In this study we have explored the interaction between CD44 (the hyaluronic acid ( The transmembrane glycoprotein CD44 isoforms are all major hyaluronic acid (HA) 1 cell surface receptors that exist on many cell types, including macrophages, lymphocytes, fibroblasts, and epithelial cells (1-6). Because of their widespread occurrence and their role in signal transduction, CD44 isoforms have been implicated in the regulation of cell growth and activation as well as cell-cell and cell-extracellular matrix interactions (1-7). One of the distinct features of CD44 isoforms is the enormous heterogeneity in the molecular masses of these proteins. It is now known that all CD44 isoforms are encoded by a single gene that contains 19 exons (8). Of the 19 exons, 12 exons can be alternatively spliced (8). Most often, the alternative splicing occurs between exons 5 and 15, leading to an insertion in tandem of one or more variant exons (v1-v10 (exon 6-exon 14) in human cells) within the membrane-proximal region of the extracellular domain (8). The variable primary amino acid sequence of different CD44 isoforms is further modified by extensive N-and O-glycosylations and glycosaminoglycan additions (9 -12). In particular, CD44v3-containing isoforms have a heparin sulfate addition at the membrane-proximal extracellular domain of the molecule that confers the ability to bind heparin sulfate-binding growth factors (9, 10). Cell surface expression of CD44v isoforms changes profoundly during tumor metastasis, particularly during the progression of various carcinomas including breast carcinomas (13-17). In fact, CD44v isoform expression has been used as an indicator of metastasis.It has been shown that interaction between the cytoskeletal protein, ankyrin, and the cytoplasmic domain of CD44 isoforms plays an important role in CD44 isoform-mediated oncogenic signaling (6,18,19). Specifically, the ankyrin-binding domain (e.g. NGGNGTVEDRKPSEL between amino acids 306 and 320 in the mouse CD44 (20) and NSGNGAVEDRKPSGL amino acids 304 and 318 in human CD44 (21)) is required for the recruitment of Src kinase and the onset of tumor cell transformation (21). Furthermore, HA binding to CD44 stimulates a concomitant activation of p185 HER2 -linked tyrosine kinase (linked to CD44s via a disulfide linkage) and results in a direct cross-talk between two different signaling pathways (e.g. proliferation versus motility/invasion) (22). In tumor cells, the transmembrane linkage between CD44 isoform and the cytoskeleton promotes invasive and metastatic-specific tumor phenotypes (e.g. matrix degradation (matrix metalloproteinases) activities (23, 24), "invadopodia" formation (membrane projections), tumor cell invasion, and migration) (23). These findings strongly suggest that the interaction between CD44 isoform and the cytoskeleton plays a pivotal role in the onset of oncogenesis and tumor progression.The Rho family proteins (e.g. Rho, Rac, and Cdc42) are members of the Ras superfamily of GTP-binding proteins structurally related to but functionally dist...

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Metastatic breast tumor Met‐1 cells express CD44v3,8–10, a major adhesion receptor that binds extracellular matrix components at its extracellular domain and interacts with the cytoskeletal protein, ankyrin, at its cytoplasmic domain. In this study, we have determined that CD44v3,8–10 and RhoA GTPases are physically associated in vivo, and that CD44v3,8–10‐bound RhoA displays GTPase activity, which can be inhibited by botulinum toxin C3‐mediated ADP‐ribosylation. In addition, we have identified a 160 kDa Rho‐Kinase (ROK) as one of the downstream targets for CD44v3,8–10‐bound RhoA GTPase. Specifically, RhoA (complexed with CD44v3,8–10) stimulates ROK‐mediated phosphorylation of certain cellular proteins including the cytoplasmic domain of CD44v3,8–10. Most importantly, phosphorylation of CD44v3,8–10 by ROK enhances its interaction with the cytoskeletal protein, ankyrin. We have also constructed two ROK cDNA constructs that encode for proteins consisting of 537 amino acids [designated as the constitutively active form of ROK containing the catalytic domain (CAT, also the kinase domain)], and 173 amino acids [designated as the dominant‐negative form of ROK containing the Rho‐binding domain (RB)]. Microinjection of the ROK's CAT domain into Met‐1 cells promotes CD44‐ankyrin associated membrane ruffling and projections. This membrane motility can be blocked by CD44 antibodies and cytochalasin D (a microfilament inhibitor). Furthermore, overexpression of a dominant‐negative form of ROK by transfection of Met‐1 cells with ROK's Rho‐binding (RB) domain cDNA effectively inhibits CD44‐ankyrin‐mediated metastatic behavior (e.g., membrane motility and tumor cell migration). These findings support the hypothesis that ROK plays a pivotal role in CD44v3,8–10‐ankyrin interaction and RhoA‐mediated oncogenic signaling required for membrane‐cytoskeleton function and metastatic tumor cell migration. Cell Motil. Cytoskeleton 43:269–287, 1999. © 1999 Wiley‐Liss, Inc.

