Regulation of CD8 T-cell signaling, metabolism, and cytotoxic activity by extracellular lysophosphatidic acid.

Lysophosphatidic acid (LPA) is an endogenous bioactive lipid that is produced extracellularly and signals to cells via cognate LPA receptors, which are G-protein coupled receptors (GPCRs). Mature lymphocytes in mice and humans express three LPA receptors, LPA2 , LPA5, and LPA6 , and work from our group has determined that LPA5 signaling by T lymphocytes inhibits specific antigen-receptor signaling pathways that ultimately impair lymphocyte activation, proliferation, and function. In this review, we discuss previous and ongoing work characterizing the ability of an LPA-LPA5 axis to serve as a peripheral immunological tolerance mechanism that restrains adaptive immunity but is subverted during settings of chronic inflammation. Specifically, LPA-LPA5 signaling is found to regulate effector cytotoxic CD8 T cells by (at least) two mechanisms: (i) regulating the actin-microtubule cytoskeleton in a manner that impairs immunological synapse formation between an effector CD8 T cell and antigen-specific target cell, thus directly impairing cytotoxic activity, and (ii) shifting T-cell metabolism to depend on fatty-acid oxidation for mitochondrial respiration and reducing metabolic efficiency. The in vivo outcome of LPA5 inhibitory activity impairs CD8 T-cell killing and tumor immunity in mouse models providing impetus to consider LPA5 antagonism for the treatment of malignancies and chronic infections.

LPA suppresses T cell function by altering the cytoskeleton and disrupting immune synapse formation.

Cancer and chronic infections often increase levels of the bioactive lipid, lysophosphatidic acid (LPA), that we have demonstrated acts as an inhibitory ligand upon binding LPAR5 on CD8 T cells, suppressing cytotoxic activity and tumor control. This study, using human and mouse primary T lymphocytes, reveals how LPA disrupts antigen-specific CD8 T cell:target cell immune synapse (IS) formation and T cell function via competing for cytoskeletal regulation. Specifically, we find upon antigen-specific T cell:target cell formation, IP3R1 localizes to the IS by a process dependent on mDia1 and actin and microtubule polymerization. LPA not only inhibited IP3R1 from reaching the IS but also altered T cell receptor (TCR)­induced localization of RhoA and mDia1 impairing F-actin accumulation and altering the tubulin code. Consequently, LPA impeded calcium store release and IS-directed cytokine secretion. Thus, targeting LPA signaling in chronic inflammatory conditions may rescue T cell function and promote antiviral and antitumor immunity.

Tissue-Nonspecific Alkaline Phosphatase Is Required for MC3T3 Osteoblast-Mediated Protection of Acute Myeloid Leukemia Cells from Apoptosis.

The bone marrow microenvironment harbors and protects leukemic cells from apoptosis-inducing agents via mechanisms that are incompletely understood. We previously showed SDF-1 (CXCL-12), a chemokine readily abundant within the bone marrow microenvironment, induces apoptosis in acute myeloid leukemia (AML) cells that express high levels of the SDF-1 receptor CXCR4. However, differentiating osteoblasts found within this niche protect cocultured AML cells from apoptosis. Additionally, this protection was abrogated upon treatment of the differentiating osteoblasts with histone deacetylase inhibitors (HDACi). In this study, we begin to characterize and target the molecular mechanisms that mediate this osteoblast protection. Quantitative RT-PCR revealed that HDACi treatment of differentiating osteoblasts (mouse MC3T3 osteoblast cell line) reduced expression of multiple genes required for osteoblast differentiation, including genes important for producing mineralized bone matrix. Interestingly, pretreating differentiating osteoblasts with cyclosporine A, a drug known to inhibit osteoblast differentiation, similarly impaired osteoblast-mediated protection of cocultured AML cells (KG1a and U937 human AML cell lines). Both HDACi and cyclosporine A reduced osteoblast expression of the key mineralization enzyme tissue-nonspecific alkaline phosphatase (TNAP; encoded by Alpl). Moreover, specifically reducing TNAP expression or activity in differentiating osteoblasts significantly impaired the ability of the osteoblasts to protect cocultured AML cells. Together, our results indicate that inhibiting osteoblast matrix mineralization by specifically targeting TNAP is sufficient to significantly impair osteoblast-mediated protection of AML cells. Therefore, designing combination therapies that additionally target the osteoblast-produced mineralized bone matrix may improve treatment of AML by reducing the protection of leukemic cells within the bone marrow microenvironment.

