Abstract 426: Targeting CXCR4 reduced T regulatory cells (Tregs) -mediated cell proliferation suppression in renal cell carcinoma (RCC) patients
Authors: Santagata Sara , Napolitano Maria , D'Alterio Crescenzo , Cecere Sabrina , De Domenico Renato , Cacciapuoti Carmela , Dimaro Salvatore , Marinelli Luciana , Longo Nicola , Pignata Sandro , Perdonà Sisto , Scala Stefania 
Published: August 2015

Introduction: Renal cell carcinoma (RCC) is an immunogenic cancer with specimens containing tumor infiltrating lymphocytes. T regulatory cells CD4+CD25+Foxp3+ (Tregs) accumulate in RCC and have been implicated in tumor immune escape. Since the chemokine receptor axis CXCR4/CXCL12/CXCR7 plays a crucial role in renal cancer biology and recent evidences demonstrate that targeting CXCR4 modulate inefficient immunotherapy, the aim of the project was to evaluate patients Tregs function from tumor tissue compared to unaffected surrounding tissue and PBL and to study the effect of CXCR4 antagonism on Tregs immuneresponse.

Materials and methods: 26 specimens from RCC, unaffected surrounding tissue and relative PBMCs plus heparinized blood from 15 healthy donors (HD) were collected. Single cells suspensions were obtained; Tregs from renal tumor, unaffected surrounding tissues and peripheral blood and circulating T effector (Teff) cells were isolated using a magnetic isolation cell kit. Treg were identified as CD4/CD25/Foxp3 and characterized for surface activation markers (CTLA-4; CXCR-4; PD-1; CD39; ICOS) by flow cytometry. Tregs suppressive activity was evaluated through in vitro co-colture of purified Tregs versus CFSE-labeled autologous Teff cells. Tregs were added at different ratios and co-cultured for 5 days in presence of anti-CD3/CD28 antibody. CXCR4 antagonism on Tregs suppressive function was evaluated through AMD3100, a selective CXCR4 antagonist, and Peptide R/Peptide R29, a new class of CXCR4 antagonists recently developed (1).

Results: Tregs were evaluated in tumor tissue, unaffected surrounding tissue and relative PBMCs from 26 RCC patients and 15 healthy donors (HD). Higher number of Tregs was detected in the RCC tumor tissues and PBMCs compared to HD (p&lt;0.001). Higher expression of activation markers CTLA-4; CXCR4; PD-1; CD39 and ICOS were detected on Treg isolated from tumor specimen compared to PBMCs Tregs cells (p&lt;0.01) and on peripheral Tregs cells compared to HD (p&lt;0.01). Treg suppression of Teff proliferation induced by intratumoral Tregs was more efficient than proliferation suppression induced by circulating and unaffected surrounding tissue isolated Tregs (p&lt;0.05). CXCR4 antagonism through AMD3100, Peptide R and R29 reverted Treg-mediated proliferation suppression (p&lt;0.05).

Conclusions: Higher number and suppressive activity was detected for Tregs isolated from primary RCC tissues compared to peritumoral unaffected tissue and PBMCs isolated Tregs. CXCR4 antagonism through AMD3100, Peptide R and R29 prevented T effector cell suppression Tregs induced. Targeting CXCL12/CXCR4 axis in renal cancer might be a strategy for immunotherapeutic intervention.

1. Portella, L., et al., PLoS One, 2013. 8(9): p. e74548

Citation Format: Sara Santagata, Maria Napolitano, Crescenzo D'Alterio, Sabrina Cecere, Renato De Domenico, Carmela Cacciapuoti, Salvatore Dimaro, Luciana Marinelli, Nicola Longo, Sandro Pignata, Sisto Perdonà, Stefania Scala. Targeting CXCR4 reduced T regulatory cells (Tregs) -mediated cell proliferation suppression in renal cell carcinoma (RCC) patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 426. doi:10.1158/1538-7445.AM2015-426