Fig 2: MEPs promote cytotoxic immune cell activity by secreted factors.A Conditioned medium from megakaryocyte-erythroid progenitor (MEP) cultures and MEP-depleted Lin-cell cultures were applied to a cytokine and chemokine array. The levels of selected proteins are shown as log ratios (MEPs vs. MEP-depleted Lin- cells, Lin-ERMAP- cells). Proteins known to be associated with CD8 + T cell and NK cell activity are indicated with a red box. B Differentially expressed genes were assessed by GO to identify associated biological pathways. CXCL16 and CCL5 (circled in black) were displayed as hubs. C CXCL16 and CCL5 levels were analyzed by specific ELISA in conditioned medium of MEPs vs. MEP-depleted Lin- cells. Statistical significance was assessed by unpaired two-tailed t-test. Significant p values are shown as **p < 0.01; ***p < 0.001.

Fig 3: CXCL16 expression is increased upon ischemic insults. (A) CXCL16 mRNA quantification in pMCAO brain. qRT-PCR analysis in contralateral and ipsilateral emisphere 24 h after pMCAO (n = 5). (B) CXCL16 mRNA quantification in in vitro OGD. qRT-PCR analysis in primary hippocampal cultures after 90 min of OGD (n = 11) and after 2 h of recovery (n = 8). Data are expressed as mRNA fold increase normalized to ß-actin. Results represent the mean ± SEM. Statistical analysis: Student’s t-test ** p < 0.001 (A); one-way ANOVA followed by Dunn’s post-hoc test * p < 0.05 (B).

Fig 4: Endogenous self-protective mechanism drived by CX3CL1–CXCL16-CCL2 upon ischemia. Ischemic insult drives irreversible brain damage in ischemic core, while in the surrounding region (ischemic penumbra) brain tissue results to be functionally compromised but potentially “salvageable”. Upon ischemic insult both pathogenic and self-protective mechanisms occur in the penumbra. CX3CL1-CXCL16-CCL2 cascade, triggered by neurons-microglia-astrocytes cross-talk, represents one endogenous self-protective mechanism that contributes to the restriction of brain damage by counteracting Glu induced excitotoxicity.

Fig 5: Increased cytotoxic immune cell activity by MEPs is mediated in part by CXCL16 and CCL5.A, B CD8 + T cells (A) and NK cells (B) were cultured in the presence of conditioned medium (CM) obtained from MEP cultures, or MEP CM depleted of CCL5 or CXCL16. Cell activity, Granzyme B expression and cell proliferation were analyzed by flow cytometry. C, D Panc02 cells were cultured for 24 h with CD8 + T cells (C) or NK cells (D) in the presence of MEP-conditioned medium as well as a ROS scavenger, sodium pyruvate (SP). The percentage of PI-positive tumor cells (representing dead cells) was analyzed by flow cytometry. Statistical significance was assessed by one-way ANOVA followed by Tukey post-hoc test. Asterisks represent significance from control, unless indicated otherwise. Significant p values are shown as *p < 0.05; **p < 0.01; ***p < 0.001.