GRK2 mediates TCR-induced transactivation of CXCR4 and TCR-CXCR4 complex formation that drives PI3Kγ/PREX1 signaling and T cell cytokine secretion.

The immune system includes abundant examples of biologically-relevant cross-regulation of signaling pathways by the T cell antigen receptor (TCR) and the G protein-coupled chemokine receptor, CXCR4. TCR ligation induces transactivation of CXCR4 and TCR-CXCR4 complex formation, permitting the TCR to signal via CXCR4 to activate a phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 protein (PREX1)-dependent signaling pathway that drives robust cytokine secretion by T cells. To understand this receptor heterodimer and its regulation, we characterized the molecular mechanisms required for TCR-mediated TCR-CXCR4 complex formation. We found that the cytoplasmic C-terminal domain of CXCR4 and specifically phosphorylation of Ser-339 within this region were required for TCR-CXCR4 complex formation. Interestingly, siRNA-mediated depletion of G protein-coupled receptor kinase-2 (GRK2) or inhibition by the GRK2-specific inhibitor, paroxetine, inhibited TCR-induced phosphorylation of CXCR4-Ser-339 and TCR-CXCR4 complex formation. Either GRK2 siRNA or paroxetine treatment of human T cells significantly reduced T cell cytokine production. Upstream, TCR-activated tyrosine kinases caused inducible tyrosine phosphorylation of GRK2 and were required for the GRK2-dependent events of CXCR4-Ser-339 phosphorylation and TCR-CXCR4 complex formation. Downstream of TCR-CXCR4 complex formation, we found that GRK2 and phosphatidylinositol 3-kinase γ (PI3Kγ) were required for TCR-stimulated membrane recruitment of PREX1 and for stabilization of cytokine mRNAs and robust cytokine secretion. Together, our results identify a novel role for GRK2 as a target of TCR signaling that is responsible for TCR-induced transactivation of CXCR4 and TCR-CXCR4 complex formation that signals via PI3Kγ/PREX1 to mediate cytokine production. Therapeutic regulation of GRK2 or PI3Kγ may therefore be useful for limiting cytokines produced by T cell malignancies or autoimmune diseases.

TCR-CXCR4 signaling stabilizes cytokine mRNA transcripts via a PREX1-Rac1 pathway: implications for CTCL.

As with many immunopathologically driven diseases, the malignant T cells of cutaneous T-cell lymphomas (CTCLs), such as Sézary syndrome, display aberrant cytokine secretion patterns that contribute to pathology and disease progression. Targeting this disordered release of cytokines is complicated by the changing cytokine milieu that drives the phenotypic changes of CTCLs. Here, we characterize a novel signaling pathway that can be targeted to inhibit the secretion of cytokines by modulating either CXCR4 or CXCR4-mediated signaling. We demonstrate that upon ligation of the T-cell antigen receptor (TCR), the TCR associates with and transactivates CXCR4 via phosphorylation of S339-CXCR4 in order to activate a PREX1-Rac1-signaling pathway that stabilizes interleukin-2(IL-2), IL-4, and IL-10 messenger RNA (mRNA) transcripts. Pharmacologic inhibition of either TCR-CXCR4 complex formation or PREX1-Rac1 signaling in primary human T cells decreased mRNA stability and inhibited secretion of IL-2, IL-4, and IL-10. Applying this knowledge to Sézary syndrome, we demonstrate that targeting various aspects of this signaling pathway blocks both TCR-dependent and TCR-independent cytokine secretion from a Sézary syndrome-derived cell line and patient isolates. Together, these results identify multiple aspects of a novel TCR-CXCR4-signaling pathway that could be targeted to inhibit the aberrant cytokine secretion that drives the immunopathogenesis of Sézary syndrome and other immunopathological diseases.

