The CML experience to elucidate the role of innate T-cells as effectors in the control of residual cancer cells and as potential targets for cancer therapy

Abstract

Considering the general view that unconventional immune effectors play a major role in antitumor immunity, we recently postulated that the distinct new innate CD8 T-cell pool (co-expressing the transcription factor Eomesodermin and innate markers such as KIR/NKG2A) may counteract tumor cells, and thereby be potential target for cancer therapy. Here, to test this assumption, we used successfully targeted anti-leukemic therapy discontinuation (TFR) in chronic myeloid leukemia (CML). Numerical and functional status of innate CD8 T-cells, iNKT cells and γδ T-cells, in comparison with NK cells, was compared longitudinally between non-relapsed patients (i.e., with > 12 months TFR) and relapsed patients (i.e., who experienced molecular recurrence during the first 12 months after TKI cessation) in a prospective pilot cohort (n=32), starting from treatment discontinuation (D0). Perforin, a key cytotoxic immune player, was expressed in a significantly higher proportion of both innate CD8 T-cell and NK-cell subsets in non-relapsed patients, compared with relapsed patients at D0. In parallel, we assessed the expression of PD-1, an exhaustion marker used as target in cancer therapy. For all T-cell subsets, surface-expression level of PD-1 decreased in non-relapsed patients compared with relapsed patients at D0. This was particularly the case when considering iNKT cells for which surface-expression level of PD-1 even decreased relative to healthy control subjects. Lastly, we found a negative correlation between the proportion of innate CD8 T-cells expressing PD-1 and those expressing perforin in non-relapsed patients at D0. The fact that this was not the case in conventional CD8 T-cells is compatible with a reprogrammed effector profile preferentially targeting innate CD8 T-cells in non-relapsed patients. All in all, our results highlight NK cells and innate CD8 T-cells harboring cytotoxic content, as well as global downregulation of PD-1-expression on effector T-cells, as potential predictive functional signatures for successful TFR in CML. Considering innate CD8 T-cells, further investigations are needed to determine whether their possible contributory role in cancer surveillance in CML could be extended to other cancers, and also whether their targeting by immune cheek-point inhibitors could enhance their anti-tumoral functions.

Keywords: PD-1; T-cell effectors; biomarkers; chronic myeloid leukemia (CML); innate CD8 T-cells; innate immunity; perforin; predictive immune signature.

Figure 1

Comparison of relative frequencies of circulating immune effector subsets between relapsed and non-relapsed patients at the moment of TKI discontinuation. (A) Frequencies of NK cells, defined as CD3(-)CD56(+) cells, among live lymphocytes from relapsed (n=4) and non-relapsed (n=18) patients. Healthy control donors (HD) mean: 17.3% ± 8.1% (n=11). (B) Frequencies of total CD8 T cells among live lymphocytes (left panel) and of EMRA, Naive, EM, and CM cells based on the expression of CD45RA and/or CCR7 among total CD8 T-cells, defined as TCR-αβ(+)CD8(+) cells (right panel), from relapsed (n=7) and non-relapsed (n=19) patients. HD means: 19% ± 5.6% of CD8-T cells among live lymphocytes; 24.4% ± 5.4% of EMRA cells, 24.6% ± 18.6% of Naive cells, 10.6% ± 5.7% of CM cells, 40.5% ± 14.3% of EM cells among total CD8 T-cells (n=11). Of note, for EM cells, differences were significant between HD and non-relapsed patients (p-value: 0.002). (C) Frequencies of innate CD8-T cells, defined as TCR-αβ(+)CD8(+) Eomes(+)KIR/NKG2A(+), among total CD8 T-cells from relapsed (n=7) and non-relapsed (n=18) patients. HD mean: 5.5% ± 7.4% (n=13). (D) Frequencies of iNKT cells, defined as CD3(+)TCR-Vα24-Jα18(+), among live lymphocytes from relapsed (n=5) and non-relapsed (n=18) patients. HD mean: 0.15% ± 0.2% (n=11). (E) Frequencies of γδ T-cells, defined as CD3(+)TCR-pan-γδ(+), among live lymphocytes from relapsed (n=5) and non-relapsed (n=18) patients. HD mean: 3.5% ± 2.9% (n=11). Representative dot plots show relative frequencies of the different populations of interest in relapsed (top left) and non-relapsed (bottom left) patients. Histograms represent cohort analysis of relative population frequencies (mean ± SD). Each dot represents a relapsed (red squares) or a non-relapsed (blue triangles) patient. Dotted lines represent relative frequency mean for each population of interest in HD. Statistical significance was determined by the two-tailed Mann-Whitney non-parametric test, ns: not significant.

