We explored the mechanism of action of CD39 antibodies that inhibit ectoenzyme CD39 conversion of extracellular ATP (eATP) to AMP and thus potentially augment eATP-P2-mediated proinfl ammatory responses. Using syngeneic and humanized tumor models, we contrast the potency and mechanism of anti-CD39 mAbs with other agents targeting the adenosinergic pathway. We demonstrate the critical importance of an eATP-P2X7-ASC-NALP3infl ammasome-IL18 pathway in the antitumor activity mediated by CD39 enzyme blockade, rather than simply reducing adenosine as mechanism of action. Effi cacy of anti-CD39 activity was underpinned by CD39 and P2X7 coexpression on intratumor myeloid subsets, an early signature of macrophage depletion, and active IL18 release that facilitated the signifi cant expansion of intratumor effector T cells. More importantly, anti-CD39 facilitated infi ltration into T cell-poor tumors and rescued anti-PD-1 resistance. Anti-human CD39 enhanced human T-cell proliferation and Th1 cytokine production and suppressed human B-cell lymphoma in the context of autologous Epstein-Barr virus-specifi c T-cell transfer. SIGNIFICANCE :Overall, these data describe a potent and novel mechanism of action of antibodies that block mouse or human CD39, triggering an eATP-P2X7-infl ammasome-IL18 axis that reduces intratumor macrophage number, enhances intratumor T-cell effector function, overcomes anti-PD-1 resistance, and potentially enhances the effi cacy of adoptive T-cell transfer.

Highlights d TCR signaling and CD8 + T effector program are altered by the absence of CD226 d Dysfunctional CD226 neg CD8 + TILs accumulate in human and mouse tumors d Eomes overexpression is involved in CD226 loss by CD8 + TILs d CD226 loss limits the efficacy of immune checkpoint blockade and CD137 agonists.

Highlights d Dysfunctional CD226-negative T cells accumulate in mouse and human tumors d CD155 induces ubiquitination via CBL-B and proteasomal degradation of CD226 d A mutation of Y319 maintains CD226 expression and improves anti-tumor immunity d ICB-therapy in melanoma patients relies on the presence of CD226 + CD8 + T cells

Patients with ataxia-telangiectasia (A-T) lack a functional ATM kinase protein and exhibit defective repair of DNA double-stranded breaks and response to oxidative stress. We show that CRISPR/Cas9-assisted gene correction combined with piggyBac (PB) transposon-mediated excision of the selection cassette enables seamless restoration of functional ATM alleles in induced pluripotent stem cells from an A-T patient carrying compound heterozygous exonic missense/frameshift mutations, and from a patient with a homozygous splicing acceptor mutation of an internal coding exon. We show that the correction of one allele restores expression of ~ 50% of full-length ATM protein and ameliorates DNA damage-induced activation (auto-phosphorylation) of ATM and phosphorylation of its downstream targets, KAP-1 and H2AX. Restoration of ATM function also normalizes radiosensitivity, mitochondrial ROS production and oxidative-stress-induced apoptosis levels in A-T iPSC lines, demonstrating that restoration of a single ATM allele is sufficient to rescue key ATM functions. Our data further show that despite the absence of a functional ATM kinase, homology-directed repair and seamless correction of a pathogenic ATM mutation is possible. The isogenic pairs of A-T and gene-corrected iPSCs described here constitute valuable tools for elucidating the role of ATM in ageing and A-T pathogenesis.

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