Proteomic and phenotypic characteristics of memory-like natural killer cells for cancer immunotherapy

Abstract

Background: Human and mouse natural killer (NK) cells have been shown to develop memory-like function after short-term exposure to the cocktail of IL-12/15/18 or to overnight co-culture with some tumor cell lines. The resulting cells retain enhanced lytic ability for up to 7 days as well as after cryopreservation, and memory-like NK cells (mlNK) have been shown to induce complete remissions in patients with hematological malignancies. No single phenotype has been described for mlNK and the physiological changes induced by the short-term cytokine or tumor-priming which are responsible for these enhanced functions have not been fully characterized. Here, we have generated mlNK by cytokine and tumor-priming to find commonalities to better define the nature of NK cell "memory" in vitro and, for the first time, in vivo.

Methods: We initiated mlNK in vitro from healthy donors with cytokines (initiated cytokine-induced memory-like (iCIML)-NK) and by tumor priming (TpNK) overnight and compared them by high-dimensional flow cytometry, proteomic and metabolomic profiling. As a potential mechanism of enhanced cytolytic function, we analyzed the avidity of binding of the mlNK to NK-resistant tumors (z-Movi). We generated TpNK from healthy donors and from cancer patients to determine whether mlNK generated by interaction with a single tumor type could enhance lytic activity. Finally, we used a replication-incompetent tumor cell line (INKmune) to treat patients with myeloid leukaemias to potentiate NK cell function in vivo.

Results: Tumor-primed mlNK from healthy donors and patients with cancer showed increased cytotoxicity against multiple tumor cell lines in vitro, analogous to iCIML-NK cells. Multidimensional cytometry identified distinct memory-like profiles of subsets of cells with memory-like characteristics; upregulation of CD57, CD69, CD25 and ICAM1. Proteomic profiling identified 41 proteins restricted to mlNK cells and we identified candidate molecules for the basis of NK memory which can explain how mlNK overcome inhibition by resistant tumors. Finally, of five patients with myelodysplastic syndrome or refractory acute myeloid leukemia treated with INKmune, three responded to treatment with measurable increases in NK lytic function and systemic cytokines.

Conclusions: NK cell "memory" is a physiological state associated with resistance to MHC-mediated inhibition, increased metabolic function, mitochondrial fitness and avidity to NK-resistant target cells.

Keywords: Adoptive cell therapy - ACT; Leukemia; Memory; Myelodysplastic Syndrome; Natural killer - NK.

Figure 1. Tumor-mlNK and cytokine-mlNK cell display their own unique phenotype but show characteristics of activation. Freshly isolated peripheral blood NK cells from HD were incubated as indicated overnight at 37 °C. The next day, cells were washed and the expression of 30 different NK cell markers was analyzed using spectral flow cytometry. viSNE analysis of multiparametric flow data from three concatenated files was performed on the indicated markers. Each point on the viSNE map represents a single cell and color depicts intensity of protein expression. Graphs show the analysis of the % expression and MeFI of the positive population on the indicated markers. Bars represent the means±SD of three different donors. Comparisons were made between rNK and the different stimulatory conditions using a one-way ANOVA with a Dunnett’s multiple comparison test. Statistical significance is indicated as *p<0.05, **p<0.01, ***p<0.001. The absence of asterisk indicates non-significance. ANOVA, analysis of variance; HD, healthy donor; iCIML, initiated cytokine-induced memory-like; mlNK, memory-like natural killer cell; viSNE, visualization stochastic neighbor embedding.

Figure 2. Tumor-mlNK and cytokine-mlNK cells share a unique phenotypical metacluster containing mature NK cells. Freshly isolated peripheral blood NK cells from HD were incubated overnight at 37 °C as indicated. The next day, cells were washed and the expression of 30 different NK cell markers was analyzed using spectral flow cytometry. After that, viSNE and FlowSOM analysis clustered on 30 NK cell phenotypic markers was performed (A) viSNE plots of the different metaclusters across all conditions. (B) Stacked bars show changes in % cell numbers of each metacluster for every condition. (C, D) Heatmaps show fluorescence intensity of the total of cells on the indicated markers of metaclusters 1–10, with metaclusters of interest highlighted in (C) TpNK and (D) iCIML-NK. Plots show the data from three concatenated donors. HD, healthy donor; iCIML, initiated cytokine-induced memory-like; NK, natural killer; viSNE, visualization stochastic neighbor embedding.

