Calreticulin exposure on malignant blasts predicts a cellular anticancer immune response in patients with acute myeloid leukemia

Abstract

Experiments performed in mice revealed that anthracyclines stimulate immunogenic cell death that is characterized by the pre-apoptotic exposure of calreticulin (CRT) on the surface of dying tumor cells. Here, we determined whether CRT exposure at the cell surface (ecto-CRT) occurs in human cancer in response to anthracyclines in vivo, focusing on acute myeloid leukemia (AML), which is currently treated with a combination of aracytine and anthracyclines. Most of the patients benefit from the induction chemotherapy but relapse within 1-12 months. In this study, we investigated ecto-CRT expression on malignant blasts before and after induction chemotherapy. We observed that leukemic cells from some patients exhibited ecto-CRT regardless of chemotherapy and that this parameter was not modulated by in vivo chemotherapy. Ecto-CRT correlated with the presence of phosphorylated eIF2α within the blasts, in line with the possibility that CRT exposure results from an endoplasmic reticulum stress response. Importantly, high levels of ecto-CRT on malignant myeloblasts positively correlated with the ability of autologous T cells to secrete interferon-γ on stimulation with blast-derived dendritic cell. We conclude that the presence of ecto-CRT on leukemia cells facilitates cellular anticancer immune responses in AML patients.

Figure 1

Malignant myleoblasts from some AML patients spontaneously express CRT on their cell surface (ecto-CRT). (a) Representative FACS analyses of one patient whose blasts do not express ecto-CRT, neither before nor after treatment (right panel), and another patient whose blasts express ecto-CRT even before treatment (left panel). (b) Percentages of blasts expressing ecto-CRT, analyzed by flow cytometry on blood samples before chemotherapy (previous) and 2–6 h after chemotherapy. Statistical analysis was performed using Wilcoxon's test on matched pairs. (c) CRT exposure (red) has been evaluated in CD45-positive blast (green) from blood samples before and after chemotherapy by immunostaining and subsequent confocal microscopy. Nuclei (blue) have been stained with DAPI. Scale bar represents 2 μm. (d) Blasts from different patients have been analyzed for their eIF2α phosphorylation state. A representative immunoblot depicting the phospho-eIF2a and is shown. A polyclonal antibody detecting a different epitope has been used to ensure equal loading

Figure 2

Ecto-CRT^pos blasts express less CD47 than ecto-CRT^neg blasts. (a) Cytofluorometric study of CD47 expression on circulating leukocytes from AML patients. The mean fluorescence intensity (MFI) of CD47 was determined by gating on CD45^High CD3⁺ lymphocytes (Ly) and CD45^low blasts (blasts) and compared using a Wilcoxon's test. (b) As in a but comparing the MFI of CD47 on CD45^low blasts between ecto-CRT^pos (n=10) and ecto-CRT^neg (n=10) AML patients. Statistical analysis was performed using the Mann–Whitney test. The dotted black bar represents the average MFI of CD47 on the entire cohort of AML patients (n=20)

Figure 3

Ecto-CRT is associated with enhanced T lymphocyte responses to autologous leukemic DCs. (a) Representative flow cytometry analysis of LPS-matured AML blast-derived DCs (^AMLDC) defined as CD11c⁺ HLA-DR⁺ double-positive cells. Expression of CD80 and CD40 on AML-DC is also depicted. (b and c) Determination of IFNγ levels in the supernatants from purified CD3⁺ T lymphocytes co-cultured with autologous ^AMLDC, autologous undifferentiated blast cells at diagnosis or medium as control. Autologous monocyte-derived DCs (mDC) from HVs were co-cultured with purified autologous CD3⁺ T lymphocytes or medium as control. Statistical analysis was performed using a Wilcoxon matched-pairs test. Results were either plotted as means±S.E.M. b or on a patient-by-patient basis c. ns, nonsignificant

Figure 4

Correlation of ecto-CRT exposure and patient evolution. (a) Overall survival. (b) Relapse-free survival for patients who achieved complete remission after intensive chemotherapy (n=15). The difference between ecto-CRT^pos and ecto-CRT^neg is not statistically significant (log-rank Mantel–Cox test; P=0.5008)