RESULTS: In this study, pro-inflammatory macrophage was selected as the target cell due to its major roles in numerous inflammatory and autoimmune disorders. We aimed to construct macrophage-targeted recombinant immunotoxins by combining HALT-1 with anti-CD64-scFv in two orientations, and to assess whether their cytotoxic activity and binding capability could be preserved upon molecular fusion. The recombinant immunotoxins, HALT-1-scFv and scFv-HALT-1, were successfully constructed and expressed in Escherichia coli (E. coli). Our data showed that HALT-1 still exhibited significant cytotoxicity against CD64+ and CD64- cell lines upon fusion with anti-CD64 scFv, although it had half cytotoxic activity as compared to HALT-1 alone. As positioning HALT-1 at N- or C-terminus did not affect its potency, the two constructs demonstrated comparable cytotoxic activities with IC50 lower in CD64+ cell line than in CD64- cell line. In contrast, the location of targeting moieties anti-CD64 scFv at C-terminal end was crucial in maintaining the scFv binding capability.

Methods: Temporal artery biopsies (TABs, n = 11) from treatment-naive GCA patients, aorta samples from GCA-related aneurysms (n = 10) and atherosclerosis (n = 10) were stained by immunohistochemistry targeting selected macrophage phenotypic markers, cytokines, matrix metalloproteinases (MMPs) and growth factors. In vitro macrophage differentiation (n = 10) followed by flow cytometry, Luminex assay and ELISA were performed to assess whether GM-CSF and M-CSF are drivers of macrophage phenotypic heterogeneity.

Results: A distinct spatial distribution pattern of macrophage phenotypes in TABs was identified. CD206+/MMP-9+ macrophages were located at the site of tissue destruction, whereas FRβ+ macrophages were located in the inner intima of arteries with high degrees of intimal hyperplasia. Notably, this pattern was also observed in macrophage-rich areas in GCA aortas but not in atherosclerotic aortas. Flow cytometry showed that GM-CSF treatment highly upregulated CD206 expression, while FRβ was expressed by M-CSF-skewed macrophages, only. Furthermore, localised expression of GM-CSF and M-CSF was detected, likely contributing to macrophage heterogeneity in the vascular wall.

METHODS: Immunohistochemistry was performed on GCA temporal artery biopsy specimens (n = 12) and aortas (n = 10) for detection of YKL-40, its receptor interleukin-13 receptor α2 (IL-13Rα2), macrophage markers PU.1 and CD206, and the tissue-destructive protein matrix metalloproteinase 9 (MMP-9). Ten noninflamed temporal artery biopsy specimens served as controls. In vitro experiments with granulocyte-macrophage colony-stimulating factor (GM-CSF)- or macrophage colony-stimulating factor (M-CSF)-skewed monocyte-derived macrophages were conducted to study the dynamics of YKL-40 production. Next, small interfering RNA-mediated knockdown of YKL-40 in GM-CSF-skewed macrophages was performed to study its effect on MMP-9 production. Finally, the angiogenic potential of YKL-40 was investigated by tube formation experiments using human microvascular endothelial cells (HMVECs).

RESULTS: YKL-40 was abundantly expressed by a CD206+MMP-9+ macrophage subset in inflamed temporal arteries and aortas. GM-CSF-skewed macrophages from GCA patients, but not healthy controls, released significantly higher levels of YKL-40 compared to M-CSF-skewed macrophages (P = 0.039). In inflamed temporal arteries, IL-13Rα2 was expressed by macrophages and endothelial cells. Functionally, knockdown of YKL-40 led to a 10-50% reduction in MMP-9 production by macrophages, whereas exposure of HMVECS to YKL-40 led to significantly increased tube formation.

METHODS: Circulating CD8+ T cells were analysed for differentiation status (CD45RO, CCR7), markers of activation (CD69 and CD25) and proliferation (Ki-67) in 14 newly diagnosed GCA patients and 18 healthy controls by flow cytometry. Proliferative capacity of CD8+ T cells upon anti-CD3 and anti-CD3/28 in vitro stimulation was assessed. Single-cell RNA sequencing of peripheral blood mononuclear cells of patients and controls (n = 3 each) was performed for mechanistic insight. Immunohistochemistry was used to detect CD3, CD8, Ki-67, TNF-α and IFN-γ in GCA-affected tissues.

RESULTS: GCA patients had decreased numbers of circulating effector memory CD8+ T cells but the percentage of Ki-67-expressing effector memory CD8+ T cells was increased. Circulating CD8+ T cells from GCA patients demonstrated reduced T cell receptor activation thresholds and displayed a gene expression profile that is concurrent with increased proliferation. CD8+ T cells were detected in GCA temporal arteries and aorta. These vascular CD8+ T cells expressed IFN-γ but not Ki-67.