Table 1.
Summary of surface markers, cytokines and chemokines expressed by polarized macrophage subsets.
Overview of the six different subsets of macrophages that were generated in vitro, and their corresponding expression of cell surface markers (relative to unpolarized (M(-) macrophages), as well as their production of cytokines and chemokines.
Fig 1.
TTI-621 triggers phagocytosis of lymphoma cells by all macrophage subsets.
Macrophage subsets were generated as described and co-cultured with DLBCL Toledo cells for two hours in the presence of TTI-621 or isotype-matched control Fc. (A) Phagocytosis was determined by scanning confocal microscopy. Representative images are shown whereby tumor cells and macrophages are stained green and red, respectively. (B) % Phagocytosis was determined by flow cytometry as the % of live, single, CD14+CD11b+ MDMs that were VPD450+. Statistical significance was calculated using a t test where * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Data shown represent n = 14 macrophage donors where each symbol/color represents the same macrophage donor.
Fig 2.
TTI-621-mediated phagocytosis lymphoma cells is dependent on FcγRII (CD32) and FcγRI (CD64).
Macrophage subsets were generated as described and co-cultured with Violet Proliferation Dye (VPD450)-labeled DLBCL Toledo cells for two hours in the presence of TTI-621 or isotype-matched control Fc. % Phagocytosis was determined by flow cytometry as the % of live, single, CD14+CD11b+ MDMs that were VPD450+. (B) Blocking F(ab’)2 against FcγRs (CD16, CD32 or CD64) were added individually or in combination as indicated during the two-hour phagocytosis assay. Data shown represent n = 3 macrophage donors. Statistical significance was carried out using a one-way ANOVA with Dunnett’s multiple comparisons test.
Fig 3.
Repolarized M(-), M(IL-4) and M(HAGG+IL-1β) MDMs have an increased phagocytic response to TTI-621.
M(-), M(IL-4) and M(HAGG+IL-1β) macrophages were generated as described, and subsequently re-polarized with IFN-γ, IL-10, LPS, R848, IFN-γ, Poly (I:C) or CpG overnight. The resulting repolarized MDM were washed and incubated with Violet Proliferation Dye (VPD450)-labeled DLBCL Toledo cells in the presence of 1 μM TTI-621 or control Fc for two hours. % Phagocytosis was determined by flow cytometry as the % of live, single, CD14+CD11b+ MDMs that were VPD450+. A summary of 4–5 independent experiments is shown. Paired t-test was performed comparing TTI-621 repolarized macrophage subset vs TTI-621 non-repolarized macrophage subset. The dotted lines indicate the phagocytic response of M(-), M(IL-4) and M(HAGG+IL-1β) macrophage in the presence of TTI-621. Statistical significance was calculated using a t test where *p<0.05, **P<0.01 and ***p<0.001.
Fig 4.
TTI-621 triggers phagocytosis of lymphoma cells by M1-like and M2-like tumor-associated macrophages (TAMs).
CD11b+ cells were isolated from DLBCL (Toledo) xenograft tumors as described and co-cultured with Violet Proliferation Dye (VPD450) labeled DLBCL Toledo cells for 2.5 hours in the presence of 1μM TTI-621, isotype-matched control Fc or left untreated. (A) Phagocytosis was assessed by flow cytometry and % phagocytosis was defined as the percentage of macrophages that were VPD450+. TAMs were defined as live, single, CD45+F480+CD11b+ cells and further defined as MHC-IIhi CD206lo M1-like and MHC-IIlo CD206hi M2-like macrophages. (B) A summary of two independent experiments is shown with human lymphoma target cells that were expanded in vitro (left panel) or purified from excised tumors (right panel). One-way ANOVA with Tukey’s multiple comparisons was performed comparing the % phagocytosis within M1- and M2-like subsets upon various treatments as well as comparing TTI-621-treated M1- and M2-like macrophages towards in vitro or in vivo expanded tumor cell targets. Data shown represent n = 8 mice. Where indicated, *p<0.05 and ***p<0.001.