Figure 2

Frequencies of perforin-expressing cells among circulating immune effector subsets between relapsed and non-relapsed patients at the moment of TKI discontinuation. (A) Frequency of perforin-expressing cells among NK cells, defined as CD3(-)CD56(+) cells, from relapsed (n=4) and non-relapsed (n=18) patients. Healthy control donors (HD) mean: 73.3% ± 16.3% (n=11). Of note, differences were significant between HD and relapsed patients (p-value: 0.0015). (B) Frequency of perforin-expressing cells among total CD8 T-cells, defined as TCR-αβ(+)CD8(+) cells (left panel), and among EMRA, Naive, EM, and CM cells based on the expression of CD45RA and/or CCR7 (right panel) from relapsed (n=7) and non-relapsed (n=19) patients. HD means: 21.2% ± 20.6% of total CD8 T-cells; 41.1% ± 24.9% of EMRA cells; 4.6% ± 7.2% of Naive cells; 21.7% ± 19.4% of EM cells; 1.9% ± 2.5% of CM cells (n=11). Of note, for CM cells, differences were significant between HD and relapsed patients (p-value: 0.0441). (C) Frequency of perforin-expressing cells among innate CD8 T-cells, defined as TCR-αβ(+)CD8(+)Eomes(+)KIR/NKG2A(+), from relapsed (n=7) and non-relapsed (n=16) patients. HD mean: 35.7% ± 14.5% (n=10). Of note, differences were significant between HD and non-relapsed patients (p-value: 0.0015). (D) Frequency of perforin-expressing cells among iNKT cells, defined as CD3(+)TCR-Vα24-Jα18(+), from relapsed (n=4) and non-relapsed (n=18) patients. HD mean: 6.2% ± 10.3% (n=11). (E) Frequency of perforin-expressing cells among γδ T-cells, defined as CD3(+)TCR-pan-γδ(+), from relapsed (n=5) and non-relapsed (n=18) patients. HD mean: 24.5% ± 17.9% (n=11). Representative overlays flow cytometry histograms show perforin expression in the different populations of interest in relapsed (red line) and non-relapsed (blue line) patients. Histograms represent cohort analysis of relative population frequencies (mean ± SD). Each dot represents a relapsed (red squares) or a non-relapsed (blue triangles) patient. Dotted lines represent frequency mean of perforin-expressing cells for each population of interest in HD. Statistical significance was determined by the two-tailed Mann-Whitney non-parametric test, ns: not significant, *p < 0.05; **p < 0.01.

Figure 3

High frequencies of perforin-expressing NK cells and innate CD8 T-cells are associated with treatment-free remission. (A) Survival without relapse over the first 12 months following TKI cessation in patients with high (blue line) and low (red line) frequency of perforin-expressing NK cells at the moment of TKI cessation. The optimal cut-off value of frequency of perforin-expressing NK cells (38.75%) was calculated using the Youden index. (B) Survival without relapse over the first 12 months following TKI cessation in patients with high (blue line) and low (red line) frequency of perforin-expressing innate CD8 T-cells at the moment of TKI cessation. The optimal cut-off value of frequency of perforin-expressing innate CD8 T-cells (45%) was calculated using the Youden index. The number of subjects at risk is shown below the curves. Statistical significance was determined by Log-Rank test.

