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Danger signaling to NK cells in AML
PMA plus ionomycin and/or anti-CD3 beads (Figure 4C). Similarly, splenocytes isolated from C1498.CRT-bearing mice contained CD4+ and CD8+ T cells that were more responsive to stimulation than their counterparts from C1498.WT-bearing mice (Figure 4D).
CRTHi/NKG2DHi vs. CRTLo/NKG2DLo, P=0.037) (Figure 5D-E).
Discussion
CRT exposure on cancer cells conveys robust prognostic information in patients with a variety of malignancies, generally reflecting the activation of clinically-relevant tumor-targeting immune responses.13 Previous work from our group demonstrated that the presence of CRT on the surface of malignant blasts from AML patients correlates not only with an increased frequency of effector memory CD4+ and CD8+ T cells but also with an increased propor- tion of circulating NK cells, suggesting that CRT exposure is linked to both adaptive and innate immunity.10 Inspired by accumulating evidence on the key role of NK cells in natural and therapy-driven immunosurveillance,25-29 we decided to extend these initial observations and character- ize the link between surface-exposed CRT and NK-cell activity in AML patients. Indeed, NK cells from patients with high CRT exposure on malignant blasts exhibited improved secretory and cytotoxic effector functions (Figure 2B and E).
As we excluded the possibility that CRT would mediate direct immunostimulatory effects on NK cells (Figure 3A and Online Supplementary Figure S2A), we thought that CRT exposure would be linked to increased levels of NKALs on the surface of malignant blasts, because both these processes have been linked to intracellular ER stress signaling.30 Indeed, the percentage of CD45+CD33+ malig- nant blasts staining positively for ecto-CRT+ correlated with the frequency of blasts staining positively for various NKALs (Figure 1D). However, we were unable to docu- ment any correlation between the NKAL expression levels and mRNA abundance of genes involved in the ER stress response (which we and others previously demonstrated to constitutively occur in AML blasts independent of ther- apy)10,12 (Online Supplementary Figure S1C). These findings suggest that NKALs and CRT are exposed on the surface of AML blasts via mechanistically distinct stress response pathways. Replication stress and the consequent DNA damage response stand out as a promising candidate for NKAL exposure in this setting.30
We also found that NK-cell activation by CRT involves a population of CD11c+CD14high cells that, upon exposure to CRT, express maturation markers (CD86 and HLA-DR), acquires improved migratory capacity as a consequence of CCR7 expression, and delivers stimulatory signals to NK cells via IL-15Rα/IL-15 trans-presentation24 and type I IFN. Consistent observations in peripheral blood of HDs and AML patients, suggest that CD11c+CD14high cells exposed to CRT have a superior capacity to migrate to secondary lymphoid organs where they can efficiently activate NK cells (Figure 3C-E). Thus, surface-exposed CRT appears to trigger the phenotypic and functional maturation of CD11c+CD14high cells leading to (i) cell contact-dependent NK-cell activation via trans-presented IL-15, as well as (ii) cell contact-independent NK-cell activation via type I IFNs. Importantly, type I IFN signaling in DCs results not only in a superior ability to drive antigen-specific T-cell priming,31 but also in IL-15 production,32 potentially sup- porting a robust adaptive and innate immune response of therapeutic relevance. Our findings and elegant preclinical data from Chen and colleagues10,33 lend robust support to
In line with this notion, PBMCs from CRTHi AML patients in complete remission contained significantly higher frequencies of both CD8+ and CD4+ T cells responding by IFN-γ secretion to PMA plus ionomycin (Online Supplementary Figure S4B-C), with a slightly sub- significant trend towards increased numbers of CD107a+GZMB+CD8+ T cells (Online Supplementary Figure S4D), compared with their CRTLo counterparts, comfort- ing previously published data from our group.10 Quantification of several cytokines essential for NK-cell homeostasis and functions (IL-21, IL-15, IFN-g and IFN-α2) and for hematopoietic stem cell (HSC) differenti- ation (IL-3 and IL-7) in the sera of AML patients in remis-
Hi
sion also revealed higher IFN-γ levels in CRT versus
CRTLo patients (Online Supplementary Figure S4E).
CRT exposure on malignant blasts and the frequency of NKG2D+ cells correlate with RFS in AML patients
To evaluate the prognostic impact of CRT exposure on malignant blasts and verify our previous results on a larg- er subgroup of our patients,10 we investigated RFS upon stratifying AML patients based on the median percentage of DAPI– blasts staining positively for surface CRT. In line with our previous observations,10 CRTHi patients exhibit- ed a significantly improved RFS compared with CRTLo patients (median: >60 vs. 14 months, P=0.027) (Figure 5A). Using a similar cutoff approach based on the median value, we also examined whether the mRNA levels of KLRK1, encoding the key NK-cell activating receptor NKG2D, would convey prognostic information in AML patients. We found that patients expressing high levels of KLRK1 (KLRK1Hi) had a significantly lower risk of relapse compared to their KLRK1Lo counterparts (median: 39 vs. 10 months, P=0.039) (Figure 5B). We validated these find- ings at the protein level by stratifying a larger group of patients based on the median frequency of CD45+CD3- CD56+NKG2D+ NK cells. Patients with a high frequency of NK cells expressing NKG2D (NKG2DHi) exhibited sig- nificantly improved RFS, compared with their NKG2DLo counterparts (median: >35 vs. 24 months, P=0.035) (Figure 5C). However, neither univariate nor multivariate Cox proportional hazard analysis confirmed these find- ings, potentially reflecting a limited follow-up of this prospectively collected patient cohort, or other confound- ing factors including disease subtype and inter-individual heterogeneity (Table 2-3). Since both CRT exposure on malignant blasts and NKG2D levels influenced RFS in our cohort of AML patients, we evaluated the combined prognostic value of ecto-CRT+ blasts and the KLRK1 mRNA levels or CD45+CD3–CD56+NKG2D+ NK-cell fre- quency by stratifying the cohort in three groups: CRTHi/KLRK11Hi or CRTHi/NKG2DHi patients, CRTLo/KLRK1Lo or CRTLo/NKG2DLo patients and patients in which the percentage of CRT+ blasts was discordant with the KLRK1 mRNA levels or the frequency of CD45+CD3-CD56+NKG2D+ NK cells (CRT/KLRK1Mix or CRT/NKG2DMix). We found that CRTHi/KLRK1Hi or CRTHi/NKG2DHi patients had superior RFS as compared with their CRTLo/KLRK1Lo or CRTLo/NKG2DLo counter- parts (CRTHi/KLRK1Hi vs. CRTLo/KLRK1Lo, P=0.050;
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