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