Briefly, unstimulated cells were maintained in a 37 °C incubator for 1 and 3 h and then fixed with 4% paraformaldehyde. NETosis was induced in 2.5 × 104 neutrophils suspended in 250 μL of EBM2 media in 8-well Millicell EZ glass chamber slides (Millipore, PEZGS0816). The cells were then stained for neutrophil markers as above and analyzed by flow cytometry. The cells were lifted by incubating for 15 min in PBS/5 mM EDTA/1 mM sodium azide plus proteinase K to cleave any beads on the surface of the cells. Neutrophils from the lungs of B16-tumor bearing mice were plated at 2.5 × 105/well in a 24 well plate and 5 µL/well of FITC-labeled zymosan A S. Replacement of testosterone effectively normalized the tumor burden in castrated male mice. The immune system is known to play a key role in controlling the growth and spread of malignancies, but whether age- and sex-dependent changes in immune cell function account for this effect remains unknown. Given the documented impact of sex on immune responses, it is hard to understand why in most ICI clinical trials a substantially larger fraction of males was included which could lead to bias in the results. Altogether, the data concerning a sex-bias in the response to current ICI treatments are ambiguous at present. The total number of cells was automatically calculated with the Keyence BioAnalyzer software and the number of neutrophils was counted by a blinded scientist. Stained cells were analyzed on an LSRII Fortessa (BD Biosciences) and data were processed using FlowJo, version 10.5 (Tree Star Inc.). To isolate murine neutrophils or NK cells from the bone marrow, spleen, or lungs, an EasySep Positive PE Selection Kit (19762 A) or Negative Selection Mouse Neutrophil Enrichment Kit (18557) was used according to the manufacturer’s protocols (Stemcell Technologies). The bone marrow cells were collected by flushing the femurs of donor mice (C57BL/6, ArTfm, or Ly5.1) under sterile conditions and were prepared as a single cell suspension using a 70 μM nylon filter (BD, Falcon, ). Seven weeks later, prior to intrahepatic ameba challenge, serum testosterone concentrations were determined indicating similar levels between male and testosterone-substituted female mice (Fig. 2B). Gradient purified liver lymphocytes from female and male C57BL/6 mice (10–12 weeks old) were stained with PE-labelled- αGalCer -CD1d tetramer (Proimmune) and FITC-labelled anti-CD4. In vitro stimulation of liver lymphocytes from testosterone treated female mice and orchiectomized male mice. Gender dependent differences were not a result of differences in the total number of NKT cells, but a result of a higher activation potential for the CD4− NKT cell subpopulation in female mice. This high level production of IFNγ in female derived NKT cells was inhibited by testosterone substitution, while the IFNγ production in male derived NKT cells was increased by orchiectomy. Statistical tests and n-counts are indicated in the figure legends. Unless stated otherwise, the visualization and statistical evaluation of the data were performed with GraphPad Prism (V10.4.2). Image analysis, including cell segmentation, classification, gating, and TME subtyping, was performed using MACSima IQ View software (definitions provided in Supplementary Table T3). All tumors were partially fixed in 4% paraformaldehyde (Carl Roth GmbH), embedded in paraffin, and sectioned at 5 µm. The UTX-KO gene set reflects the intersection of Kdm6a-KO, Kdm6a-Chip data, and cut&seq data according to Cheng et al. 25 The detailed composition of this gene set is provided in Supplementary Table T2. Hallmarks, M2 chemical and genetic perturbations (M2cgp), M2 – canonical pathways Reactome and Biocarta, M7 – immunology gene set collections, NK cell sets by Cui et al. 54, and the UTX-KO gene set were analyzed. PolyA enrichment was performed using oligo dT beads, followed by strand-specific library construction with the Stranded mRNA Prep kit (Illumina) according to the manufacturer’s instructions.48 Paired-end sequencing was carried out on a NextSeq 1000 instrument (Illumina) with a P2 flow cell and a P200 cartridge (Illumina). However, the extent to which the actions of these hormones underlie sex-specific vulnerabilities to disease has remained unclear. However, the reason for the rise in melanoma incidence in aging males remains to be elucidated. In addition, numerous studies have confirmed that sex is an independent prognostic factor even after adjustment for Breslow thickness, histological subtype, body site, age, ulceration, vascular invasion, mitotic rate, sentinel lymph node positivity, and detection and diagnostic delay8–14. Interestingly, the rate of malignant melanoma is similar in men and women aged 15–45 years, but after age 50, the rate in males increases dramatically4, and nearly two-thirds of melanoma deaths are male2. In the United States, the incidence of melanoma is rising faster than any other preventable cancer1.