Histone Deacetylase Inhibitors Target the Leukemic Microenvironment by Enhancing a Nherf1-Protein Phosphatase 1α-TAZ Signaling Pathway in Osteoblasts.

Disrupting the protective signals provided by the bone marrow microenvironment will be critical for more effective combination drug therapies for acute myeloid leukemia (AML). Cells of the osteoblast lineage that reside in the endosteal niche have been implicated in promoting survival of AML cells. Here, we investigated how to prevent this protective interaction. We previously showed that SDF-1, a chemokine abundant in the bone marrow, induces apoptosis of AML cells, unless the leukemic cells receive protective signals provided by differentiating osteoblasts (8, 10). We now identify a novel signaling pathway in differentiating osteoblasts that can be manipulated to disrupt the osteoblast-mediated protection of AML cells. Treating differentiating osteoblasts with histone deacetylase inhibitors (HDACi) abrogated their ability to protect co-cultured AML cells from SDF-1-induced apoptosis. HDACi prominently up-regulated expression of the Nherf1 scaffold protein, which played a major role in preventing osteoblast-mediated protection of AML cells. Protein phosphatase-1α (PP1α) was identified as a novel Nherf1 interacting protein that acts as the downstream mediator of this response by promoting nuclear localization of the TAZ transcriptional modulator. Moreover, independent activation of either PP1α or TAZ was sufficient to prevent osteoblast-mediated protection of AML cells even in the absence of HDACi. Together, these results indicate that HDACi target the AML microenvironment by enhancing activation of the Nherf1-PP1α-TAZ pathway in osteoblasts. Selective drug targeting of this osteoblast signaling pathway may improve treatments of AML by rendering leukemic cells in the bone marrow more susceptible to apoptosis.

ß-Arrestin1 and distinct CXCR4 structures are required for stromal derived factor-1 to downregulate CXCR4 cell-surface levels in neuroblastoma.

CXC chemokine receptor 4 (CXCR4) is a G protein-coupled receptor (GPCR) located on the cell surface that signals upon binding the chemokine stromal derived factor-1 (SDF-1; also called CXCL 12). CXCR4 promotes neuroblastoma proliferation and chemotaxis. CXCR4 expression negatively correlates with prognosis and drives neuroblastoma growth and metastasis in mouse models. All functions of CXCR4 require its expression on the cell surface, yet the molecular mechanisms that regulate CXCR4 cell-surface levels in neuroblastoma are poorly understood. We characterized CXCR4 cell-surface regulation in the related SH-SY5Y and SK-N-SH human neuroblastoma cell lines. SDF-1 treatment caused rapid down-modulation of CXCR4 in SH-SY5Y cells. Pharmacologic activation of protein kinase C similarly reduced CXCR4, but via a distinct mechanism. Analysis of CXCR4 mutants delineated two CXCR4 regions required for SDF-1 treatment to decrease cell-surface CXCR4 in neuroblastoma cells: the isoleucine-leucine motif at residues 328 and 329 and residues 343-352. In contrast, and unlike CXCR4 regulation in other cell types, serines 324, 325, 338, and 339 were not required. Arrestin proteins can bind and regulate GPCR cell-surface expression, often functioning together with kinases such as G protein-coupled receptor kinase 2 (GRK2). Using SK-N-SH cells which are naturally deficient in ß-arrestin1, we showed that ß-arrestin1 is required for the CXCR4 343-352 region to modulate CXCR4 cell-surface expression following treatment with SDF-1. Moreover, GRK2 overexpression enhanced CXCR4 internalization, via a mechanism requiring both ß-arrestin1 expression and the 343-352 region. Together, these results characterize CXCR4 structural domains and ß-arrestin1 as critical regulators of CXCR4 cell-surface expression in neuroblastoma. ß-Arrestin1 levels may therefore influence the CXCR4-driven metastasis of neuroblastoma as well as prognosis.

