The B-Cell Chemoattractant Factor CXCL13 Is Expressed in the Malignant Lymphocyte of the Sezary Syndrome.

Sezary Syndrome (SS) is a rare and aggressive form of Cutaneous T-Cell Lymphoma (CTCL) characterised by a distinct metastatic pattern mainly involving blood and skin. Our expression analyses performed by microarrays demonstrated that many chemokines resulted up-regulated in this type of lymphoma. Since these chemoattractant molecules play a critical role in cellular recruitment and homing to tissues and in the metastatic process of several tumors, we focused our attention on one of them named CXCL13, a lymphoid chemokine involved in B-cell compartmental homing within secondary lymphoid organs. Peripheral Blood Mononuclear cells (PBMCs) were isolated from blood obtained from SS patients and controls by Ficoll-Hypaque density gradient centrifugation (Sigma Aldrich). SS cells and healthy resting CD4+ lymphocytes were purified by positive selection using an anti-human-CD4 conjugated dynabeads (Oxoid). Total RNA was extracted using the Trizol reagent (Life Technologies). Quantitative-Real Time RT-PCR analysis was performed on CD4+ sorted from 14 SS patients and 3 controls. CXCL13 primers were designed by means of the Primer Express software package (Applied Biosystems). The qRT-PCR were performed with a SYBR Green I dye chemistry and AmpliTaq Gold DNA Polymerase on an ABI PRISM 7000 machine (Applied Biosystems). Immunohistochemistry analyses for CXCL13 were performed on formalin-fixed, paraffin-embedded skin biopsies from 15 SS, 15 MF, 6 MF-B cell rich patients using streptoavidin-biotin peroxidase labeling method (DAKO). Sections were counterstained with hematoxylin. Plasma CXCL13 levels were determined using a CXCL13 ELISA kit (BD Pharmingen). Results can be summarized as follow: qRT-PCR analysis revealed that 6 out 13 of SS patients showed an high mRNA levels of CXCL13; Immunohistochemistry analysis showed that CXCL13 is abundantly expressed by neoplastic skin-infiltrating lymphocytes of 9 out 15 SS skin biopsies. Conversely, CXCL13 is weakly expressed on scattered neoplastic skin-infiltrating lymphocytes of 1 out 15 MF and 1 out 6 MF-B cell rich biopsies. Plasma CXCL13 concentrations in SS patients ( n = 10) were 1362 ± 134 pg/mL. Conversely, those in MF patients ( n = 10) and healthy donors ( n = 5) were 70 ± 43 and 13 ± 10 pg/mL, respectively. Compared with healthy controls, plasma CXCL13 levels were significantly higher in patients with SS (p