Although phosphoantigen-specific Vγ2Vδ2 T cells appear to play a role in antimicrobial and anticancer immunity, mucosal immune responses and effector functions of these γδ T cells during infection or phospholigand treatment remain poorly characterized. In this study, we demonstrate that the microbial phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) plus IL-2 treatment of macaques induced a prolonged major expansion of circulating Vγ2Vδ2 T cells that expressed CD8 and produced cytotoxic perforin during their peak expansion. Interestingly, HMBPP-activated Vγ2Vδ2 T cells underwent an extraordinary pulmonary accumulation, which lasted for 3–4 mo, although circulating Vγ2Vδ2 T cells had returned to baseline levels weeks prior. The Vγ2Vδ2 T cells that accumulated in the lung following HMBPP/IL-2 cotreatment displayed an effector memory phenotype, as follows: CCR5+CCR7−CD45RA−CD27+ and were able to re-recognize phosphoantigen and produce copious amounts of IFN-γ up to 15 wk after treatment. Furthermore, the capacity of massively expanded Vγ2Vδ2 T cells to produce cytokines in vivo coincided with an increase in numbers of CD4+ and CD8+ αβ T cells after HMBPP/IL-2 cotreatment as well as substantial perforin expression by CD3+Vγ2− T cells. Thus, the prolonged HMBPP-driven antimicrobial and cytotoxic responses of pulmonary and systemic Vγ2Vδ2 T cells may confer immunotherapeutics against infectious diseases and cancers.

Nanoscale imaging of an in vivo antigenspecific T-cell immune response has not been reported. Here, the combined nearfield scanning optical microscopy-and fluorescent quantum dot-based nanotechnology was used to perform immunofluorescence imaging of antigen-specific T-cell receptor (TCR) response in an in vivo model of clonal T-cell expansion. The near-field scanning optical microscopy/quantum dot system provided a best-optical-resolution (Ͻ50 nm) nanoscale imaging of V␥2V␦2 TCR on the membrane of nonstimulated V␥2V␦2 T cells. Before Ag-induced clonal expansion, these nonstimulating V␥2V␦2 TCRs appeared to be distributed differently from their ␣␤ TCR counterparts on the cell surface. Surprisingly, V␥2V␦2 TCR nanoclusters not only were formed but also sustained on the membrane during an in vivo clonal expansion of V␥2V␦2 T cells after phosphoantigen treatment or phosphoantigen plus mycobacterial infection. IntroductionT-cell receptors (TCRs) play a crucial role in recognition of antigens and development of immune responses. Whereas immune events for TCR-mediated recognition, signaling, and activation are well described, 1-4 nanoscale imaging of immunobiology of antigenspecific TCR during the in vivo clonal T-cell expansion has not been studied. Because TCRs trigger downstream signaling and activation after antigen recognition, some unique TCR nanostructures may develop after TCR contact on Ag/antigen-presenting cell (APC) and thus contribute to selected functions, such as clonal expansion, effector function, contracting (clonal exhaustion), or differentiation. Whereas this presumption can be tested by imaging or visualization of antigen-specific TCR during the in vivo T-cell response, conventional imaging techniques using fluorescent or confocal microscopy do not have nanoscale optical resolution power to reveal individual TCRs and their dynamics during clonal expansion-maturation. [1][2][3][4] Nanotechology-based imaging may make it possible to reveal TCR nanostructures in the context of T-cell recognition of antigens and therefore provide new insight into T-cell response or ultimately immunity. 5 Nanotechnology is emerging as a multidisciplinary tool to advance life science and medicine. 6,7 However, nanoscale imaging or dissecting of functional biologic molecules in cells remain challenging. Near-field scanning optical microscopy (NSOM) has proved to be a useful nanotechnology tool for studying hard and flat materials, but its application in biomedical research is still limited. [8][9][10][11] Complicated natures of cell membranes or biologic molecules make it difficult for NSOM to generate high spatial resolution images. Whereas home-made NSOM operating in liquid can yield images of biologic molecules, the current commercial NSOM instruments are all designed for in-air imaging, 12-15 posing a challenge for nanoscale imaging of cell membrane proteins. Although NSOM combined with some common fluorescent materials were used for imaging, 16 the absence of highly photostable fluorophores for use in NSOM is perhap...

