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

Sézary 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:

1. qRT-PCR analysis revealed that 6 out 13 of SS patients showed an high mRNA levels of CXCL13;

2. 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.

3. 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<0.001) and with MF (p=0.04). In this study we report that both circulating and skin-infiltrating neoplastic lymphocytes of SS patients abundantly express CXCL13. Furthermore, this chemokine is also detectable at high level on plasma of SS patients. Conversely, CXCL13 is not observable in healthy controls as well as in Mycosis Fungoides, a variant of low grade of CTCL. These findings indicate that CXCL13 could play a role in pathobiology of Sézary Syndrome and that the expression of this chemokine could be used as diagnostic marker for this kind of tumor