CXCR4 chemokine receptor signaling induces apoptosis in acute myeloid leukemia cells via regulation of the Bcl-2 family members Bcl-XL, Noxa, and Bak.

The CXCR4 chemokine receptor promotes survival of many different cell types. Here, we describe a previously unsuspected role for CXCR4 as a potent inducer of apoptosis in acute myeloid leukemia (AML) cell lines and a subset of clinical AML samples. We show that SDF-1, the sole ligand for CXCR4, induces the expected migration and ERK activation in the KG1a AML cell line transiently overexpressing CXCR4, but ERK activation did not lead to survival. Instead, SDF-1 treatment led via a CXCR4-dependent mechanism to apoptosis, as evidenced by increased annexin V staining, condensation of chromatin, and cleavage of both procaspase-3 and PARP. This SDF-1-induced death pathway was partially inhibited by hypoxia, which is often found in the bone marrow of AML patients. SDF-1-induced apoptosis was inhibited by dominant negative procaspase-9 but not by inhibition of caspase-8 activation, implicating the intrinsic apoptotic pathway. Further analysis showed that this pathway was activated by multiple mechanisms, including up-regulation of Bak at the level of mRNA and protein, stabilization of the Bak activator Noxa, and down-regulation of antiapoptotic Bcl-XL. Furthermore, adjusting expression levels of Bak, Bcl-XL, or Noxa individually altered the level of apoptosis in AML cells, suggesting that the combined modulation of these family members by SDF-1 coordinates their interplay to produce apoptosis. Thus, rather than mediating survival, SDF-1 may be a means to induce apoptosis of CXCR4-expressing AML cells directly in the SDF-1-rich bone marrow microenvironment if the survival cues of the bone marrow are disrupted.

Osteoblasts protect AML cells from SDF-1-induced apoptosis.

The bone marrow provides a protective environment for acute myeloid leukemia (AML) cells that often allows leukemic stem cells to survive standard chemotherapeutic regimens. Targeting these leukemic stem cells within the bone marrow is critical for preventing relapse. We recently demonstrated that SDF-1, a chemokine abundant in the bone marrow, induces apoptosis in AML cell lines and in patient samples expressing high levels of its receptor, CXCR4. Here we show that a subset of osteoblast lineage cells within the bone marrow can protect AML cells from undergoing apoptosis in response to the SDF-1 naturally present in that location. In co-culture systems, osteoblasts at various stages of differentiation protected AML cell lines and patient isolates from SDF-1-induced apoptosis. The differentiation of the osteoblast cell lines, MC3T3 and W-20-17, mediated this protection via a cell contact-independent mechanism. In contrast, bone marrow-derived mesenchymal cells, the precursors of osteoblasts, induced apoptosis in AML cells via a CXCR4-dependent mechanism and failed to protect AML cells from exogenously added SDF-1. These results indicate that osteoblasts in the process of differentiation potently inhibit the SDF-1-driven apoptotic pathway of CXCR4-expressing AML cells residing in the bone marrow. Drugs targeting this protective mechanism could potentially provide a new approach to treating AML by enhancing the SDF-1-induced apoptosis of AML cells residing within the bone marrow microenvironment.

G alpha i2 and ZAP-70 mediate RasGRP1 membrane localization and activation of SDF-1-induced T cell functions.

