CD56 as a marker of an ILC1-like population with NK cell properties that is functionally impaired in AML

Abstract

An understanding of natural killer (NK) cell physiology in acute myeloid leukemia (AML) has led to the use of NK cell transfer in patients, demonstrating promising clinical results. However, AML is still characterized by a high relapse rate and poor overall survival. In addition to conventional NKs that can be considered the innate counterparts of CD8 T cells, another family of innate lymphocytes has been recently described with phenotypes and functions mirroring those of helper CD4 T cells. Here, in blood and tissues, we identified a CD56+ innate cell population harboring mixed transcriptional and phenotypic attributes of conventional helper innate lymphoid cells (ILCs) and lytic NK cells. These CD56+ ILC1-like cells possess strong cytotoxic capacities that are impaired in AML patients at diagnosis but are restored upon remission. Their cytotoxicity is KIR independent and relies on the expression of TRAIL, NKp30, NKp80, and NKG2A. However, the presence of leukemic blasts, HLA-E-positive cells, and/or transforming growth factor-β1 (TGF-β1) strongly affect their cytotoxic potential, at least partially by reducing the expression of cytotoxic-related molecules. Notably, CD56+ ILC1-like cells are also present in the NK cell preparations used in NK transfer-based clinical trials. Overall, we identified an NK cell-related CD56+ ILC population involved in tumor immunosurveillance in humans, and we propose that restoring their functions with anti-NKG2A antibodies and/or small molecules inhibiting TGF-β1 might represent a novel strategy for improving current immunotherapies.

Graphical abstract

Figure 1.

Identification of a CD56⁺ILC1-like population with NK properties that is impaired in AML patients at diagnosis. (A-B) Representative density plots of the extracellular flow cytometry panel used to identify the cNK cell subsets (A) and the ILC subsets (B) in peripheral blood (PB) mononuclear cells (PBMCs; ILC1 as CRTH2⁻ c-Kit⁻ CD56⁻, ILC2 as CRTH2⁺ c-Kit^+/− CD56^+/−, ILCP as CRTH2⁻ c-Kit⁺ CD56^+/−, and cNKs as CD16⁻ CD56^bright and CD16⁺ CD56^dim).^, Lineage markers used for the “helper” ILC staining include CD3, CD4, CD8, CD14, CD15, CD16, CD19, CD20, CD33, CD34, CD203c, and FcεRIα; same lineage markers, except for CD16, were used for the cNK staining. (C) CD127, CD56, CD16, CRTH2, c-Kit fluorescence intensity on ILC and NK subsets were concatenated from 5 HDs and analyzed with t-distributed stochastic neighbor embedding (t-SNE). c-Kit, CD127, CD56, CD16 expression levels on ILC1, CD56⁺ ILC1-like cells and NKs are represented in panel D. Representative gating (E) and quantification of CD56⁺ ILC1-like cell and cNK proportions among lymphocytes in blood from HDs and AML patients at diagnosis (F) (CD56⁺ ILC1-like cells: HD, n = 47; AML patients: n = 60; cNKs: HD: n = 12, AML: n = 18). (G) Summary of the results of the total ILC proportions and ILC1, ILC2, ILCP, and CD56⁺ ILC1-like cell subset frequencies among the total ILCs in HD peripheral blood (N = 47, age median 48, interquartile range 31 to 64). (H) Correlation between ILC subsets’ relative frequencies in blood and age (cord blood: n = 9, children: n = 6, [3 to 12] years old, adults: n = 47, age mean 48). (I) CD56⁺ ILC1-like cell relative frequencies among the total ILCs in tissues from healthy adults (n = 3-18). Spearman correlations were used in panel H. One dot = 1 donor. Mann-Whitney unpaired U tests were used in panel F. ****P < .0001. BM, bone marrow; LN, lymph node. ns, not significant.

Figure 1.

