Extensive cross-regulation of immune function has been reported between the T lymphocyte antigen receptor (TCR) that mediates T cell immune activation, and CXCR4, a chemokine receptor that stimulates cell migration and gene expression. Yet the molecular mechanisms responsible for the integration of CXCR4 and TCR signal transduction pathways are still mostly unknown. The CXCR4 ligand, SDF-1, is constitutively expressed in many tissues, including lymph nodes and gut. CXCR4 is widely expressed and critically regulates multiple diseases, including HIV-1/AIDS and cancer. At the beginning of the last funding cycle, we showed that SDF-1 signals in T cells by inducing the formation of CXCR4-TCR heterodimeric complexes, which are required for SDF-1 to stimulate migration, increase cytosolic Ca2+, and activate the ERK MAP kinase pathway and gene expression. During the last cycle of this grant, we found that CXCR4-TCR complexes signal in regulatory T cells (Tregs) to potently increase IL-10 secretion via a signaling pathway that specifically requires a particular phospholipase isoform, PLC-23. We also found that CXCR4-TCR signaling via PLC-23 critically cross- regulates and antagonizes naive T cell activation in response to ligation of the TCR, even in the absence of SDF-1. IL-10 is an immune modulator and inhibitor of T cell-mediated autoimmunity including inflammatory bowel disease (IBD). Thus, our results indicate that CXCR4-TCR signaling via PLC-23 is a potent promoter of immune tolerance that both enhances functions of Tregs while modulating activation of naive T cells. Assessing the physiological significance of these observations is important, however, until now this has been difficult since targeting either CXCR4 or the TCR disrupts multiple immune functions including T cell development and homeostatic T cell migration patterns. Since PLC-23 is required for many immune effects of CXCR4-TCR complexes but not for T cell migration, we here propose to address the molecular mechanisms and in vivo immune impact of CXCR4-TCR signaling via PLC-23 by studying PLC-23-/- mice. Our Central Hypothesis is that signaling by the CXCR4-TCR heterodimer depends on PLC-23 in order to activate the Ras- ERK pathway to promote Treg functions, and to antagonize TCR signaling, thereby inhibiting autoimmunity.
Our Specific Aims are to: 1) Characterize the molecular mechanisms by which the CXCR4-TCR heterodimer uses PLC-23 for signaling, 2) Determine the role of CXCR4-TCR heterodimer signaling via PLC-23 in regulating Tregs and preventing autoimmune disease in vivo, and 3) Characterize the role of the CXCR4-TCR heterodimer and PLC-23 in modulating TCR signaling and T cell immune responses in vivo. Together, the proposed studies will address key questions regarding the in vivo immune impact of CXCR4-TCR heterodimers and PLC-23. These studies also have the potential to characterize PLC-23 as a novel pharmacologic target that could be used to selectively enhance CXCR4-TCR heterodimer signaling and thereby Treg function and tolerance without disrupting the normal in vivo migration patterns of immune cells.

Public Health Relevance

CXCR4 is a molecule on T lymphocytes that participates in regulating the human immune system, and CXCR4 dysfunction contributes to many diseases including autoimmunity, immune-deficiency, cancer, and AIDS. Currently little is known about the chemical mechanisms that determine how CXCR4 works, especially how CXCR4 uses these chemical mechanisms to regulate the immune system in a live animal or human. This lack of knowledge prevents the development of effective drugs for many diseases. This project aims to change that, by studying the chemical mechanisms used by CXCR4 and by showing how these chemical mechanisms regulate the immune system in mice, when mice have autoimmune disease or a viral infection.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Cellular and Molecular Immunology - A Study Section (CMIA)
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Marino, Pamela
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Mayo Clinic, Rochester
United States
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Kremer, Kimberly N; Clift, Ian C; Miamen, Alexander G et al. (2011) Stromal cell-derived factor-1 signaling via the CXCR4-TCR heterodimer requires phospholipase C-*3 and phospholipase C-ýý1 for distinct cellular responses. J Immunol 187:1440-7
Kremer, Kimberly N; Kumar, Ashok; Hedin, Karen E (2011) G alpha i2 and ZAP-70 mediate RasGRP1 membrane localization and activation of SDF-1-induced T cell functions. J Immunol 187:3177-85
Kumar, Ashok; Kremer, Kimberly N; Dominguez, Daniel et al. (2011) Gýý13 and Rho mediate endosomal trafficking of CXCR4 into Rab11+ vesicles upon stromal cell-derived factor-1 stimulation. J Immunol 186:951-8
Kumar, Ashok; Kremer, Kimberly N; Sims, Olivia L et al. (2009) Measuring the proximity of T-lymphocyte CXCR4 and TCR by fluorescence resonance energy transfer (FRET). Methods Enzymol 460:379-97
Kremer, Kimberly N; Kumar, Ashok; Hedin, Karen E (2007) Haplotype-independent costimulation of IL-10 secretion by SDF-1/CXCL12 proceeds via AP-1 binding to the human IL-10 promoter. J Immunol 178:1581-8
Kumar, Ashok; Humphreys, Troy D; Kremer, Kimberly N et al. (2006) CXCR4 physically associates with the T cell receptor to signal in T cells. Immunity 25:213-24
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Kremer, Kimberly N; Humphreys, Troy D; Kumar, Ashok et al. (2003) Distinct role of ZAP-70 and Src homology 2 domain-containing leukocyte protein of 76 kDa in the prolonged activation of extracellular signal-regulated protein kinase by the stromal cell-derived factor-1 alpha/CXCL12 chemokine. J Immunol 171:360-7
Hedin, Karen E (2002) Chemokines: new, key players in the pathobiology of pancreatic cancer. Int J Gastrointest Cancer 31:23-9