RasGRP1, a Ras guanine-nucleotide exchange factor, critically mediates T cell development and function and controls immunodeficiency and autoimmunity. In this study, we describe a unique mechanism of mobilization and activation of RasGRP1 in response to SDF-1, a chemokine that signals via the G protein-coupled receptor CXCR4. Depletion of RasGRP1 impaired SDF-1-stimulated human T cell migration, expression of the activation marker CD69, and activation of the ERK MAPK pathway, indicating that RasGRP1 mediates SDF-1 functions. SDF-1 treatment caused RasGRP1 to localize to the plasma membrane to activate K-Ras and to the Golgi to activate N-Ras. These events were required for cellular migration and for ERK activation that mediates downstream transcriptional events in response to SDF-1. SDF-1-dependent localization of RasGRP1 did not require its diacylglycerol-binding domain, even though diacyglycerol was previously shown to mediate localization of RasGRP1 in response to Ag stimulation. This domain was, however, required for activity of RasGRP1 after its localization. Intriguingly, SDF-1 treatment of T cells induced the formation of a novel molecular signaling complex containing RasGRP1, Gαi2, and ZAP-70. Moreover, SDF-1-mediated signaling by both Gi proteins and ZAP-70 was required for RasGRP1 mobilization. In addition, RasGRP1 mobilization and activation in response to SDF-1 was dependent on TCR expression, suggesting that CXCR4 heterodimerizes with the TCR to couple to ZAP-70 and mobilize RasGRP1. These results increase understanding of the molecular mechanisms that mediate SDF-1 effects on T cells and reveal a novel mechanism of RasGRP1 regulation. Other G protein-coupled receptors may similarly contribute to regulation of RasGRP1.

Gα13 and Rho mediate endosomal trafficking of CXCR4 into Rab11+ vesicles upon stromal cell-derived factor-1 stimulation.

CXCR4, like other G protein-coupled receptors, signals via heterotrimeric guanine nucleotide-binding proteins (G proteins) to regulate gene transcription, migration, development, growth, and transformation. We describe a formerly uncharacterized function of a G protein: a role in receptor trafficking. We previously showed that CXCR4 and the TCR physically associate and form a heterodimer upon stromal cell-derived factor-1 or CXCL12 (SDF-1) stimulation in human T cells to prolong ERK activation and, thereby, lead to gene upregulation and cytokine secretion. The CXCR4-TCR heterodimers occur on the cell surface and in an intracellular compartment in response to SDF-1. Neither the intracellular compartment to which the CXCR4-TCR heterodimers localize nor the mechanism for localization has been elucidated. In this article, we characterize molecular mechanisms required for postendocytic trafficking of CXCR4. Upon SDF-1 stimulation, CXCR4 localizes to Rab11(+) vesicles, a recycling compartment near the microtubule organizing center and Golgi apparatus. This trafficking requires the CXCR4 C-terminal tail domain but not the CXCR4 ubiquitination sites. The TCR also constitutively localizes to this Rab11(+) compartment. Trafficking of CXCR4 into the Rab11(+), TCR-containing endosomes requires actin polymerization. Furthermore, inhibiting Rho activation or depleting Gα13 prevented trafficking of CXCR4 into the Rab11(+) endosomes without hindering the ability of CXCR4 to endocytose. These results indicated that, upon SDF-1 treatment, Gα13 and Rho mediate the actin polymerization necessary for trafficking CXCR4 into the Rab11(+), recycling endosomal compartment, which also contains constitutively recycling TCR and, thus, CXCR4-TCR heterodimers. To our knowledge, this is the first report of Gα13 as a mediator of receptor trafficking.

Stromal cell-derived factor-1 signaling via the CXCR4-TCR heterodimer requires phospholipase C-ß3 and phospholipase C-γ1 for distinct cellular responses.

