Large extracellular vesicles derived from human regulatory macrophages (L-EVMreg) attenuate CD3/CD28-induced T-cell activation in vitro

Abstract

Macrophages belong to the innate immune system, and we have recently shown that in vitro differentiated human regulatory macrophages (Mreg) release large extracellular vesicles (L-EVMreg) with an average size of 7.5 μm which regulate wound healing and angiogenesis in vitro. The aim of this study was to investigate whether L-EVMreg also affect the CD3/CD28-mediated activation of T-cells. Mreg were differentiated using blood monocytes and L-EVMreg were isolated from culture supernatants by differential centrifugation. Activation of human T-cells was induced by CD3/CD28-coated beads in the absence or presence of Mreg or different concentrations of L-EVMreg. Inhibition of T-cell activation was quantified by flow cytometry and antibodies directed against the T-cell marker granzyme B. Phosphatidylserine (PS) exposure on the surface of Mreg and L-EVMreg was analyzed by fluorescence microscopy. Incubation of human lymphocytes with CD3/CD28 beads resulted in an increase of cell size, cell granularity, and number of granzyme B–positive cells (P < 0.05) which is indicative of T-cell activation. The presence of Mreg (0.5 × 106 Mreg/ml) led to a reduction of T-cell activation (number of granzyme B–positive cells; P < 0.001), and a similar but less pronounced effect was also observed when incubating activated T-cells with L-EVMreg (P < 0.05 for 3.2 × 106 L-EVMreg/ml). A differential analysis of the effects of Mreg and L-EVMreg on CD4+ and CD8+ T-cells showed an inhibition of CD4+ T-cells by Mreg (P < 0.01) and L-EVMreg (P < 0.05 for 1.6 × 106 L-EVMreg/ml; P < 0.01 for 3.2 × 106 L-EVMreg/ml). A moderate inhibition of CD8+ T-cells was observed by Mreg (P < 0.05) and by L-EVMreg (P < 0.01 for 1.6 × 106 L-EVMreg/ml and 3.2 × 106 L-EVMreg/ml). PS was restricted to confined regions of the Mreg surface, while L-EVMreg showed strong signals for PS in the exoplasmic leaflet. L-EVMreg attenuate CD3/CD28-mediated activation of CD4+ and CD8+ T-cells. L-EVMreg may have clinical relevance, particularly in the treatment of diseases associated with increased T-cell activity. Key messages Mreg release large extracellular vesicles (L-EVMreg) with an average size of 7.5 µm L-EVMreg exhibit phosphatidylserine positivity L-EVMreg suppress CD4+ and CD8+ T-cells L-EVMreg hold clinical potential in T-cell-related diseases

Effects of temporal IFNγ exposure on macrophage phenotype and secretory profile: exploring GMP-Compliant production of a novel subtype of regulatory macrophages (MregIFNγ0) for potential cell therapeutic applications

“…Prior research conducted by the authors has been dedicated to characterizing Mreg for several years [ 8 , 12 , 16 ], demonstrating their ability to secrete pro-angiogenic factors, particularly under hypoxic conditions [ 8 ]. Building upon these findings, a GMP-compliant protocol for the production of Mreg was developed and a phase I/II study employing Mreg for treating patients with chronic limb threatening ischemia (CLTI) is under preparation.…”

“…Different research groups have demonstrated in recent years that distinct macrophage subtypes can be differentiated in vitro by the administration of various cytokines/ factors (e.g., IL-4, IL-13, IFNγ, LPS), signifying the potential of these in vitro-differentiated cell types in the context of cell therapy [10,11]. We and others have shown that the administration of IFNγ on day 6 of the differentiation period can generate an anti-inflammatory, T-cell suppressive, and potentially pro-angiogenic macrophage subtype known as regulatory macrophages (Mreg) [7,8,[12][13][14][15]. These Mreg cells have already been successfully employed in a clinical study to reduce rejection reactions in kidney transplantations [4,6].…”

“…The authors have previously elucidated the production of Mreg capable of suppressing T-cell activation and promoting pro-angiogenic responses [ 7 , 8 ]. Presently, we are preparing for a clinical phase I/II study employing pro-angiogenic Mreg in treating patients with chronic limb threatening ischemia (CLTI).…”