Functional enrichment performed with time course transcriptome expression profile ( Supplemental Table S1 ) on ToppCell Atlas database revealed that this signature mainly characterized natural killer-T (NK-T) lymphocyte cell type ( Figure 2A ), with specific upregulation of IL21R, ADGR1, S1PR5, AUTS2, BCR, BPGM, ANKRD16, EPHA4, OCLN, CHIC1, and CEP41 ( Figure 2B ). These results suggested that during the natural evolution of the disease, the molecular signature of an NK/T lymphocyte cell population could be detected in the PBMC.

A single-cell RNA-seq analysis was performed on the PBMC sample collected on March 2017 where the patient had evident molecular progression with a blood BCR::ABL^-IS level at 2%. She was out of therapy at that time ( Figure 1 ), and her blood counts were normal.

Single-cell transcriptome of 9,036 PBMC could be demultiplexed after Cell Ranger pipeline. A mean number of 9,054 transcripts has been found to be expressed by cell. After preprocessing of single-cell transcriptome of PBMCs, dimension reduction by PCA ( Figure 3A ) and UMAP identified three main topological cell clusters including T-lymphocytes (expressing CD3E), B-lymphocytes (expressing MS4A1), and monocytes expressing CD68 ( Figure 3B ). UMAP dimension reduction conjugated to unsupervised clustering identified 11 distinct cell communities ( Figure 3C ), which expressed distinct patterns of markers ( Figure 3D ). Clusters 7–10 were found to be less represented quantitatively in the blood of the patient ( Figure 3D ).

Clusters 0 and 1, which were the largest ones, seemed to share expression of markers ( Figure 3D ) corresponding to T lymphocytes ( Figures 3B, C ). In terms of expression specificity, cluster 5 shared some markers with clusters 0 and 1( Figure 3D ). Interestingly, among the cluster of T-lymphocytes ( Figures 3B, C ), we could observe another cluster (cluster C3) that expressed a distinct signature from the three other T-lymphocyte clusters (0, 1, and 5, Figure 3D ). Among the best markers characterized during single-cell transcriptome analysis, a high expression of NKG7 was revealed in individual cluster C3 ( Figure 4A ), which correspond to a subpopulation of CD3E+ T lymphocytes ( Figure 4B ). Differential expression analysis between the distinct cell clusters showed that a high expression of natural killer effective markers characterized cluster C3 ( Figure 4C ), such as GNLY (granulysin), GZMH (granzyme H), and CTSW (cathepsin W). In addition, a high expression of CCL5 chemokine and FGFBP2 secreted protein (cytotoxic marker) was found in cluster C3 of natural killer cells from this patient ( Figure 4C ). These molecular results on single-cell transcriptome performed on PBMC suggested therefore the presence of an immunologically competent cells in the blood sample analyzed.

Interestingly, functional enrichment of the gene signature on ToppCell atlas signature highlighted that activation of genes involved in natural killer/T lymphocytes following BCR::ABL quantification. NK cells play important roles in innate immunity against virus or tumors by secreting cytotoxic granules (13). In the case reported here, the transcriptomic profile was compatible with both NK and a NK/T-cell population, the latter known to exhibit anti-leukemia activity (14, 15). The deficient function of the invariant NK/T cells in CML and their restoration on TKI therapy has previously been reported (16, 17). The role of NK cells in CML immune surveillance has also been suggested in patients remaining in deep molecular response after TKI discontinuation (11).

We then analyzed genes expressed in NK cell clusters among NK-T lymphocyte population in single-cell transcriptome, while the patient was in molecular recurrence and without TKI therapy but without hematological relapse. This NK cell cluster was positive for NKG7 expression and cells highly expressed effectors of cytotoxicity genes. Granulysin is an antimicrobial peptide (AMP) highly active against cancer cells such as melanoma (18). Cathepsin W (lymphopain) is a papain-like cysteine protease of unknown function that is specifically expressed in NK cells and to a lesser extent in cytotoxic T cells (CTL), and its expression in NK-92 (NK cell model) is linked to the cytotoxicity observed against K562 (19). Granzyme H is well-known cytotoxic effector of NK cells (20). NKG7+ positive NK cell cluster also highly expressed FGFBP2-secreted protein that is known to be a marker of the cytotoxicity of lymphocytes (21).

These molecular findings could also be linked to the immuno-molecular data obtained from the whole transcriptome analyses. Indeed, transcriptome experiments showed that the IL21R expression (receptor to interleukin 21) followed the BCR::ABL^-IS quantification analyses during TKI interruption period. IL-21 has been shown to participate to the NK cell expansion of K562 cell line engineered to express membrane-bound IL-21 feeder in combination with IL-2 (22, 23).

S1P (5) receptor found in NK cell network is also known to be essential for human NK cell trafficking (24, 25). Another interesting receptor expression was that of GPR56, which is an inhibitory receptor of NK cells, negatively regulating effector functions such as production of inflammatory cytokines and cytolytic proteins, degranulation, and target cell killing through association with the tetraspanin CD81 (26).

Overall, the molecular analyses reported during the long-term TKI discontinuation in this patient allowed to detect major modulation of genes involved with NK cell activity. Interestingly, several genes that we identified to be part of the immunological pathway seemed to be activated with a successful TFR after TKI discontinuation (10, 27–29). Importantly we have identified for the first time NKG7 expression in single cell transcriptome from CML PBMC, this gene being a major regulator of granule exocytosis in cancer (30). Its expression deserves further evaluation in patients with CML.