The CXCR4 chemokine receptor is a G protein-coupled receptor that signals in T lymphocytes by forming a heterodimer with the TCR. CXCR4 and TCR functions are consequently highly cross regulated, affecting T cell immune activation, cytokine secretion, and T cell migration. The CXCR4-TCR heterodimer stimulates T cell migration and activation of the ERK MAPK and downstream AP-1-dependent cytokine transcription in response to stromal cell-derived factor-1 (SDF-1), the sole chemokine ligand of CXCR4. These responses require Gi-type G proteins as well as TCR ITAM domains and the ZAP70 tyrosine kinase, thus indicating that the CXCR4-TCR heterodimer signals to integrate G protein-coupled receptor-associated and TCR-associated signaling molecules in response to SDF-1. Yet, the phospholipase C (PLC) isozymes responsible for coupling the CXCR4-TCR heterodimer to distinct downstream cellular responses are incompletely characterized. In this study, we demonstrate that PLC activity is required for SDF-1 to induce ERK activation, migration, and CXCR4 endocytosis in human T cells. SDF-1 signaling via the CXCR4-TCR heterodimer uses PLC-ß3 to activate the Ras-ERK pathway and increase intracellular calcium ion concentrations, whereas PLC-γ1 is dispensable for these outcomes. In contrast, PLC-γ1, but not PLC-ß3, is required for SDF-1-mediated migration via a mechanism independent of LAT. These results increase understanding of the signaling mechanisms employed by the CXCR4-TCR heterodimer, characterize new roles for PLC-ß3 and PLC-γ1 in T cells, and suggest that multiple PLCs may also be activated downstream of other chemokine receptors to distinctly regulate migration versus other signaling functions.

ARHGEF1 deficiency reveals Gα13-associated GPCRs are critical regulators of human lymphocyte function.

Primary antibody deficiencies are the most common immunodeficiencies in humans; however, identification of the underlying genetic and biochemical basis for these diseases is often difficult, given that these deficiencies typically involve complex genetic etiologies. In this issue of the JCI, Bouafia et al. performed whole-exome sequencing on a pair of siblings with primary antibody deficiencies and identified genetic mutations that result in a deficiency of ARHGEF1, a hematopoietic intracellular signaling molecule that transmits signals from GPCRs. ARHGEF1-deficient lymphocytes from the affected siblings exhibited important functional deficits that indicate that loss of ARHGEF1 accounts for the observed primary antibody deficiency, which manifests in an inability to mount antibody responses to vaccines and pathogens. Thus, this report demonstrates an important role for ARHGEF1 in GPCR signal transduction required for appropriate adaptive immune responses in humans.

LPA5 Is an Inhibitory Receptor That Suppresses CD8 T-Cell Cytotoxic Function via Disruption of Early TCR Signaling.

Persistent T cell antigen receptor (TCR) signaling by CD8 T cells is a feature of cancer and chronic infections and results in the sustained expression of, and signaling by, inhibitory receptors, which ultimately impair cytotoxic activity via poorly characterized mechanisms. We have previously determined that the LPA5 GPCR expressed by CD8 T cells, upon engaging the lysophosphatidic acid (LPA) bioactive serum lipid, functions as an inhibitory receptor able to negatively regulate TCR signaling. Notably, the levels of LPA and autotaxin (ATX), the phospholipase D enzyme that produces LPA, are often increased in chronic inflammatory disorders such as chronic infections, autoimmune diseases, obesity, and cancer. In this report, we demonstrate that LPA engagement selectively by LPA5 on human and mouse CD8 T cells leads to the inhibition of several early TCR signaling events including intracellular calcium mobilization and ERK activation. We further show that, as a consequence of LPA5 suppression of TCR signaling, the exocytosis of perforin-containing granules is significantly impaired and reflected by repressed in vitro and in vivo CD8 T cell cytolytic activity. Thus, these data not only document LPA5 as a novel inhibitory receptor but also determine the molecular and biochemical mechanisms by which a naturally occurring serum lipid that is elevated under settings of chronic inflammation signals to suppress CD8 T cell killing activity in both human and murine cells. As diverse tumors have repeatedly been shown to aberrantly produce LPA that acts in an autocrine manner to promote tumorigenesis, our findings further implicate LPA in activating a novel inhibitory receptor whose signaling may be therapeutically silenced to promote CD8 T cell immunity.

