Mechanism of CTLA-4 Inhibition of Antigen-mediated NFkB Signaling in T Cells

Brian Schaefer, Ph.D.

Uniformed Services University of the Health Sciences

Funded in December, 2004: $120000 for 3 years
LAY SUMMARY . ABSTRACT . BIOGRAPHY . HYPOTHESIS . FINDINGS . SELECTED PUBLICATIONS .

LAY SUMMARY

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How Do Tumors Block the Transmission of Distress Signals to Immune Cells to Prevent Attack?

These investigators will use cellular imaging to define the molecular processes that tumor cells use to block signals that ordinarily stimulate immune T cells to attack the tumor.  The findings could lead to the development of new approaches to therapies that prevent this signal blockade.

Adaptive immune T cells attack invaders, and evidence suggests that T cells also initially recognize tumor cells that take over normal cells.  In both situations, T cell responses are governed by both stimulatory and inhibitory signals.  Scientists speculate that the balance between these two opposing signals is key and that excess inhibitory signaling weakens T cell activation.  The excess inhibitory signaling, leading to failure to stimulate full-scale T cell activation, may emanate from a specific molecule called CTL-4.  Moreover, the investigators hypothesize that redistribution of proteins within this molecule is critical for the release of excessive inhibitory signaling.  The investigators will use fluorescence microscopy to examine this protein redistribution with the CTL-4 molecule.  They also will examine how the CTL-4 exerts its influence on nearby normal cells, inhibiting their distress signals to T cells, using fluorescence resonance energy transfer (FRET)

Significance: These findings may lead to new approaches to developing therapies that are aimed at these CTL-4 molecular targets to quiet their inhibitory signaling, which allows tumor cells to grow unabated by immune attack.

ABSTRACT

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Mechanism of CTLA-4 Inhibition of Antigen-mediated NFkB Signaling in T Cells

The T cell transmembrane molecule CTLA-4 negatively regulates antigen-mediated T cell signals. The cytoplasmic signal transduction cascade that activates the transcription factor NF-kB is a crucial target of CTLA-4 inhibition. In spite of many studies of CTLA-4 activity, the mechanism of CTLA-4 inhibition of NF-kB activation remains largely undefined. Current evidence suggests that CTLA-4 blocks antigen-mediated activation of NF-kB through two distinct mechanisms: physical displacement of the costimulatory molecule, CD28, from the central T cell/APC interaction zone, and delivery of an inhibitory signal that interferes with cytoplasmic signal transduction events in antigen activation of NF-kB. However, CTLA-4 competition with CD28 has not been directly observed, and the molecular mechanisms of CTLA-4 inhibitory signaling are controversial and remain poorly understood.

We propose experiments employing direct microscopic observation of molecular redistribution events in T cells to establish the mechanistic basis of both of the above processes, and to reveal to what extent each mechanism contributes to CTLA-4 inhibition of NF-kB activation. We will employ multiparameter 3-dimensional live cell imaging and analysis of protein-protein interactions by fluorescence resonance energy transfer (FRET) to accomplish these goals. Once completed, these studies will represent a basic science foundation for development of small molecule inhibitors of CTLA-4 signaling, by identifying the potential cytoplasmic targets of CTLA-4 inhibition of NF-kB activation. Such drugs would likely have substantial therapeutic benefit for a variety of human cancers by enhancing T cell mediated anti-tumor immunity.

INVESTIGATOR BIOGRAPHIES

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Brian Schaefer, Ph.D.

Assistant Professor, Microbiology and Immunology

HYPOTHESIS

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Hypothesis:
The T cell transmembrane molecule CTLA-4 negatively regulates antigen-mediated T cell signals. The cytoplasmic signal transduction cascade that activates the transcription factor NF-kB is a crucial target of CTLA-4 inhibition. We propose that CTLA-4 blocks antigen-mediated activation of NF-kB through two distinct mechanisms:

1. Physical displacement of the costimulatory molecule, CD28, from the central T cell/APC interaction zone by direct competition with CTLA-4 for B7 ligands on the antigen-presenting cell (APC).

2. Delivery of an inhibitory signal that interferes with cytoplasmic signal transduction events in antigen activation of NF-kB.

Goals:
In spite of many studies of CTLA-4 activity, the mechanism of CTLA-4 inhibition of NF-kB activation remains largely undefined. We will employ direct microscopic observation to examine sub-cellular protein redistribution events that are crucial to the function of CTLA-4. These experiments will reveal to what extent CTLA-4 inhibition of NF-kB activation is the result of physical blockade of CD28 interaction with B7 at the cell surface vs. the generation of inhibitory signals within the cytoplasm. Ultimately, this work will identify candidate target molecules against which small molecule inhibitors of CTLA-4 activity could be designed. Such drugs would likely have substantial therapeutic benefit in combating human cancer.

In a second approach, we are examining the effects of CTLA-4 expression on NF-kB activation in the secondary lymphoid organs of living mice. These experiments will demonstrate to what extent CTLA-4 expression down modulates NF-kB activation in vivo.  In addition, we expect these experiments to show whether CTLA-4 alters the kinetics of in vivo antigen-driven NF-kB activation.

Methods:
To attempt to define the molecular mechanisms of CTLA-4 inhibition of T cell activation, we will examine sub-cellular protein redistribution events that are crucial to the function of CTLA-4.  We will use fluorescence microscopy for advanced, 3-dimensional live cell imaging of the redistribution of fluorescently tagged T cell signaling proteins during the process of T cell activation.  We will also use the technique of fluorescence resonance energy transfer (FRET) to define close molecular interactions that are of critical importance for this inhibitory phenomenon.

For in vivo studies of CTLA-4 regulation of NF-kB activation, we are developing a novel in vivo imaging method that combines detection of bioluminescence and quantum dot fluorescence, in order to detect functional, antigen-driven interactions between T cells and antigen presenting cells. 

 

FINDINGS

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Lay Results:
In research which we continue to pursue, we are evaluating the contribution of a molecule called CTLA-4 on the function of T cells, a cell type that is critical for the function of the human immune system.  Our data are helping to establish at a molecular level how CTLA-4 regulates the strength and duration of the T cell response to antigen.

Scientific Results: 
In ongoing studies, we are employing confocal imaging of live and fixed cells to characterize the subcellular localization and dynamic behavior of CTLA-4 and other costimulatory ligands, following antigen stimulation of T cells.  We are also assessing the effects of CTLA-4 ligation on redistribution of and signal transmission by T cell signaling molecules related to NF-kB signaling. In order to assess the effects of CTLA-4 on in vivo activation of NF-kB in T cells, we are using a sensitive in vivo imaging system (IVIS) to monitor activation of an NF-kB promoter in response to antigen stimulation.  By repeatedly monitoring the responses in the same individual animals, we are determining how CTLA-4 affects the magnitude and kinetics of CTLA-4 activation in vivo.

SELECTED PUBLICATIONS

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Rossman J.S., Stoicheva N.G., Langel F.D., Patterson G.H., Lippincott-Schwartz J., and Schaefer B.C. POLKADOTS are foci of functional interactions in T-Cell receptor-mediated signaling to NF-kB.. Mol Biol Cell. 2006 May;17(5):2166-76.

Schaefer B.C., Kappler J.W., Kupfer A., and Marrack P. Complex and dynamic redistribution of NF-kB signaling intermediates in response to T cell receptor stimulation. Proc Natl Acad Sci U S A. 2004 Jan 27;101(4):1004-9.