Effective, Long-Term, Neutrophil Depletion Using a Murinized Anti-Ly-6G 1A8 Antibody

Abstract

Neutrophils are crucial innate immune cells but also play key roles in various diseases, such as cancer, where they can perform both pro- and anti-tumorigenic functions. To study the function of neutrophils in vivo, these cells are often depleted using Ly-6G or Gr-1 depleting antibodies or genetic "knockout" models. However, these methods have several limitations, being only partially effective, effective for a short term, and lacking specificity or the ability to conditionally deplete neutrophils. Here, we describe the use of a novel murinized Ly-6G (1A8) antibody. The murinized Ly-6G antibody is of the mouse IgG2a isotype, which is the only isotype that can bind all murine Fcγ receptors and C1q and is, therefore, able to activate antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) pathways. We show that this mouse-Ly-6G antibody shows efficient, long-term, and near-complete (>90%) neutrophil depletion in the peripheral blood of C57Bl6/J, Balb/c, NXG and SCID mice for up to at least four weeks, using a standardized neutrophil depletion strategy. In addition, we show that neutrophils are efficiently depleted in the blood and tumor tissue of IMR32 tumor-bearing SCID mice, analyzed six weeks after the start of the treatment.

Keywords: cancer; immunotherapy; in vivo neutrophil targeting; neutrophil depletion; neutrophils.

Figure 1

Mouse-Ly-6G efficiently depletes neutrophils in old C57Bl6/J mice. (a) Experimental set-up; i.p. injections with the depletion-antibodies or vehicle control (PBS) were performed three times a week for four weeks. Blood was drawn via cheek puncture before injection with the antibodies; (b) FACS dot plots of a representative PBS-treated mouse, showing the gating strategy to retrieve the number of neutrophils per 5000 beads. First, the latex beads and CD45⁺ cells were selected. From the CD45⁺ population, Siglec-F and CD115 positive cells were excluded. Based on SSC/FSC profile, the neutrophils were selected and purity was confirmed using Gr-1 and CD11b positivity; (c–e) Longitudinal analysis of the number of CD45⁺ Siglec-F⁻ CD115⁻ SSC^high Gr-1⁺ CD11b⁺ neutrophils per 5000 beads in the peripheral blood (n = four mice per subgroup) showing (c) inefficient depletion with 50 μg rat-Ly-6G antibody; (d) efficient short-term depletion with 25 μg mouse-Ly-6G spanning the first week, and (e) efficient long-term depletion with 100 μg mouse-Ly-6G. Data is presented as mean with standard error of the mean (SEM). Statistics: one-way ANOVA with Bonferroni correction, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

Figure 2

Mouse-Ly-6G efficiently depleted neutrophils in C57Bl6/J, Balb/c, NXG, and SCID mice. (a) Experimental set-up; i.p. injections with the depletion antibodies were performed three times a week for four weeks. Blood was drawn via cheek puncture once a week before injection with the antibodies; (b,c) Longitudinal analysis of the number of CD45⁺ Siglec-F⁻ CD115⁻ SSC^high Gr-1⁺ CD11b⁺ neutrophils in the peripheral blood (n = five mice per group) per 5000 latex beads, showing; (b) Significant, almost complete, neutrophil depletion in all mouse strains tested (C57Bl6/J, Balb/c, NXG, and SCID) upon treatment with 100 μg mouse-Ly-6G antibody, and (c) Significantly better neutrophil depletion in 100 μg mouse-Ly-6G treated C57Bl6/J mice than when treated with 100 μg rat-Ly-6G antibody. Data is presented as mean with SEM. Statistics: one-way ANOVA with Bonferroni correction. Repeated measures one-way ANOVA with Bonferroni correction was used to compare groups in Figure 2c, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

Figure 3

Mouse-Ly-6G efficiently depletes neutrophils in the blood and tumor of IMR32 tumor-bearing SCID mice (a) Experimental set-up; 2.5 × 10⁶ IMR32 cells were s.c. injected in SCID mice in a 1:2 mix with Vitrogel Hydrogel Matrix. Tumors were established for 28 days, after which mice were randomized over the different treatment groups, and treatment was started. I.p. injections with PBS, IgA ch14.18, and mouse-Ly-6G were performed three times a week for six weeks. The SIRPα-D1 fusion protein was injected i.p. every nine days. Blood was drawn via cheek puncture once a week before injection with the antibodies; (b) Longitudinal analysis of the percentage of SSC^high Ly-6C^low Ly-6G⁺ CD11b⁺ neutrophils in the peripheral blood (n = 2–5 mice per group) of the CD45+ leukocyte population, showing complete neutrophil depletion upon treatment with 100 μg mouse-Ly-6G antibody. Of note; blood at day 70 was collected from the orbit and processed in a similar fashion as the tumor material, meaning cells were fixed at a later time point, possibly resulting in some neutrophil cell death; (c) Intratumoral analysis of the percentage of SSC^high Ly-6C^low Ly-6G⁺ CD11b⁺ neutrophils of the live (TO-PRO-3 negative) cells, CD45+ leukocytes, and CD11b+ myeloid populations. Data showed increased tumor infiltration when the mice were treated with IgA ch14.18/SIRPα-D1 fusion, which was completely ablated when 100 μg mouse Ly-6G was added. Data is presented as mean with SEM. Statistics: one-way ANOVA with Bonferroni correction. Repeated measures one-way ANOVA with Bonferroni correction was used to compare groups in Figure 3b, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

Figure 4

Mouse-Ly-6G efficiently depletes neutrophils in FcRγ-deficient mice. (a) Experimental set-up; FcRγ^−/− mice (n = 5) were i.p. injected three times a week with 100 μg mouse-Ly-6G antibody, and blood was drawn via cheek puncture before the start of the experiment, and at days 5, 9, and 14; (b): FcRγ^−/− mice show depletion of the number of CD45⁺ Siglec-F⁻ CD115⁻ SSC^high Gr-1⁺ CD11b⁺ neutrophils in the peripheral blood (shown per 5000 latex beads) at all time points. Data is presented as mean with SEM. Statistics: one-way ANOVA with Bonferroni correction, ** = p < 0.01, *** = p < 0.001.