Figure 4

Comparison of PD1 surface-expression level on circulating immune effector T-cell subsets between relapsed and non-relapsed patients at the moment of TKI discontinuation. (A) PD-1 cell-surface MFI (mean fluorescence intensity) levels among total CD8 T-cells, defined as TCR-αβ(+)CD8(+) cells (left panel), and among EMRA cells, Naive cells, EM cells, and CM cells based on the expression of CD45RA and/or CCR7 (right panel) from relapsed (n=7) and non-relapsed (n=19) patients. Healthy control donors (HD) means: 274.6 ± 168.8 of total CD8 T-cells; 256.1 ± 168.8 of EMRA cells; 56.4 ± 40.2 of Naive cells; 545.3 ± 201.5 of EM cells; 278.7 ± 139 of CM cells (n=11). Of note, differences were significant between HD and non-relapsed patients for total CD8-T cells, EMRA cells and EM cells (p-values: 0.0031; 0.0164; 0.0037 respectively). (B) PD-1 cell-surface MFI on innate CD8 T-cells, defined as TCR-αβ(+)CD8(+)Eomes(+)KIR/NKG2A(+), from relapsed (n=7) and non-relapsed (n=16) patients. HD mean: 253.4 ± 125.1 (n=10). (C) PD-1 cell-surface MFI on iNKT cells, defined as CD3(+)TCR-Vα24-Jα18(+), from relapsed (n=5) and non-relapsed (n=11) patients. HD mean: 385.1 ± 113 (n=18). Of note, differences were significant between HD and relapsed patients (p-value: 0.0051). (D) PD-1 cell-surface MFI on γδ T-cells, defined as CD3(+)TCR-pan-γδ(+), from relapsed (n=5) and non-relapsed (n=17) patients. HD mean: 230.1 ± 93.2 (n=11). Representative overlays flow cytometry histograms show PD-1 expression in the different populations of interest in relapsed (red line) and non-relapsed (blue line) patients. Histograms represent cohort analysis of PD-1 cell-surface MFI (mean ± SD). Each dot represents a relapsed (red squares) or a non-relapsed (blue triangles) patient. Dotted lines represent frequency mean of PD-1 cell-surface MFI for each population of interest in HD. Statistical significance was determined by the two-tailed Mann-Whitney non-parametric test, ns: not significant, *p < 0.05; **p < 0.01.

Figure 5

Low PD-1 MFI on total CD8 T-cells, iNKT innate CD8 T-cells are associated with treatment-free remission. (A) Survival without relapse over the first 12 months following TKI cessation in patients with low (blue line) and high (red line) PD-1 MFI on total CD8 T-cells at the moment of TKI cessation. The optimal cut-off value of PD-1 MFI on total CD8 T-cells (151) was calculated using the Youden index. (B) Survival without relapse over the first 12 months following TKI cessation in patients with low (blue line) and high (red line) PD-1 MFI on iNKT cells at the moment of TKI cessation. The optimal cut-off value of PD-1 MFI on iNKT cells (433.5) was calculated using the Youden index. (C) Survival without relapse over the first 12 months following TKI cessation in patients with low (blue line) and high (red line) PD-1 MFI on innate CD8 T-cells at the moment of TKI cessation. The optimal cut-off value of PD-1 MFI on total CD8 T-cells (131.5) was calculated using the Youden index. The number of subjects at risk is shown below the curves. Statistical significance was determined by Log-Rank test.

Figure 6

Correlation analysis between percentages of perforin-expressing cells and PD-1 cell-surface MFI levels among CD8 T-cell subpopulations in non-relapsed patients at the moment of TKI discontinuation. (A) Correlation plots showing negative correlation between PD-1 cell-surface MFI and perforin-expressing innate CD8-T cells, defined as TCR-αβ(+)CD8(+)Eomes(+)KIR/NKG2A(+), (n=16). Representative dot plots show frequencies of PD1 versus perforin-expressing innate CD8 T cells from two non-relapsed patients (left). (B) Correlation plots showing no correlation between PD-1 cell-surface MFI and percentages of perforin-expressing cells among total CD8-T cells, defined as TCR-αβ(+)CD8(+), EMRA cells, defined as TCR-αβ(+)CD8(+)CCR7(-)CD45RA(+),and EM cells, defined as TCR-αβ(+)CD8(+)CCR7(-)CD45RA(-), frequencies (n= 19). Spearman correlation test.