Abstract A82: Neutrophils recruit T-regulatory cells into the tumor via secretion of CCL17 – a new mechanism of tumor immune surveillance.

Abstract

Abstract Introduction: Regulatory T cells (T-regs) play an important role in immunological self-tolerance and are functionally immune-suppressive subsets of T cells. It has been repeatedly shown that T-regs are able to migrate in response to CCL22 and CCL17, and that depletion or inhibition of T-regs can enhance anti-tumor immunity. However, there is still limited data on the mechanisms of accumulation of T-regs within the tumor microenvironment. We have recently described the characteristics of “native” pro-tumorigemic N2 tumor-associated Neutrophils (TAN), and found that TAN significantly up-regulated many chemokines compared to bone-marrow (BM) naïve neutrophils. One of the highly up-regulated genes in TAN is CCL17. Aims: We aimed to study the role of TAN in the secretion of CCL17 and the recruitment of T-regs into the tumor microenvironment. We hypothesized that the secretion of CCL17 by N2 TAN has an important role in the recruitment of T-regs, thus inhibiting the T-cytotoxic cell activity in the tumor. Material and Methods: CCL17 production by N1 and N2 TAN and by BM naive Neutrophils was evaluated using ELISA, RT-PCR and IHC staining. We conducted in vivo and in vitro migration assays, and used depletion of TAN to reveal the role of CCL17 secreted from TAN in chemoattraction of T-regs. Results: N2-TAN were more capable of producing CCL17 ex-vivo than N1-TAN or BM neutrophils. The level of CCL17 secreted from TAN increased during tumor development. In vitro migration assay using CD4+CD25+ cells purified from spleens of naïve mice revealed that migration of T-cells towards TAN were 5.27±1.76 times higher compared to BM neutrophils (p<0.05). Furthermore, migration was inhibited by neutralizing anti-mouse CCL17. In vivo migration assay using the air pouch model strengthen these results, demonstrating that more CD4+CD25+ cells migrated into the pouch when injecting TAN compared to BM neutrophils, again inhibited using anti-CCL17 mAb, suggesting that TAN-induced T-regs chemoattraction is CCL17 dependent. Systemic neutrophil depletion after tumor establishment reduced T-reg migration into the tumor. Conclusions: Our results show that TAN from established tumors produced CCL17, increasing T-regs migration into the tumor. These results suggest a new indirect mechanism by which TAN may inhibit the anti-tumor immune activity, promoting tumor growth. Citation Format: Inbal Mishalian, rachel Bayuh, Lida Zolotarov, Liran Levy, Zvi Gregorio Fridlender. Neutrophils recruit T-regulatory cells into the tumor via secretion of CCL17 – a new mechanism of tumor immune surveillance. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A82.