Activation of the Innate Immune Checkpoint CLEC5A on Myeloid Cells in the Absence of Danger Signals Modulates Macrophages’ Function but Does Not Trigger the Adaptive T Cell Immune Response

Figure 1

Monoclonal α-CLEC5A Ab clone # 283834 binds to U937 wild-type (WT) but not to CLEC5A-deficient (CLEC5A KO) U937 cells. (a) WT (A) or CLEC5A KO (B) U937 cell line was first differentiated with 10 nM PMA for 3 days and then incubated 15 min either with a PE-labelled α-CLEC5A Ab or with its corresponding isotype ctrl. α-CLEC5A Ab (grey line) vs isotype (grey shadow) binding in FACS is shown as a histogram overlay. (b) CRISPR/Cas9-mediated knockout of CLEC5A at exon 2 in U937 cells was confirmed by sequencing of genomic DNA around the targeted locus. Alignment of CLEC5A WT locus and clone 2.11 CLEC5A KO shows a 17 bp insertion (a, allele 1) and 43 bp deletion (b, allele 2) leading in both cases to a premature stop codon () leading to translation termination. (c) The U937 WT and CLEC5A KO cells were differentiated with PMA as described above. RNA from the differentiated cells was extracted, reverse transcribed, and screened for mRNA levels by real-time qPCR for CLEC5A and β-actin (housekeeping gene) as a reference. Values represent the from three technical replicates; .

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