stimulation,
• MTOC reorganizes MTOC reorganizes
•A population of Intracellular CTLA-4 A population of Intracellular CTLA-4 polarizes to a site facing contact sitepolarizes to a site facing contact site
• PKCPKCθθ, , CDCD28 and a population of 28 and a population of CTLA-4 accumulate at T cell-APC CTLA-4 accumulate at T cell-APC interfaceinterface
Approach: Approach:
Immunoflurescence co-staining with PKCImmunoflurescence co-staining with PKCθθ and CD28/CTLA-4 and CD28/CTLA-4
Results: Results:
A population of CTLA-4 localizes on T cell surface at immunological A population of CTLA-4 localizes on T cell surface at immunological synapse independently from its intracellular polarized populationsynapse independently from its intracellular polarized population
To better localize CTLA-4 population at the synapse …To better localize CTLA-4 population at the synapse …
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
Model: Trafficking of CTLA-4
Intracellular polarized CTLA-4
B7CD28Surface CTLA-4
APC
T cell
Non-polarized CTLA-4
As APC and T cell interact:As APC and T cell interact:
• CTLA-4 rapidly localize to a site CTLA-4 rapidly localize to a site facing contact interface from uropodfacing contact interface from uropod
• intracellular polarized CTLA-4 intracellular polarized CTLA-4 translocates to surface synapsetranslocates to surface synapse
Regulatory check point to control Regulatory check point to control CTLA-4 induced inhibitory signalCTLA-4 induced inhibitory signal
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 Test MCC and its variants for :variants for :
IL-2 productionIL-2 production T cell-APC conjugates formation T cell-APC conjugates formation
Results: Identified 2 agonists and 2 weak agonists for further experiments
More on MCC and its variants
Conclusion: Conclusion:
Both agonist peptides and weak Both agonist peptides and weak agonist peptides were equally agonist peptides were equally capable of inducing MTOC capable of inducing MTOC reorientationreorientation
Test their ability to reorganize MTOCTest their ability to reorganize MTOC
Step 2: test effect of TCR signal strength on surface CTLA-4 expression
Approach:Approach:
• stimulate naïve TCR transgenic T cells stimulate naïve TCR transgenic T cells with MCC, restimulate with different with MCC, restimulate with different peptides along with APCpeptides along with APC
• immunofluorescence staining and flow immunofluorescence staining and flow cytometry cytometry
Must:Must:
• use10uM peptide to ensure constant use10uM peptide to ensure constant degree of conjugate formation for alldegree of conjugate formation for all
• measure total CTLA-4 to ensure measure total CTLA-4 to ensure constant protein synthesis for allconstant protein synthesis for all
Conclusion:Conclusion:
CTLA-4 expression on T cell surface is CTLA-4 expression on T cell surface is proportional to the TCR signal strengthproportional to the TCR signal strength
Figure 7. TCR signal strength determines translocation of CTLA-4 to T cell-APC interface
Summary of ResultsCD28 CTLA- 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 slow Rapid
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 instantaneous Polarization follows the kinetics of MTOC reorientation
Effect of TCR signal strength on trafficking
None Stronger TCR signals induce higher CTLA-4 surface expression
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
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
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
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
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