Efficient and specific Ly6G⁺cell depletion: A change in the current practices toward more relevant functional analyses of neutrophils

Abstract

Neutrophils orchestrate the innate immune response against microorganisms and are increasingly recognized to modulate cancer development in primary tumors and metastases.To address their function in vivo, different approaches are used, the most common ones relying on antibody-mediated neutrophil depletion. By comparing the effects of two widely used antibodies, we demonstrate a strong efficacy but a lack of specificity for anti-Gr1. In contrast, anti-Ly6G lacks neutrophil-depletion capacity in C57BL/6 mice, which can be explained by an insufficient celerity of neutrophil clearance that is counterbalanced by exacerbated mobilization of immature cells. When combined with a secondary antibody, anti-Ly6G treatment results in specific and efficient neutrophil depletion. Using a mouse model of lung adenocarcinoma, we demonstrate the efficacy of this new approach to diminish primary tumor growth and propose the existence of a local intercellular communication between neutrophils and alveolar macrophages that fosters regulatory T cell proliferation in lung cancer.3

Time-resolved single-cell transcriptomics uncovers dynamics of cardiac neutrophil diversity in murine myocardial infarction

“…Also, how neutrophil temporal heterogeneity impacts repair of the infarcted heart remains to be determined. This will require precise, well controlled and validated timed depletion strategies (44). Gene expression manipulation using e.g.…”

Mouse Models and Tools for the in vivo Study of Neutrophils

“…Furthermore, 2 days posttreatment, neutrophil counts rise again in 1A8-treated mice while it is not the case in RB6-8C5-treated mice (55). Finally, 1A8 efficiency has recently been questioned, as 1A8-treated 24 weeks old C57BL/6J mice showed no neutrophil depletion (57). The same observation was conducted with bone marrow neutrophils from BALB/c mice (58).…”

“…The same observation was conducted with bone marrow neutrophils from BALB/c mice (58). To circumvent this low efficacy, it was recently suggested that combining 1A8 treatment with a secondary antibody injection could lead to a 90% depletion of blood neutrophils (57). Nevertheless, this statement needs to be tempered since the 1A8-induced neutrophil depletion was still efficient in 9 and 24 weeks old BALB/c and FVB/N mice, and in 9 weeks old C57BL/6J mice (57).…”

“…To circumvent this low efficacy, it was recently suggested that combining 1A8 treatment with a secondary antibody injection could lead to a 90% depletion of blood neutrophils (57). Nevertheless, this statement needs to be tempered since the 1A8-induced neutrophil depletion was still efficient in 9 and 24 weeks old BALB/c and FVB/N mice, and in 9 weeks old C57BL/6J mice (57). Furthermore, 1A8-induced neutrophil depletion was also questioned in PbNK65-infected C57BL/6 mice and CFA-challenged BALB/c mice (58) and further demonstrates the need to verify the depletion efficacy when using this antibody.…”

“…Besides using the hMRP8 promoter to control Crerecombinase expression, the latter was also inserted in the neutrophil-specific ly6G locus (77). These mice were recently crossed with iDTR mice as a novel model for neutrophil depletion (57). Unfortunately, upon DT injection, no drop in blood neutrophil counts was reported (57).…”