IntroductionHuman ␥␦ T cells appear to belong to nonclassical T cells that contribute to both innate and adaptive immune responses. Circulating V␥2V␦2 (also termed V␥9V␦2) T cells exist only in primates and, in humans, constitute 60% to 95% of total blood ␥␦ T cells. V␥2V␦2 T cells in primates can be activated by nonpeptidic phosphorylated metabolites of isoprenoid biosynthesis (eg, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate [HMBPP], isopentenyl pyrophosphate [IPP], and its isomer dimethylallyl pyrophosphate [DMAPP]). [1][2][3] We have recently shown that HMBPP is associated with antigen-presenting cell (APC) membrane and specifically recognized by V␥2V␦2 T-cell receptor (TCR) expressed on V␥2V␦2 T cells. 4 Although HMBPP produced by mycobacteria/other microbes is a potent activator for V␥2V␦2 T cells, these ␥␦ T cells possess both innate and adaptive immune features. [5][6][7][8] The finding that "unprimed" V␥2V␦2 T cells can recognize and react to wide ranges of nonpeptide phospholigands with the capability of "naive" production of cytokines has been interpreted as a pattern recognition-like feature of innate immune cells. On the other hand, the capacity of V␥2V␦2 T cells to undergo major clonal expansion in primary infection and to mount rapid recall-like expansion upon reinfection has been proposed as adaptive immune response of these ␥␦ T cells. 8 Consistent with these memory-type responses is the demonstration of memory phenotypes of V␥2V␦2 T cells in the blood of humans. 9 Accumulating evidence suggests that V␥2V␦2 T cells play a role in mediating immunity against microbial pathogens 8 and tumors. 10 Foxp3-expressiong CD4 ϩ CD25 ϩ regulatory T cells (Tregs) control immune responses to self-antigens and foreign antigens and play a major role in maintaining the balance between immunity and tolerance. 11-14 Murine CD4 ϩ CD25 ϩ regulatory T cells are induced by transforming growth factor ␤ (TGF-␤), although TGF-␤ plus IL-6 favors the development of Th17 cells. 15 [23][24][25][26] We and others have also shown that IL-2 plus phospholigand treatment can induce remarkable expansion of V␥2V␦2 T cells in nonhuman primates. 1,27,28 We therefore took advantage of the IL-2-based in vivo model systems to assess potential interplay or mutual regulation between V␥2V␦2 T cells and Tregs during early mycobacterial infection in nonhuman primates. We found that phosphoantigenactivated V␥2V␦2 T cells were able to down-regulate IL-2-induced expansion of Tregs, and antagonize Treg-driven suppression of in vivo immune responses. Methods AnimalsFour-to 8-year-old, 3-to 4-kg cynomolgus macaques (Macaca fascicularis) were used in this study. A total of 18 monkeys were divided into 3 groups, 6 for each. All animals were maintained and used in accordance with the guidelines of the institutional animal care and use committee of all participating institutions. Animals were anesthetized with 10 mg/kg ketamine HCl (Fort Dodge Animal Health, Fort Dodge, IA) intramuscularly for all blood sampling and treatments. EDTA-anticoagulated...

Tiam1 (T-lymphoma invasion and metastasis 1) is one of the known guanine nucleotide (GDP/GTP) exchange factors (GEFs) for Rho GTPases (e.g., Rac1) and is expressed in breast tumor cells (e.g., SP-1 cell line). Immunoprecipitation and immunoblot analyses indicate that Tiam1 and the cytoskeletal protein, ankyrin, are physically associated as a complex in vivo. In particular, the ankyrin repeat domain (ARD) of ankyrin is responsible for Tiam1 binding. Biochemical studies and deletion mutation analyses indicate that the 11–amino acid sequence between amino acids 717 and 727 of Tiam1 (717GEGTDAVKRS727L) is the ankyrin-binding domain. Most importantly, ankyrin binding to Tiam1 activates GDP/GTP exchange on Rho GTPases (e.g., Rac1).Using an Escherichia coli–derived calmodulin-binding peptide (CBP)–tagged recombinant Tiam1 (amino acids 393–728) fragment that contains the ankyrin-binding domain, we have detected a specific binding interaction between the Tiam1 (amino acids 393–738) fragment and ankyrin in vitro. This Tiam1 fragment also acts as a potent competitive inhibitor for Tiam1 binding to ankyrin. Transfection of SP-1 cell with Tiam1 cDNAs stimulates all of the following: (1) Tiam1–ankyrin association in the membrane projection; (2) Rac1 activation; and (3) breast tumor cell invasion and migration. Cotransfection of SP1 cells with green fluorescent protein (GFP)–tagged Tiam1 fragment cDNA and Tiam1 cDNA effectively blocks Tiam1–ankyrin colocalization in the cell membrane, and inhibits GDP/GTP exchange on Rac1 by ankyrin-associated Tiam1 and tumor-specific phenotypes. These findings suggest that ankyrin–Tiam1 interaction plays a pivotal role in regulating Rac1 signaling and cytoskeleton function required for oncogenic signaling and metastatic breast tumor cell progression.

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