T淋巴細胞的遷移失常，而導致過度累積在發炎處，是造成慢性發炎的主要因素。T淋巴細胞遷移是由趨化因子(chemokine) 與其受體所調控的一個高度協調的過程。了解T淋巴細胞遷移的機制有助於釐清許多發炎相關疾病的病因。趨化因子受體CXCR4及其配體(ligand) – 基質細胞衍生因子(stromal cell-derived factor 1; SDF-1) 信號調控細胞遷移及附著力。ZAP70蛋白即是由CXCR4/SDF-1信號所活化的酪氨酸激酶(tyrosine kinase)。先前研究顯示，在SDF-1趨化物梯度下爬行的細胞，其ZAP70蛋白會不平均的分布在細胞前後兩端。一部分的ZAP70蛋白聚集在移動中的T細胞偽足前端，調控著T細胞趨化移動的方向性，另一部分則是聚集在尾端。在本篇研究中發現，SDF-1刺激會促成ZAP70和CXCR4形成複合體。並且在Jurkat與人類T淋巴細胞中觀察到hZAP70-EGFP融合蛋白的分布位置與CXCR4相近，且部分重疊。因此，我們認為在ZAP70蛋白聚集到細胞前緣促成偽足形成的過程，可能需要CXCR4。為了探討在細胞爬行的過程中，CXCR4與ZAP70位置的動態變化，我們建構hCXCR4與螢光蛋白DsRed結合的融合蛋白。在轉染至T細胞後，利用RT-PCR確認其表現。 hCXCR4-DsRed 的表現增加T細胞對SDF-1的穿透性的移動能力。表現hCXCR4-DsRed的細胞，在SDF-1刺激下，活化的ZAP70、ERK及1整合素增加。當hCXCR4-DsRed及hZAP70-EGFP融合蛋白同時轉染至T細胞，在爬行的細胞中，hCXCR4-DsRed的分布位置和hZAP70-EGFP重疊。透過cell^R 曠時影像系統觀察這些爬行的細胞可見，hCXCR4-DsRed首先聚集在細胞膜上，之後hZAP70-EGFP聚集到相近的位置，造成新偽足的形成。綜合以上結果，我們認為在SDF-1刺激下，CXCR4透過與ZAP70調控新偽足的形成，進而控制T細胞趨化移動的方向性。

Aberrant lymphocytes accumulation due to disregulation of T cell migration is a major cause of chronic inflammation. T lymphocyte migration is a highly coordinated process that is regulated by chemokines and its receptors. Identifying the mechanism of T lymphocyte migration may contribute to clarify the etiology of many inflammation- related disorders. Chemokine receptor CXCR4 and its ligand stromal cell-derived factor 1 (SDF-1) regulate cell migration and adhesion. ZAP70 protein is a tyrosine kinase activated by CXCR4/SDF-1 signaling. Our previous studies showed that ZAP70 protein accumulated unevenly in migrating cells in a chemoattractant SDF-1 gradient. A part of them is localized close to the protruding membrane of lamellipodia along with direction of migration, and the others are localized at the trailing edge. In this study, we found that ZAP70 bound to CXCR4 after SDF-1 stimulation. ZAP70 protein was co-localized with CXCR4 by CXCR4 antibody-induced patching of Jurkat and primary T cells. Therefore, we hypothesized that CXCR4 plays a role in the recruitment of ZAP70 protein at lemallipodia. To investigate the interaction between CXCR4 and ZAP70, we have constructed hCXCR4-hDsRed fusion protein to study the dynamic localization of ZAP70 and CXCR4 during T lymphocyte migration. The expression of pDsRed-hCXCR4 in both Jurkat and P116 T cells was confirmed by RT-PCR. Overexpression of CXCR4 increased the transmigration towards SDF-1 and induced ZAP70, ERK phosphorylation and 1-integrin activation, suggesting that the hCXCR4-DsRed protein is functional. We then co-expressed both hCXCR4-DsRed and hZAP70-EGFP fusion proteins in T cells and found that the distribution of hCXCR4-DsRed overlapped with hZAP70-EGFP during cell migration. By using cell^R time-lapse imaging system, we observed that hCXCR4-DsRed first located at the cell membrane then recruited hZAP70-EGFP and protruding lamellipodium was formed. In conclusion, CXCR4 plays a role in regulating the formation of lemellipodia and controlling the directionality of T cell migration through interaction with ZAP70.