Identification of a CD56⁺ILC1-like population with NK properties that is impaired in AML patients at diagnosis. (A-B) Representative density plots of the extracellular flow cytometry panel used to identify the cNK cell subsets (A) and the ILC subsets (B) in peripheral blood (PB) mononuclear cells (PBMCs; ILC1 as CRTH2⁻ c-Kit⁻ CD56⁻, ILC2 as CRTH2⁺ c-Kit^+/− CD56^+/−, ILCP as CRTH2⁻ c-Kit⁺ CD56^+/−, and cNKs as CD16⁻ CD56^bright and CD16⁺ CD56^dim).^, Lineage markers used for the “helper” ILC staining include CD3, CD4, CD8, CD14, CD15, CD16, CD19, CD20, CD33, CD34, CD203c, and FcεRIα; same lineage markers, except for CD16, were used for the cNK staining. (C) CD127, CD56, CD16, CRTH2, c-Kit fluorescence intensity on ILC and NK subsets were concatenated from 5 HDs and analyzed with t-distributed stochastic neighbor embedding (t-SNE). c-Kit, CD127, CD56, CD16 expression levels on ILC1, CD56⁺ ILC1-like cells and NKs are represented in panel D. Representative gating (E) and quantification of CD56⁺ ILC1-like cell and cNK proportions among lymphocytes in blood from HDs and AML patients at diagnosis (F) (CD56⁺ ILC1-like cells: HD, n = 47; AML patients: n = 60; cNKs: HD: n = 12, AML: n = 18). (G) Summary of the results of the total ILC proportions and ILC1, ILC2, ILCP, and CD56⁺ ILC1-like cell subset frequencies among the total ILCs in HD peripheral blood (N = 47, age median 48, interquartile range 31 to 64). (H) Correlation between ILC subsets’ relative frequencies in blood and age (cord blood: n = 9, children: n = 6, [3 to 12] years old, adults: n = 47, age mean 48). (I) CD56⁺ ILC1-like cell relative frequencies among the total ILCs in tissues from healthy adults (n = 3-18). Spearman correlations were used in panel H. One dot = 1 donor. Mann-Whitney unpaired U tests were used in panel F. ****P < .0001. BM, bone marrow; LN, lymph node. ns, not significant.

Figure 2.

Transcriptomic signature of ex vivo CD56⁺ILC1-like cells in peripheral blood from HDs. (A) Principal component analysis (PCA) of ex vivo fluorescence-activated cell–sorted ILC and NK subsets from HDs peripheral blood (n = 3). (B) Heat map of z scores of the expression levels of genes encoding ILC/NK transcription factors (n = 3). (C) Heat map of log counts per million (CPM) of the 100 most differentially expressed genes between CD56⁺ ILC1-like cells and ILC1, ILC2, ILCP, CD56^bright NKs or CD56^dim NKs. The GO pathway to which each gene belongs is represented at the left of each heat map: Metabolic process GO0008152 (“Metabolism"); Lymphocyte activation GO0046649 (“Activation”); Leukocyte migration GO0050900 (“Migration”); Immune effector process GO0002252 (“Effector”). Max, maximum; Min, minimum.

Figure 3.

CD56⁺ILC1-like cells are cytotoxic effectors regulated by the NKp30, NKp80, TRAIL, and HLA-E pathways. (A) Extracellular flow cytometry analysis of NK receptor expression in ILCP, CD56⁺ ILC1-like cells, and cNKs (n = 4-18). (B) Intracellular flow cytometry was performed using HD PBMCs to assess CD56⁺ ILC1-like cell production of granzyme A (n = 15), granzyme B (n = 15), granzyme K (n = 12), granzyme M (n = 12), perforin (n = 12), and granulysin (n = 12). (C) Extracellular flow cytometry was performed after a 4-hour coculture of ILC/NK-enriched PBMCs with K562 (ratio E:T 1:1), anti-CD107a, and Golgistop to assess CD56⁺ ILC1-like cell degranulation (n = 16). (D) Specific lysis of the K562 tumor cell line by CD56⁺ ILC1-like cells, cNKs, and helper ILCs (results in duplicate). (E) Specific lysis of the U937, K562, and BJAB tumor cell lines by CD56⁺ ILC1-like cells (results in duplicate). (F) Specific lysis by CD56⁺ ILC1-like cells of K562 (ratio E:T 20:1), BJAB (ratio E:T 20:1), and U937 (ratio E:T 10:1) tumor cell lines in the presence of anti-DNAM-1, anti-NKp30, and anti-NKp80 blocking antibodies or TRAIL decoy receptor. (G) Specific lysis of wild-type (WT) or HLA-E–transfected 721.221 tumor cell lines by CD56⁺ ILC1-like cells (results in triplicate). One dot = 1 donor. Statistical tests used for analyses: panel B: Mann-Whitney unpaired U test; panel C: Wilcoxon paired t test, panel G: multiple Holm-Sidak t tests. *P < .05, **P < .01, ***P < .001, ****P < .0001.

Figure 4.

