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. 2021 Sep 16;9(9):e002688. doi: 10.1136/jitc-2021-002688

Figure 5.

Figure 5

Neutrophil depletion abrogates the survival advantage of CFA treatment. (A) Mice bearing P815 tumors were treated with intratumoral CFA or PBS for controls. Mice additionally received either 0.2 mg anti-GR-1(RB6-8C5) or anti-isotype control antibody (LTF-2) by intraperitoneal injection every 2–3 days, beginning 3 days prior to CFA /PBS treatment. For statistical analysis, mice were stratified by treatment and by neutrophil status (neutrophil depleted or non-depleted). No interaction between neutrophil depletion and treatment was observed (p=0.230), indicating that treatment had no effect on the survival of neutrophil-depleted mice, and neutrophil depletion was found to significantly reduce survival (p=0.00016, HRs: non-depleted=1, GR-1-depleted=4.28). The median survival of CFA+anti-GR-1 mice was 5 days post-treatment compared with 13 days and 14 days for CFA and CFA+isotype control mice, respectively. The median survival of PBS+anti-GR-1 mice was 6 days post-treatment compared with 10 days for both PBS and PBS+isotype control mice. CFA n=4, PBS n=3, CFA+anti-isotype n=5, PBS+anti-isotype n=5, CFA+anti-GR-1 n=10, PBS+anti-GR-1 n=10. Data pooled from two separate experiments. (B) Assessment of neutrophil depletion in the spleen and bone marrow. Bone marrow cells from a non-depleted control mouse and a GR-1-depleted mouse were processed and stained with 7-AAD, anti-CD45.2-Alexa fluor 488, anti-CD11b-PE-Cy7, anti-Ly6G-PE, and anti-Ly6C-Brilliant violet 421 (BV421). Cells have been gated for CD45.2 positive, live (7-AAD negative) singlet cells. The histograms show that the proportion of myeloid cells (CD11b+) were similar in both non-depleted control mice (top panels) and GR-1-depleted mice (bottom panels). From the myeloid population, distinct populations of both neutrophils (Ly6G+Ly6C+) and monocytes (Ly6G- Ly6C+) could be identified in the non-depleted control mice. However, the neutrophil population is absent from the GR-1-depleted mouse, indicating that neutrophils had been successfully depleted using the anti-GR-1 antibody (RB6-8C5) (C). Assessment of neutrophil depletion in mice with P815 tumors. neutrophils were depleted from mice bearing P815 tumors starting 3 days prior to CFA treatment using 0.2 mg anti-GR-1 antibody (RB6-8C5), which was re-administered every 2–3 days. FNAs were taken from P815 tumors 3 days post-CFA treatment to ensure successful depletion of neutrophils by flow cytometry. FNA samples were pooled into groups from GR-1-depleted and isotype control mice. The neutrophil population (CD11b+Ly6G+Ly6C+) is present in the isotype control mice. CFA, complete Freund’s adjuvant; FNA, fine-needle aspirate; PBS, phosphate-buffered saline; 7-AAD, 7-aminoactinomycin-D.