Histone deacetylase inhibitors reduce differentiating osteoblast-mediated protection of acute myeloid leukemia cells from cytarabine.

The bone marrow microenvironment protects acute myeloid leukemia (AML) cells during chemotherapy and is a major factor in relapse. Here, we examined which type(s) of bone marrow cells are responsible for the relapse of AML following treatment with cytarabine (Ara-C), and we identified a means to inhibit this protection. To determine the protective cell type(s), AML cells were treated with Ara-C, and AML cell survival in the presence or absence of osteoblast lineage cells was assessed. Cultured AML cells and patient bone marrow isolates were each significantly protected from Ara-C-induced apoptosis by co-culture with differentiating osteoblasts. Moreover, pretreating differentiating osteoblasts with the histone deacetylase inhibitors (HDACi) vorinostat and panobinostat abrogated the ability of the differentiating osteoblasts to protect AML cells. Together, our results indicate that differentiating osteoblasts have the potential to promote residual AML in the bone marrow following standard chemotherapy and act via a mechanism requiring HDACi-sensitive gene expression. Using HDACi to target the leukemic microenvironment in combination with Ara-C could potentially improve treatment of AML. Moreover, other strategies for manipulating bone marrow osteoblasts may also help eradicate AML cells and reduce relapse.

IQGAP1 promotes CXCR4 chemokine receptor function and trafficking via EEA-1+ endosomes.

IQ motif-containing GTPase-activating protein 1 (IQGAP1) is a cytoskeleton-interacting scaffold protein. CXCR4 is a chemokine receptor that binds stromal cell-derived factor-1 (SDF-1; also known as CXCL12). Both IQGAP1 and CXCR4 are overexpressed in cancer cell types, yet it was unclear whether these molecules functionally interact. Here, we show that depleting IQGAP1 in Jurkat T leukemic cells reduced CXCR4 expression, disrupted trafficking of endocytosed CXCR4 via EEA-1(+) endosomes, and decreased efficiency of CXCR4 recycling. SDF-1-induced cell migration and activation of extracellular signal-regulated kinases 1 and 2 (ERK) MAPK were strongly inhibited, even when forced overexpression restored CXCR4 levels. Similar results were seen in KMBC and HEK293 cells. Exploring the mechanism, we found that SDF-1 treatment induced IQGAP1 binding to α-tubulin and localization to CXCR4-containing endosomes and that CXCR4-containing EEA-1(+) endosomes were abnormally located distal from the microtubule (MT)-organizing center (MTOC) in IQGAP1-deficient cells. Thus, IQGAP1 critically mediates CXCR4 cell surface expression and signaling, evidently by regulating EEA-1(+) endosome interactions with MTs during CXCR4 trafficking and recycling. IQGAP1 may similarly promote CXCR4 functions in other cancer cell types.

Measuring the proximity of T-lymphocyte CXCR4 and TCR by fluorescence resonance energy transfer (FRET).

Multiprotein complexes play an important role in nearly all cell functions; therefore, the characterization of protein-protein interactions in living cells constitutes an important step in the analysis of cellular signaling pathways. Using fluorescence resonance energy transfer (FRET) as a "molecular ruler" is a powerful approach for identifying biologically relevant molecular interactions with high spatiotemporal resolution. Here, we describe two methods that use FRET to detect a physical interaction between the T-cell antigen receptor (TCR) and the CXCR4 chemokine receptor in living T lymphocytes. These FRET approaches use two different sets of chromophores. We discuss the design strategies, control experiments, and pitfalls involved in using these FRET approaches. Although there is no perfect pair of chromophores for FRET, the two FRET methods described here provide complementary and reliable insight into the molecular interactions between these receptor molecules.

Haplotype-independent costimulation of IL-10 secretion by SDF-1/CXCL12 proceeds via AP-1 binding to the human IL-10 promoter.

