Figure 3.

The mechanism of natural killer cell-stimulatory effects of calreticulin and its impact on CD11c⁺CD14^high cell phenotype. (A, B) The effect of recombinant human calreticulin (rCRT, Sino Biological Inc.) on effector functions of natural killer (NK) cells isolated from healthy donors (HDs) (n=8) or NK cells in HD peripheral blood mononuclear cells (PBMC) mixture (n=8). Purified NK cells (A) or whole PBMC (B) were pre-incubated with 5 μg/mL of rCRT overnight and subsequently stimulated by PMA + Ionomycin or K562 cell line for 4 hours (h). The percentage of responding (CD107a⁺PRF1⁺CD45⁺CD3⁻CD56⁺) NK cells was determined by flow cytometry. Alternatively, NK cells were purified from rCRT-pre-incubated PBMC and their capacity to kill K562 cell line was tested in cytotoxicity assay. The percentage of dead (AnnV⁺DAPI⁺) K562 cells was determined by flow cytometry after 4 h (B). NK cells/PBMCs without rCRT and unstimulated NK cells/PBMC were used as a negative controls; ns: not significant. (C) The expression of maturation-associated molecules (CD86 and HLA-DR) and CCR7 on CD11c⁺CD14^high cells in HD PBMCs (n=8) incubated with rCRT (5 μg/mL) overnight versus control PBMCs without rCRT as determined by flow cytometry. The expression of individual markers is shown as mean fluorescence intensity (MFI). Flow cytometry was also used for the detection of IL-15Rα⁺CD11c⁺CD14^high cells in rCRT-pre-incubated versus control PBMCs. (D) The frequency of CCR7+ and IL-15Rα⁺CD11c⁺CD14^high cells in CRT^Hi versus CRT^Lo AML patients upon the restoration of normal hematopoiesis (n=16) determined by flow cytometry. Boxplots: lower quartile, median, upper quartile; whiskers, minimum, maximum. (E) Quantitative RT-PCR-assisted quantification of IFNA1 and IFNB1 expression levels in PBMCs from 20 CRT^Hi versus 21 CRT^Lo acute myeloid leukemia (AML) patients at recovery of normal hematopoiesis. Boxplots: lower quartile, median, upper quartile; whiskers, minimum, maximum.