CD56⁺ILC1-like cell cytotoxicity is impaired in AML patients at diagnosis. (A) TRAIL, NKp30, and NKp80 expression in CD56⁺ ILC1-like cells and cNKs in HDs (CD56⁺ ILC1-like: n = 12-18, cNKs: n = 11-15) and AML patients at diagnosis (CD56⁺ ILC1-like: n = 11-20, cNKs: n = 8-10) by extracellular flow cytometry. (B) Intracellular flow cytometry was performed using PBMCs from AML patients at diagnosis to assess granzyme A (n = 4), granzyme B (n = 4), granzyme K (n = 4), perforin (n = 4), and granulysin (n = 7) expression in the CD56⁺ ILC1-like cells and cNKs. The results are compared with the values obtained in the HDs. (C-D) CD107a expression in CD56⁺ ILC1-like cells is assessed by flow cytometry after a 4-hour incubation of ILC/NK-enriched PBMCs from HDs (n = 16) or AML patients at diagnosis (n = 4-13) with medium, the K562 tumor cell line, or blasts at a ratio of 1:1. Representative density plot of CD107a expression is shown in panel C, and the summary results are shown in panel D (1 dot = 1 donor). (E) Representative density plot of HLA-E expression in primary leukemic AML blasts. (F) Summary of HLA-E expression in primary leukemic AML blasts (n = 5). (G) Flow cytometry analysis of CD94, NKG2A, and NKG2C in CD56⁺ ILC1-like cells from AML patients at diagnosis (n = 3-10) and HDs (n = 5-17). One dot = 1 donor. Statistical tests used: panels A-B,D,G: Mann-Whitney U test. **P < .01, ***P < .001, ****P < .0001.

Figure 5.

CD56⁺ILC1-like cell cytotoxicity is restored in AML patients during remission and is modulated by blasts and TGF-β1. (A) Comparison of peripheral blood (PB) CD56⁺ ILC1-like cell relative frequencies in AML patients at diagnosis and during remission (n = 12). (B) Comparison of PB CD56⁺ ILC1-like cell frequencies in paired AML patient samples at diagnosis and during remission (n = 7). (C) Comparison of CD56⁺ ILC1-like cell AML patients at diagnosis or during remission to determine the degranulation capacity after a 4-hour coculture of ILC/NK-enriched PBMCs with K562 (ratio E:T 1:1), anti-CD107a, and Golgistop (remission: n = 3). (D) Comparison of TRAIL, NKp30, and NKp80 expression in PB CD56⁺ ILC1-like cells from AML patients at diagnosis (n = 10) and during remission (n = 5). (E-F) PBMCs from AML patients at diagnosis were depleted of CD33⁺ blasts and cultured for 24 hours in complete medium. (E) Extracellular flow cytometry was performed to assess TRAIL, NKp30, and NKp80 expression in CD56⁺ ILC1-like cells (n = 6-7). Correlation between TRAIL expression after the 24 h culture and blast frequencies in PB (n = 4, panel F). (G) PBMCs from HDs were enriched in ILC/NK cells and cultured for 24 hours with medium only or supplemented with rhTGF-β1 at 5 ng/mL (n = 8). TRAIL, NKp30, and NKp80 expression was assessed by flow cytometry after the culture. (H) Heat map of z scores of expression levels of genes encoding TGF-β receptors in ILCs and cNKs (n = 3). (I) Free/total TGF-β1 ratio in sera from HDs and AML patients. Sera with concentrations above the limit of detection of the assay are shown (HDs: n = 9, AML patients: n = 11). (J) Kaplan-Meier overall survival analysis based on TGF-β1 expression in AML patients from The Cancer Genome Atlas program (TCGA) (n = 125). We excluded patients presenting a t(15;17) translocation (ie, patients with acute promyelocytic leukemia as classified according to the 2017 European LeukemiaNet recommendations³⁶⁾ from our analysis since this condition represents a distinct AML pathophysiological entity. Statistical tests used: panels C-D: Mann-Whitney unpaired U test; panels E,G: Wilcoxon paired t test; panel F: Spearman correlation; panel J: difference in overall survival (OS) between AML patients with high (n = 64) or low (n = 61) TGF-β1 expression based on TCGA data. *P < .05, ***P < .001.

Figure 6.

CD56⁺ILC1-like cells are present in NK-cell preparations used for NK-cell transfer therapy. (A) Representative density plots of the extracellular flow cytometry panel used to identify the ILC subsets in CD3⁻ CD56⁺-enriched fractions of HD PBMCs. (B) Proportions of total ILCs (Lineage⁻CD127⁺), CD56^bright CD16⁻, and CD56^dim CD16⁺ NKs in NK-cell transfer therapy products from HD PBMCs (n = 10). (C) NK marker expression on PB CD56⁺ ILC1-like cells before and after the CD3⁻ CD56⁺ enrichment (n = 5).