Abstract 2158: ART1 tumor expression mediates immune resistance in non-small cell lung cancer by elimination of P2 × 7R+ CD8 tissue resident memory T cells and conventional type I dendritic cells
Authors: Wennerberg Erik , Hung Clarey , Valeta Amanda , McGraw Timothy , Demaria Sandra , Stiles Brendon 
Published: September 2020

Abstract
Extracellular NAD+ (eNAD) is released from stressed and dying cells where it acts as a proinflammatory mediator. However, in the presence of mono-ADP-ribosyltransferases (ARTs) eNAD serves as a substrate for ADP-ribosylation of the P2 × 7 receptor (P2RX7), resulting in NAD-induced cell death (NICD). P2RX7 is expressed on several immune cells including CD103+ conventional type I dendritic cells (cDC1s) which are essential for initiation of tumor-specific immune responses as well as on tissue resident memory T cells (TRMs), of which high tumor infiltration has been associated with improved survival in non-small cell lung cancer (NSCLC) [1]. NICD of P2 × 7R+ TRMs was recently identified as a regulator of TRM tissue homeostasis [2]. CD38 is expressed on activated immune cells and can reduce eNAD levels by converting eNAD into ADPR. We tested the hypothesis that ART1 expression in NSCLC constitutes a mechanism of immune resistance by mediating NICD of P2 × 7R+ TRMs and P2 × 7R+ cDC1s.
ART1 expression in human NSCLC samples was determined by immunohistochemistry scoring of NSCLC tumors using a tissue microarray (TMA) (n=493). In addition, matched tumor and adjacent normal lung tissue samples were analyzed for ART1 expression by qPCR (n=40) and for infiltration of TRM and cDC1s by flow cytometry. A mouse KP1 lung tumor cell line with high ART1 expression was established from KRASG12D/P53−/− mice and transduced with a doxycycline-inducible shRNA targeting ART1 (ART1KD). KP1 cells and their derivatives were intravenously injected into syngeneic C57BL/6 mice to generate orthotopic lung tumors. Lung tumor nodules were enumerated two weeks after tumor inoculation using H&E-stained FFPE lung sections and lung-derived cell suspensions were analyzed by flow cytometry for infiltration of T cell and DC subsets.
Increased expression of ART1 in tumor compared to normal lung was observed in 55% of NSCLC samples by TMA analysis, and confirmed by qPCR. The frequency of P2RX7+ CD8 TRMs (p&lt;0.05) and P2 × 7R+ cDC1s (p&lt;0.01) were reduced in ART1+ tumors compared to matched normal lung tissue. In line with the hypothesis that CD38 protects P2 × 7R+ cells from ART1-mediated NICD, P2 × 7R+ CD8 TRMs and P2 × 7R+ cDC1s present within ART1+ tumors had elevated expression of CD38. In mice, knockdown of ART1 in KP1 tumors resulted in significantly delayed growth in immunocompetent mice (p&lt;0.001) but not in nude or CD8-depeleted mice. In an orthotopic lung model, ART1KD resulted in a 75% decrease in lung metastatic nodules (p&lt;0.01) and was associated with increased infiltration of CD8 T cells (P&lt;0.0001) and cDC1s (p&lt;0.01). In vitro experiments confirmed that CD38- P2RX7+ CD8 T cells were susceptible to ADP-ribosylation and NICD when co-cultured with ART1high lung tumor cells, while P2 × 7R+ CD8 T cells that co-expressed CD38 were resistant to NICD.
In conclusion, we show that ART1 is overexpressed in NSCLC and identify ART1-mediated NICD of P2 × 7R+ TRMs and cDC1s as a possible novel immune escape mechanism in NSCLC.
Furthermore, we describe a novel role of CD38 expression on immune cells in its protection against NICD.
1. Nizard, M., et al., Induction of resident memory T cells enhances the efficacy of cancer vaccine. Nat Commun, 2017. 8: p. 15221.
2. Stark, R., et al., T RM maintenance is regulated by tissue damage via P2RX7. Sci Immunol, 2018. 3(30).
Citation Format: Erik Wennerberg, Clarey Hung, Amanda Valeta, Timothy McGraw, Sandra Demaria, Brendon Stiles. ART1 tumor expression mediates immune resistance in non-small cell lung cancer by elimination of P2 × 7R+ CD8 tissue resident memory T cells and conventional type I dendritic cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2158.