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Danger signaling to NK cells in AML
NK cells isolated from PBMCs pre-incubated with rCRT were able to kill an increased amount of K562 cells com- pared to NK cells isolated from control PBMC (Figure 3B). These results suggest that CRT stimulate NK cells indi- rectly, via mechanisms that involve other cellular compo- nents of the PBMC mixture.
Previous in vitro studies support a role for APCs, mainly DCs, in NK-cell activation.23 We therefore decided to focus on the phenotype of APCs exposed to rCRT. We found that incubating PBMCs from healthy donors (HD) with rCRT induced the upregulation of the chemotaxis- associated receptor C-C motif chemokine receptor 7 (CCR7) and the maturation-associated molecules CD86 and HLA-DR on CD11c+CD14high cells and increased the frequency of CD11c+CD14high expressing interleukin 15 receptor subunit alpha (IL15RA, best known as IL-15Rα) (Figure 3C),which is crucial for the activatory trans-pre- sentation of IL-15 to NK cells.24 Inspired by these data, we
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investigated the relationship between CRT exposed on malignant blasts and the phenotype of APCs in AML patients in remission. We found that CRTHi patients har- bor a significantly higher percentage of CD11c+CD14high cells expressing CCR7 and IL-15Rα compared to their CRTLo counterparts (Figure 3D), suggesting that these cells have an increased capacity to migrate to secondary lymphoid organs, where they can efficiently activate NK cells. In vitro assays suggested a prominent role for human myeloid over plasmocytoid DCs in NK-cell acti- vation upon exposure to rCRT (Online Supplementary Figure S3A). Of note, also mouse PBMCs or bone mar- row-derived DCs exposed to rCRT upregulated activa- tion markers including CD54, CD86, and MHC class II molecules, and secreted increased amounts of IL-12 (Online Supplementary Figure S3B-D).
We have previously shown that the PMBCs of CRTHi AML patients who are in complete remission and have
C
P=0.001 P=0.02
D P=0.01 P=0.03
E
Figure 3. The mechanism of natural killer cell-stimulatory effects of calreticulin and its impact on CD11c+CD14high 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 stim- ulated 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 cytom- etry. 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 neg- ative controls; ns: not significant. (C) The expression of maturation-associated molecules (CD86 and HLA-DR) and CCR7 on CD11c+CD14high 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+CD14high cells in rCRT-pre-incubated versus control PBMCs. (D) The frequency of CCR7+ and IL-15Rα+CD11c+CD14high cells in CRTHi versus CRTLo 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 CRTHi versus 21 CRTLo acute myeloid leukemia (AML) patients at recovery of normal hematopoiesis. Boxplots: lower quartile, medi- an, upper quartile; whiskers, minimum, maximum.
P=0.02
P=0.04
P=0.01
P=0.005
P=0.009
P=0.003
P=0.02
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