Costimulation by the chemokine, stromal cell-derived factor-1 (SDF-1)/CXCL12, has been shown to increase the amount of IL-10 secreted by TCR-stimulated human T cells; however, the molecular mechanisms of this response are unknown. Knowledge of this signaling pathway may be useful because extensive evidence indicates that deficient IL-10 secretion promotes autoimmunity. The human IL-10 locus is highly polymorphic. We report in this study that SDF-1 costimulates IL-10 secretion from T cells containing all three of the most common human IL-10 promoter haplotypes that are identified by single-nucleotide polymorphisms at -1082, -819, and -592 bp (numbering is relative to the transcription start site). We further show that SDF-1 primarily costimulates IL-10 secretion by a diverse population of CD45RA(-) ("memory") phenotype T cells that includes cells expressing the presumed regulatory T cell marker, Foxp3. To address the molecular mechanisms of this response, we showed that SDF-1 costimulates the transcriptional activities in normal human T cells of reporter plasmids containing 1.1 kb of all three of the common IL-10 promoter haplotypes. IL-10 promoter activity was ablated by mutating two nonpolymorphic binding sites for the AP-1 transcription factor, and chromatin immunoprecipitation assays of primary human T cells revealed that SDF-1 costimulation enhances AP-1 binding to both of these sites. Together, these results delineate the molecular mechanisms responsible for SDF-1 costimulation of T cell IL-10 secretion. Because it is preserved among several human haplotypes and in diverse T cell populations including Foxp3(+) T cells, this pathway of IL-10 regulation may represent a key mechanism for modulating expression of this important immunoregulatory cytokine.

CXCR4 physically associates with the T cell receptor to signal in T cells.

SDF-1alpha (CXCL12) signaling via its receptor, CXCR4, stimulates T cell chemotaxis and gene expression. The ZAP-70 tyrosine kinase critically mediates SDF-1alpha-dependent migration and prolonged ERK mitogen-activated protein (MAP) kinase activation in T cells. However, the molecular mechanism by which CXCR4 or other G protein-coupled receptors activate ZAP-70 has not been characterized. Here we show that SDF-1alpha stimulates the physical association of CXCR4 and the T cell receptor (TCR) and utilizes the ZAP-70 binding ITAM domains of the TCR for signal transduction. This pathway is responsible for several of the effects of SDF-1alpha on T cells, including prolonged ERK MAP kinase activity, increased intracellular calcium ion concentrations, robust AP-1 transcriptional activity, and SDF-1alpha costimulation of cytokine secretion. These results suggest new paradigms for understanding the effects of SDF-1alpha and other chemokines on immunity.

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.

Stimulation of T lymphocytes with the ligand for the CXCR4 chemokine receptor stromal cell-derived factor-1alpha (SDF-1alpha/CXCL12), results in prolonged activation of the extracellular signal-regulated kinases (ERK) ERK1 and ERK2. Because SDF-1alpha is unique among several chemokines in its ability to stimulate prolonged ERK activation, this pathway is thought to mediate special functions of SDF-1alpha that are not shared with other chemokines. However, the molecular mechanisms of this response are poorly understood. In this study we show that SDF-1alpha stimulation of prolonged ERK activation in Jurkat T cells requires both the ZAP-70 tyrosine kinase and the Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) scaffold protein. This pathway involves ZAP-70-dependent tyrosine phosphorylation of SLP-76 at one or more of its tyrosines, 113, 128, and 145. Because TCR activates ERK via SLP-76-mediated activation of the linker of activated T cells (LAT) scaffold protein, we examined the role of LAT in SDF-1alpha-mediated ERK activation. However, neither the SLP-76 proline-rich domain that links to GADS and LAT, nor LAT, itself are required for SDF-1alpha to stimulate SLP-76 tyrosine phosphorylation or to activate ERK. Together, our results describe the distinct mechanism by which SDF-1alpha stimulates prolonged ERK activation in T cells and indicate that this pathway is specific for cells expressing both ZAP-70 and SLP-76.