Interleukin-15-activated natural killer cells kill autologous osteoclasts via LFA-1, DNAM-1 and TRAIL, and inhibit osteoclast-mediated bone erosion <i>in vitro</i>

Osteoclasts reside on bone and are the main bone resorbing cells playing an important role in bone homeostasis, while natural killer (NK) cells are bone-marrow-derived cells known to play a crucial role in immune defence against viral infections. Although mature NK cells traffic through bone marrow as well as to inflammatory sites associated with enhanced bone erosion, including the joints of patients with rheumatoid arthritis, little is known about the impact NK cells may have on mature osteoclasts and bone erosion. We studied the interaction between human NK cells and autologous monocyte-derived osteoclasts from healthy donors in vitro. We show that osteoclasts express numerous ligands for receptors present on activated NK cells. Co-culture experiments revealed that interleukin-15-activated, but not resting, NK cells trigger osteoclast apoptosis in a dose-dependent manner, resulting in drastically decreased bone erosion. Suppression of bone erosion requires contact between NK cells and osteoclasts, but soluble factors also play a minor role. Antibodies masking leucocyte function-associated antigen-1, DNAX accessory molecule-1 or tumour necrosis factor-related apoptosis-inducing ligand enhance osteoclast survival when co-cultured with activated NK cells and restore the capacity of osteoclasts to erode bone. These results suggest that interleukin-15-activated NK cells may directly affect bone erosion under physiological and pathological conditions

TTI-621 (SIRPαFc), a CD47-blocking cancer immunotherapeutic, triggers phagocytosis of lymphoma cells by multiple polarized macrophage subsets

<div><p>Tumor-associated macrophages (TAMs) are heterogeneous and can adopt a spectrum of activation states between pro-inflammatory and pro-tumorigenic in response to the microenvironment. We have previously shown that TTI-621, a soluble SIRPαFc fusion protein that blocks the CD47 “do-not-eat” signal, promotes tumor cell phagocytosis by IFN-γ-primed macrophages. To assess the impact of CD47 blockade on diverse types of macrophages that are found within the tumor microenvironment, six different polarized human macrophage subsets (M(-), M(IFN-γ), M(IFN-γ+LPS), M(IL-4), M(HAGG+IL-1β), M(IL-10 + TGFβ)) with distinct cell surface markers and cytokine profiles were generated. Blockade of CD47 using TTI-621 significantly increased phagocytosis of lymphoma cells by all macrophage subsets, with M(IFN-γ), M(IFN-γ+LPS) and M(IL-10 + TGFβ) macrophages having the highest phagocytic response. TTI-621-mediated phagocytosis involves macrophage expression of both the low- and high-affinity Fcγ receptors II (CD32) and I (CD64), respectively. Moreover, macrophages with lower phagocytic capabilities (M(-), M(IL-4), M(HAGG+IL-1β)) could readily be re-polarized into highly phagocytic macrophages using various cytokines or TLR agonists. In line with the in vitro study, we further demonstrate that TTI-621 can trigger phagocytosis of tumor cells by diverse subsets of isolated mouse TAMs ex vivo. These data suggest that TTI-621 may be efficacious in triggering the destruction of cancer cells by a diverse population of TAMs found in vivo and support possible combination approaches to augment the activity of CD47 blockade.</p></div

Summary of surface markers, cytokines and chemokines expressed by polarized macrophage subsets.

<p>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.</p

TTI-621 triggers phagocytosis of lymphoma cells by all macrophage subsets.

<p>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.</p

TTI-621 triggers phagocytosis of lymphoma cells by M1-like and M2-like tumor-associated macrophages (TAMs).

<p>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-II^hi CD206^lo M1-like and MHC-II^lo CD206^hi 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.</p

Repolarized M(-), M(IL-4) and M(HAGG+IL-1β) MDMs have an increased phagocytic response to TTI-621.

<p>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.</p