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I. Truxova et al.
bone-marrow derived DCs and tumor cells were stained with panels of fluorescent antibodies to evaluate the abundance, phe- notype and function of immune cell subsets (Online Supplementary Table S1-2). Briefly, cells were incubated with primary antibodies or appropriate isotype controls for 20 min at 4 °C. For the analysis of CRT levels on AML blasts, PBMCs were labeled with anti- CD45 PerCP (Exbio) and anti-CD33 PE monoclonal antibodies (BioLegend). Malignant blasts from AML patients were defined as CD45+ cells expressing high levels of CD33 (CD33high). Surface CRT staining was performed by a three-step procedure: (1) incu- bation with primary CRT-specific antibody (Enzo Life Sciences), (2) incubation with an APC-conjugated secondary antibody (Jackson Immunoresearch Laboratories) and (3) incubation with Annexin V-FITC (Exbio) and 4′,6-diamidino-2-phenylindole (DAPI, from Molecular Probes) to assess the cell viability. Surface- exposed CRT levels were analyzed only on live (AnnV–DAPI–) and dying (AnnV+/DAPI–) but not dead (DAPI+) cells. Flow cytometry data were acquired on the LSRFortessa analyzer (BD Biosciences) and analyzed with the FlowJo software package (Tree Star, Inc.).
Statistical analysis
Survival analyses were performed by using log-rank tests upon patient stratification into two groups based on the median cutoff of continuous variables. Univariate and multivariate Cox propor- tional hazard analysis was performed to assess the association of clinicopathological or immunological parameters with relapse-free survival (RFS). Variables that were intrinsically correlated were not included in multivariate Cox regressions. Fisher’s exact tests, Student’s t-tests, and the Wilcoxon and Mann-Whitney tests were used to test for association between variables, P-values are report- ed (considered not significant when >0.05).
Results
CRT exposure on malignant blasts is associated with increased NK-cell frequency and upregulation of ligands for activating NK-cell receptors
We previously demonstrated a link between CRT expo- sure on malignant blasts and clinically-relevant anticancer immunity in AML patients.10 To extend these findings, we examined the potential impact of CRT on the plasma membrane (ecto-CRT) of CD45+CD33+ malignant blasts on the frequency and phenotype of NK cells from AML patients prior to the initiation of anthracycline-based chemotherapy and at the recovery of normal hematopoiesis. Patients were stratified based on the median percentage of DAPI–ecto-CRT+ blasts at diagnosis into a CRTHi and CRTLo group. In baseline conditions (prior to induction chemotherapy), we were unable to identify statistically significant differences in the frequen- cy and absolute numbers of circulating CD45+CD3–CD56+ NK cells between these two groups of patients (Figure 1A-B). Conversely, upon complete remis- sion and recovery of nonmalignant hematopoiesis, CRTHi AML patients had significantly higher frequency and absolute numbers of CD45+CD3–CD56+ NK cells in the circulation as compared to their CRTLo counterparts (Figure 1A-B). These results are in line with previously published data from our group.10 Of note, CRTHi AML patients did not display increased frequency of CD45+CD3–CD56+ NK cells in the bone marrow as com- pared to their CRTLo counterparts (Online Supplementary Figure S1A).
As NK-cell activation is modulated by the balance
between stimulatory and inhibitory signals delivered by
multiple ligand/receptor interactions,14 we next analyzed
the levels of common activating (NKp30, NKp46, NKp80,
NKG2D, DNAM-1 and CD16) and inhibitory (CD158e1,
CD158bj, CD158ah, NKG2A, ILT2) NK-cell receptors by
flow cytometry. With the exception of ILT2+ cells (which
were less represented in the circulation of CRTHi AML
patients upon remission), we failed to detect significant
differences in the percentage of NK cells staining positive-
ly for these receptors between CRTHi and CRTLo AML
patients, neither prior to induction chemotherapy nor
upon complete remission (Figure 1C and Online
Supplementary Figure S1B). Because CRT exposure relies 21
on ER stress responses, and different stress response pathways may also modulate the expression of ligands for NK-cell receptors,22 we decided to evaluate the poten- tial connection between CRT exposure and the levels of multiple NK-cell ligands on the surface of CD45+CD33+ blasts, namely major histocompatibility complex (MHC) class I polypeptide-related sequence A (MICA), MICB, UL16 binding protein 2 (ULBP2), ULBP5, ULBP6, poliovirus receptor (PVR, also known as CD155), nectin cell adhesion molecule 2 (NECTIN2, also known as CD112 and PVRL2), and B7-H6, by flow cytometry. We found that the percentage of DAPI–ecto-CRT+ blasts pos- itively correlates with the percentage of AML blasts stain- ing positively for MICA, MICB, CD155 and CD112 (Figure 1D). In the attempt to identify a potential connec- tion between the exposure of NK-cell-activating ligands (NKALs) and ER stress, we retrieved normalized MICA, ULBP2, PVR and NECTIN2 expression levels for 173 AML patients from The Cancer Genome Atlas (TCGA) public database and analyzed their correlation with the expression levels of genes involved in the ER stress response, namely activating transcription factor 4 (ATF4), DNA damage inducible transcript 3 (DDIT3) and HSP family A (Hsp70) member 5 (HSPA5). However, linear regression analysis showed limited degrees of correlation (Online Supplementary Figure S1C), suggesting the involve- ment of other stress response mechanisms in the expo- sure of NKALs by malignant blasts. Altogether, these findings indicate that malignant blasts from AML patients display different danger signals on their surface, and this influences the abundance of circulating NK cells.
CRT exposure on malignant blasts correlates with improved NK-cell effector functions in AML patients in remission
Since the ability of surface-exposed CRT to deliver acti- vatory signals to NK cells had not been previously inves- tigated, we set out to address this possibility. To this aim, we evaluated degranulation and IFN-g production by NK cells from CRTHi and CRTLo AML patients upon non-spe- cific stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin by flow cytometry (Online Supplementary Figure S1D). We failed to detect statistically significant differences in the frequency of NK cells responding to stimulation with IFN-g production (IFN-g+CD45+CD3-CD56+ cells) and degranulation (CD107a+GZMB+CD45+CD3–CD56+ cells) between CRTHi and CRTLo AML patients prior to induction chemotherapy (Figure 2A). On the contrary, upon remis- sion and recovery of non-malignant hematopoiesis, CRTHi patients exhibited significantly improved NK-cell secretory and cytotoxic effector functions compared to
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