Ait-Oufella H, Sage AP, Mallat Z, Tedgui A (2014) Adaptive (T and B cells) immunity and control by dendritic cells in atherosclerosis. Circ Res 114: 1640-1660

Alsayed Y, Ngo H, Runnels J, Leleu X, Singha UK, Pitsillides CM, Spencer JA, Kimlinger T, Ghobrial JM, Jia X, Lu G, Timm M, Kumar A, Cote D, Veilleux I, Hedin KE, Roodman GD, Witzig TE, Kung AL, Hideshima T, Anderson KC, Lin CP, Ghobrial IM (2007) Mechanisms of regulation of CXCR4/SDF-1 (CXCL12)-dependent migration and homing in multiple myeloma. Blood 109: 2708-2717

Au-Yeung BB, Deindl S, Hsu LY, Palacios EH, Levin SE, Kuriyan J, Weiss A (2009) The structure, regulation, and function of ZAP-70. Immunol Rev 228: 41-57

Biddison WE (2001) Preparation and culture of human lymphocytes. Curr Protoc Cell Biol Chapter 2: Unit 2 2

Bleul CC, Fuhlbrigge RC, Casasnovas JM, Aiuti A, Springer TA (1996) A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 (SDF-1). J Exp Med 184: 1101-1109

Chan AC, Iwashima M, Turck CW, Weiss A (1992) ZAP-70: a 70 kd protein-tyrosine kinase that associates with the TCR zeta chain. Cell 71: 649-662

Chen W, Zhu C (2013) Mechanical regulation of T-cell functions. Immunol Rev 256: 160-176

Cho HH, Kyoung KM, Seo MJ, Kim YJ, Bae YC, Jung JS (2006) Overexpression of CXCR4 increases migration and proliferation of human adipose tissue stromal cells. Stem Cells Dev 15: 853-864

Cope AP, Schulze-Koops H, Aringer M (2007) The central role of T cells in rheumatoid arthritis. Clin Exp Rheumatol 25: S4-11

Friedman RS, Jacobelli J, Krummel MF (2005) Mechanisms of T cell motility and arrest: deciphering the relationship between intra- and extracellular determinants. Semin Immunol 17: 387-399

Fuss IJ, Kanof ME, Smith PD, Zola H (2009) Isolation of whole mononuclear cells from peripheral blood and cord blood. Curr Protoc Immunol Chapter 7: Unit7 1

Garcia-Bernal D, Parmo-Cabanas M, Dios-Esponera A, Samaniego R, Hernan PdlOD, Teixido J (2009) Chemokine-induced Zap70 kinase-mediated dissociation of the Vav1-talin complex activates alpha4beta1 integrin for T cell adhesion. Immunity 31: 953-964

Gouwy M, Struyf S, Berghmans N, Vanormelingen C, Schols D, Van Damme J (2011) CXCR4 and CCR5 ligands cooperate in monocyte and lymphocyte migration and in inhibition of dual-tropic (R5/X4) HIV-1 infection. Eur J Immunol 41: 963-973

Guyon A (2014) CXCL12 chemokine and its receptors as major players in the interactions between immune and nervous systems. Front Cell Neurosci 8: 65

Heckmann D, Laufs S, Maier P, Zucknick M, Giordano FA, Veldwijk MR, Eckstein V, Wenz F, Zeller WJ, Fruehauf S, Allgayer H (2011) A Lentiviral CXCR4 overexpression and knockdown model in colorectal cancer cell lines reveals plerixafor-dependent suppression of SDF-1alpha-induced migration and invasion. Onkologie 34: 502-508

Huttenlocher A, Horwitz AR (2011) Integrins in cell migration. Cold Spring Harb Perspect Biol 3: a005074

Kahn J, Byk T, Jansson-Sjostrand L, Petit I, Shivtiel S, Nagler A, Hardan I, Deutsch V, Gazit Z, Gazit D, Karlsson S, Lapidot T (2004) Overexpression of CXCR4 on human CD34+ progenitors increases their proliferation, migration, and NOD/SCID repopulation. Blood 103: 2942-2949

Kremer KN, Kumar A, Hedin KE (2011) G alpha i2 and ZAP-70 mediate RasGRP1 membrane localization and activation of SDF-1-induced T cell functions. J Immunol 187: 3177-3185

Kumar A, Humphreys TD, Kremer KN, Bramati PS, Bradfield L, Edgar CE, Hedin KE (2006) CXCR4 physically associates with the T cell receptor to signal in T cells. Immunity 25: 213-224

Kummer C, Petrich BG, Rose DM, Ginsberg MH (2010) A small molecule that inhibits the interaction of paxillin and alpha 4 integrin inhibits accumulation of mononuclear leukocytes at a site of inflammation. J Biol Chem 285: 9462-9469

