Association between immune checkpoint expression and the cytokine profile of M2-like macrophages induced by efferocytosis

Background. We previously developed a protocol for generating macrophages with pronounced anti-inflammatory and reparative/neuroregenerative activity, polarized by efferocytosis (M2(Eff)). Due to their unique secretome, these cells are promising candidates for cell therapy of neuroinflammatory diseases. However, the expression of the inhibitory receptors PD-1 and TIM-3 on M2(Eff) and its relationship with cytokine production remain unclear.

Aim. To characterize PD-1 and TIM-3 expression on M2(Eff) macrophages and determine their role in the regulation of proand anti-inflammatory cytokine production (IL-10 and TNF-α).

Materials and methods. Macrophages were differentiated from peripheral blood monocytes of healthy donors. PD-1 and TIM-3 expression, as well as IL-10 and TNF-α production, were assessed by flow cytometry. To evaluate the contribution of PD-1 and TIM-3 to cytokine secretion, cells were pre-treated with blocking antibodies, followed by assessment of intracellular IL-10 and TNF-α levels.

Results. M2(Eff) exhibited higher PD-1 and TIM-3 levels compared to M1 cultures, primarily due to cells co-expressing both markers. M2(Eff) also had higher levels of PD-1 and lower levels of TIM-3 compared to “classical” M2 macrophages polarized by IL-4. The proportion of IL-10-positive cells was higher in M2(Eff) cultures expressing PD-1, as well as in TIM-3-expressing cells after lipopolysaccharide stimulation, compared to the negative populations. TNF-α levels were higher in TIM-3-positive macrophages compared to TIM-3-negative ones. PD-1 blockade during lipopolysaccharide stimulation increased the proportion of TNF-α-expressing cells, whereas TIM-3 blockade had no such effect.

Conclusion. M2(Eff) macrophages induced by apoptotic cells display a unique PD-1 and TIM-3 expression profile. Moreover, PD-1 and TIM-3 are associated with IL-10 and TNF-α production.

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