Macrophage-derived Fgl2 dampens antitumor immunity through regulation of FcγRIIB+CD8+ T cells in melanoma
Kelsey B. Bennion, Julia Miranda R.Bazzano, Danya Liu, Maylene Wagener, Chrystal M. Paulos, Mandy L. Ford
Kelsey B. Bennion, Julia Miranda R.Bazzano, Danya Liu, Maylene Wagener, Chrystal M. Paulos, Mandy L. Ford
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Research Article Immunology Oncology

Macrophage-derived Fgl2 dampens antitumor immunity through regulation of FcγRIIB+CD8+ T cells in melanoma

Abstract

Cancer immunotherapy has emerged as a promising therapeutic modality but heterogeneity in patient responsiveness remains. Thus, greater understanding of the immunologic factors that dictate response to immunotherapy is critical to improve patient outcomes. Here, we show that fibrinogen-like protein 2 (Fgl2) is elevated in the setting of melanoma in humans and mice and plays a functional role in inhibiting the CD8+ T cell response. Surprisingly, the tumor itself is not the major cellular source of Fgl2. Instead, we found that macrophage-secreted Fgl2 dampens the CD8+ T cell response through binding and apoptosis of FcγRIIB+CD8+ T cells. This regulation was CD8+ T cell autonomous and not via an antigen-presenting cell intermediary, as absence of Fcgr2b from the CD8+ T cells rendered T cells insensitive to Fgl2 regulation. Fgl2 is robustly expressed by macrophages in 10 cancer types in humans and in 6 syngeneic tumor models in mice, underscoring the clinical relevance of Fgl2 as a therapeutic target to promote T cell activity and improve patient immunotherapeutic response.

Authors

Kelsey B. Bennion, Julia Miranda R.Bazzano, Danya Liu, Maylene Wagener, Chrystal M. Paulos, Mandy L. Ford

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Figure 7

Macrophage-secreted Fgl2 induces apoptosis of WT but not Fcgr2b–/– tumor-specific CD8+ T cells.

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Macrophage-secreted Fgl2 induces apoptosis of WT but not Fcgr2b–/– tumor...
(A) Schematic showing that WT and Fgl2–/– mice were challenged with B16 cells and 14 days later, CD11b+ splenocytes were MACS enriched with CD11b microbeads. Two days prior to CD11b+ cell isolation, splenocytes from WT or Fcgr2b–/– OT-I transgenic mice were stimulated with OVA257–264 (SIINFEKL). Upon CD11b+ isolation as described above, 2-day-stimulated OT-I T cells were cocultured with WT or Fgl2–/– CD11b+ cells. Twenty-four hours later, cell supernatant was collected for Fgl2 ELISA and OT-I T cells were collected for caspase 3/7 and 7AAD flow staining. (B) Representative flow plots showing similar frequency of CD11b+ cells isolated from WT or Fgl2–/– mice. (C) Pie graphs showing the frequency of CD11c+, F4/80+, and CD14+ cells within the CD11b+ cell isolate (n = 5, average value is shown). (D) Representative flow plots showing caspase 3/7 and 7AAD staining of WT and Fcgr2b–/– OT-I cocultured with WT or Fgl2–/– CD11b+ cells. Bar graphs showing frequency of caspase 3/7+7AAD+ (E) WT (n = 16, pooled data from 2 experiments) vs. (F) Fcgr2b–/– OT-I (n = 12, pooled data from 2 experiments) cocultured with WT or Fgl2–/– CD11b+ cells. (G) Bar graph showing Fgl2 protein concentration of WT CD11b+ cells incubated with WT or Fcgr2b–/– OT-I, with no detectable protein signal in the Fgl2–/– CD11b+ cells incubated with WT or Fcgr2b–/– OT-I (n = 8, representative data from 2 experiments). Mann-Whitney nonparametric, unpaired t test was used when comparing 2 groups; Kruskall-Wallis nonparametric, 1-way ANOVA was used when comparing more than 2 groups. Summary data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001.