Le Clainche C, Carlier MF (2008) Regulation of actin assembly associated with protrusion and adhesion in cell migration. Physiol Rev 88: 489-513

Lin YP, Cheng YJ, Huang JY, Lin HC, Yang BC (2010) Zap70 controls the interaction of talin with integrin to regulate the chemotactic directionality of T-cell migration. Mol Immunol 47: 2022-2029

Liu X, Duan B, Cheng Z, Jia X, Mao L, Fu H, Che Y, Ou L, Liu L, Kong D (2011) SDF-1/CXCR4 axis modulates bone marrow mesenchymal stem cell apoptosis, migration and cytokine secretion. Protein Cell 2: 845-854

Luster AD, Alon R, von Andrian UH (2005) Immune cell migration in inflammation: present and future therapeutic targets. Nat Immunol 6: 1182-1190

Matalon O, Reicher B, Barda-Saad M (2013) Wiskott-Aldrich syndrome protein--dynamic regulation of actin homeostasis: from activation through function and signal termination in T lymphocytes. Immunol Rev 256: 10-29

Moll NM, Ransohoff RM (2010) CXCL12 and CXCR4 in bone marrow physiology. Expert Rev Hematol 3: 315-322
Moser B, Loetscher P (2001) Lymphocyte traffic control by chemokines. Nat Immunol 2: 123-128

Okabe S, Fukuda S, Broxmeyer HE (2002) Activation of Wiskott-Aldrich syndrome protein and its association with other proteins by stromal cell-derived factor-1alpha is associated with cell migration in a T-lymphocyte line. Exp Hematol 30: 761-766

Ottoson NC, Pribila JT, Chan AS, Shimizu Y (2001) Cutting edge: T cell migration regulated by CXCR4 chemokine receptor signaling to ZAP-70 tyrosine kinase. J Immunol 167: 1857-1861

Peacock JW, Jirik FR (1999) TCR activation inhibits chemotaxis toward stromal cell-derived factor-1: evidence for reciprocal regulation between CXCR4 and the TCR. J Immunol 162: 215-223

Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, Borisy G, Parsons JT, Horwitz AR (2003) Cell migration: integrating signals from front to back. Science 302: 1704-1709

Roifman CM, Dadi H, Somech R, Nahum A, Sharfe N (2010) Characterization of zeta-associated protein, 70 kd (ZAP70)-deficient human lymphocytes. J Allergy Clin Immunol 126: 1226-1233

Rose DM, Grabovsky V, Alon R, Ginsberg MH (2001) The affinity of integrin alpha(4)beta(1) governs lymphocyte migration. J Immunol 167: 2824-2830

Severson C, Hafler DA (2010) T-cells in multiple sclerosis. Results Probl Cell Differ 51: 75-98

Stutte S, Quast T, Gerbitzki N, Savinko T, Novak N, Reifenberger J, Homey B, Kolanus W, Alenius H, Forster I (2010) Requirement of CCL17 for CCR7- and CXCR4-dependent migration of cutaneous dendritic cells. Proc Natl Acad Sci U S A 107: 8736-8741
Tan W, Martin D, Gutkind JS (2006) The Galpha13-Rho signaling axis is required for SDF-1-induced migration through CXCR4. J Biol Chem 281: 39542-39549

Teicher BA, Fricker SP (2010) CXCL12 (SDF-1)/CXCR4 pathway in cancer. Clin Cancer Res 16: 2927-2931

Ticchioni M, Charvet C, Noraz N, Lamy L, Steinberg M, Bernard A, Deckert M (2002) Signaling through ZAP-70 is required for CXCL12-mediated T-cell transendothelial migration. Blood 99: 3111-3118

van Buul JD, Voermans C, van Gelderen J, Anthony EC, van der Schoot CE, Hordijk PL (2003) Leukocyte-endothelium interaction promotes SDF-1-dependent polarization of CXCR4. J Biol Chem 278: 30302-30310

Wu CY (2011) The role of ZAP70 in regulating the chemotactic directionality of T lymphocyte migration. National Chang Kong University, Tainan, Taiwan

Zenewicz LA, Antov A, Flavell RA (2009) CD4 T-cell differentiation and inflammatory bowel disease. Trends Mol Med 15: 199-207

Zlotnik A, Yoshie O (2000) Chemokines: a new classification system and their role in immunity. Immunity 12: 121-127

Zweemer AJ, Toraskar J, Heitman LH, AP IJ (2014) Bias in chemokine receptor signalling. Trends Immunol 35: 243-252