Figure 3. Memory-like NK cells exhibit a unique proteomic profile characterized by the upregulation of mitochondrial survival proteins. Freshly isolated NK cells from three healthy donors were incubated overnight at 37°C as indicated. The next day, cells were prepared for mass spectrometry for proteome-wide protein profiling by removing dead cells and positively selecting NK cells to remove stimulatory agents. (A) Hierarchical clustering analysis using the Manhattan distance measure using all protein z-score values across all samples was carried out. (B) Venn diagram generated by the intersection list of the proteins significantly (p<0.001) upregulated by more than four log compared with NK cells incubated in medium alone. (C, D) Graph showing the average Log ratio from rNK in TpNK, iCIML-NK and IL-15 treated NK cells corresponding to (C) mitochondrial survival proteins and (D) lytic function related proteins. iCIML, initiated cytokine-induced memory-like; NK, natural killer; viSNE, visualization stochastic neighbor embedding.

Figure 4. Tumor-priming increases spare respiratory capacity of NK cells Freshly isolated NK cells were incubated overnight at 37°C as indicated. The next day, cells were prepared by removing dead cells and positively selecting NK cells to remove stimulatory agents. After that, (A–D) glycolytic function and (E–H) mitochondrial respiration were measured according to manufacturer’s protocol in Seahorse XF platform (Agilent). Data were normalized to cell count and cell viability per well using normalization function. Bars represent the means±SD of five different donors. Comparisons were made between all conditions using a one-way ANOVA with a Tukey multiple comparison test. Statistical significance is indicated as *p<0.05, **p<0.01. The absence of asterisk indicates non-significance. ANOVA, analysis of variance; NK, natural killer.

Figure 5. Tumor-primed NK cells from healthy donors and cancer patients lyse hematological and solid tumor cells. (A) Representative example of flow cytometry plots for cytotoxicity assay against 786O. Top plot shows the gating strategy for rNK cells and bottom plot for TpNK cells. NK cells and target cells are gated in P1; in the second plot, target cells are gated as PKH67⁺; in the third plot, live cells are gated as Topro⁻. Cell counts are obtained from the ‘Live cells gate’. (B, C) Freshly isolated peripheral blood NK cells from HD were incubated as indicated overnight at 37 °C. (B) The next day, cells were harvested and set-up for a 4h-cytotoxicity assay against hematological cancer cell lines (Raji, U266, MDS-L and K562), renal cell carcinoma (786-O and ACHN) and ovarian cancer (SKOV3 and OVCAR) at E:T ratio 5:1. Bars represent the means±SD of 3–6 different donors represented by the individual dots. (C) Surface and intracellular expression of different markers was analyzed in NK cells the next day by flow cytometer. Bars represent the means±SD of 2–3 different donors. (D) Freshly isolated peripheral blood NK cells from patients were incubated overnight at 37°C as indicated. The ovarian cancer patient NK cells were isolated from the ascites and were tested in hypoxia (1% O₂). The next day, cells were harvested and set-up for a 4h-cytotoxicity assay as described against hematological cancer cell lines (Raji, K562 and MDS-L), renal cell carcinoma (786-O) and ovarian cancer (SKOV3) at E:T ratio 5:1. To measure avidity (E–H), NK cells from HD were stimulated overnight as indicated. The next day, chips were coated and target cells were seeded. NK cells were prepared by removing overnight stimulatory agents and labeled using CellTrace Far Red dye, before placing into the chips. (E, F) Avidity measure of (E) Raji and (F) SKOV3 at 10 min. (G) Avidity measure of Raji at various timepoints. (H) Avidity of Raji conjugation was compared with lytic function. For measuring cytotoxicity, rNK and TpNK cells from the same donor were set-up for a 4h-cytotoxicity assay against Raji at E:T ratio 5:1. Bars represent the means±SD of three different donors. (B, C, H) Data were tested for normality and comparisons were made between rNK and TpNK cells using the paired t-test. Statistical significance is indicated as *p<0.05, **p<0.01, ***p<0.001. The absence of asterisk indicates non-significance. E:T, effector and target; HD, healthy donor; NK, natural killer.

Figure 6. INKmune generates memory-like NK cells in vivo in patients with myelodysplastic syndrome and acute myeloid leukemia. (A) Infographic representing treatment course. Patients receive three infusions of INKmune on days 1, 8 and 15. Blood samples are taken on days 1, 8, 15, 29, 43, 73 and 119. The sample from patient AML#01 on day 140 is from the bone marrow. (B) Percentage of CD69+NK cells in each patient on the indicated dates. (C, D) Percentage of specific lysis of (C) Raji and (D) K562 mediated by the patients NK cells on the indicated days. (E, F) Histograms show the % expression of CD57, CD2 and granzyme B on the indicated days. Heatmap represents median fluorescence intensity of the positive population normalized to the lowest value for each column for patients (E) MDS#01 and (F) AML#01. MDS, myelodysplastic syndrome; NK, natural killer.