A neutrophil response linked to tumor control in immunotherapy I

Neutrophils accumulate in solid tumors and their abundance correlates with poor prognosis. Neutrophils are not homogenous, however, and could play different roles in cancer therapy. Here, we investigated the role of neutrophils in immunotherapy leading to tumor control. We show that successful therapies acutely expanded tumor neutrophil numbers. This expansion could be attributed to a Sell-hi state, rather than to other neutrophils that accelerate tumor progression. Therapy-elicited neutrophils acquired an interferon gene signature, also seen in human patients, and appeared essential for successful therapy, as loss of the interferon-responsive transcription factor IRF1 in neutrophils led to failure of immunotherapy. The neutrophil response depended on key components of antitumor immunity, including BATF3-dependent DCs, IL12 and IFNγ. In addition, we found that a therapy-elicited systemic neutrophil response positively correlated with disease outcome in lung cancer patients. Thus, we establish a crucial role of a neutrophil state in mediating effective cancer therapy. KP tumors were induced in C57BL6/J mice, and allowed to grow for 31 days before treating, or not, with aCD40. Two days after aCD40 treatment, the lungs of these mice were perfused, and tumor nodules were macroscopically dissected from the lungs and digested as above to generate single cell suspensions. Cells were then stained with a combination of DNA-tagged TotalSeq-A antibodies and Hashtag antibodies (listed in table S2), stained with a fluorochrome-labelled CD11b antibody for sorting, and labelled with 7AAD to identify live cells. CD11b+ cells were sorted, using a BD FACSAria sorter, into PBS (no Ca or Mg) with 0.04% BSA, at a concentration of 1000-1500 cells/µL. After this, cells were processed with Chromium Next GEM Single Cell 3’ Kit v3.1, 4 rxns (PN-1000269) before loading on a Chromium Next GEM Chip G Single Cell Kit, 16 rxns (PN-1000127). In collaboration with the Single Cell Core Facility at Harvard Medical School, standard steps were followed for library preparation, quality control and amplification. Sequencing was performed in collaboration with the Biopolymers Facility at Harvard Medical School, using the NovaSeqSP platform (1,600,000,000 reads, 20k reads/cell).