A neutrophil-based approach to elicit natural killer cells into an immunologically "cold" tumor
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Abstract
BACKGROUND: Cancer ranks as the second most common cause of death worldwide, following cardiovascular disease. Immunotherapies such as immune checkpoint inhibitors have been approved for several cancers. However, efficacy is observed in a minority of patients and specific types of solid tumors that may be related in part to the absence of immune cell infiltration into the immunosuppressive environment of solid tumors, resulting in immunologically “cold” tumors. Mysore et al. showed that a neutrophil-targeting antibody conjugated to a tumor antigen, herein referred to as antibody-antigen conjugate, or AAC, leads to anti-tumor immunity in a T cell-dependent prophylactic melanoma model. Engaging neutrophils in this manner resulted in neutrophil-derived antigen-presenting cells (APCs) that promoted T cell activation. It is well documented that adequate T cell activation and maturation by APCs in secondary lymphoid organs combined with intratumoral APCs that release chemokines to recruit cytotoxic CD8+ T cells are critical for anti-tumor immunity. Another type of cancer-fighting immune cell referred to as a natural killer or NK cell directly kills cancer cells. The intratumoral accumulation of NK cells is known to be particularly important for eradicating cancers that evade cytotoxic CD8+ T cell-mediated killing by downregulating MHC-I. Here, we determined whether AAC treatment of a mouse with an established tumor leads to infiltration of cytotoxic T cells and NK cells into the tumor. Moreover, we evaluated the phenotype of NK cells, since different phenotypic subsets play diverse roles in anti-tumor immunity.
METHODS: C57Bl/6 mice expressing the uniquely human FcγRIIIB and FcγRIIA selectively on the neutrophils of mice lacking their endogenous FcγRs (γ-/- mice) were inoculated subcutaneously with B16F10 melanoma cells expressing ovalbumin (Ova), a model T cell-dependent “tumor-associated” antigen, followed by the intravenous injection of naïve CD8+ T cells recognizing the Ova peptide SIINFEKL in the context of MHC-I, and subsequently with an intravenous injection of anti-FcgRIIIB-Ova conjugate (AAC-Ova) or its isotype control. Tumors were measured bi-weekly over 12 days, and immune cell composition in the tumors, spleen, and draining inguinal lymph nodes of study animals were analyzed by flow cytometry at the end of the study period.
RESULTS: AAC treatment of FcR humanized mice with an established melanoma resulted in an increase in CD8+ T lymphocyte and NK cell infiltration into the tumor. However, the results were not statistically significant due to the small number of animals and the high level of variability within independent experimental replicates.
CONCLUSIONS: Our data show a trend towards increased NK cell infiltration into the tumor and draining lymph node after AAC versus control treatment. A trend is also evident for increased intratumoral T lymphocyte infiltration after AAC versus control treatment, though it is less pronounced. A greater sample size is required to determine whether the results reach statistical significance and are therefore biologically meaningful.
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2024