Anti-Ly6G binding and trafficking mediate positive neutrophil selection to unleash the anti-tumor efficacy of radiation therapy.

The anti-Ly6G antibody is used to deplete Ly6G <sup>pos</sup> neutrophils and study their role in diverse pathologies. However, depletion is never absolute, as Ly6G <sup>low</sup> neutrophils resistant to depletion rapidly emerge. Studying the functionality of these residual neutrophils is necessary to interpret anti-Ly6G-based experimental designs. In vitro, we found anti-Ly6G binding induced Ly6G internalization, surface Ly6G paucity, and primed the oxidative burst of neutrophils upon TNF α co-stimulation. In vivo, we found neutrophils resistant to anti-Ly6G depletion exhibited anti-neutrophil-cytoplasmic-antibodies. In the pre-clinical Kras <sup>Lox-STOP-Lox-G12D/WT</sup> ; Trp53 <sup>Flox/Flox</sup> mouse lung tumor model, abnormal neutrophil accumulation and aging was accompanied with an N2-like SiglecF <sup>pos</sup> polarization and ly6g downregulation. Consequently, SiglecF <sup>pos</sup> neutrophils exposed to anti-Ly6G reverted to Ly6G <sup>low</sup> and were resistant to depletion. Noting that anti-Ly6G mediated neutrophil depletion alone had no anti-tumor effect, we found a long-lasting rate of tumor regression (50%) by combining anti-Ly6G with radiation-therapy, in this model reputed to be refractory to standard anticancer therapies. Mechanistically, anti-Ly6G regulated neutrophil aging while radiation-therapy enhanced the homing of anti-Ly6G-boundSiglecF <sup>neg</sup> neutrophils to tumors. This anti-tumor effect was recapitulated by G-CSF administration prior to RT and abrogated with an anti-TNFα antibody co-administration. In summary, we report that incomplete depletion of neutrophils using targeted antibodies can intrinsically promote their oxidative activity. This effect depends on antigen/antibody trafficking and can be harnessed locally using select delivery of radiation-therapy to impair tumor progression. This underutilized aspect of immune physiology may be adapted to expand the scope of neutrophil-related research