Blockade of inhibitory killer cell immunoglobulin-like receptors and IL-2 triggering reverses the functional hypoactivity of tumor-derived NK-cells in glioblastomas

Abstract

Killer cell immunoglobulin-like receptors (KIRs) comprise a group of highly polymorphic inhibitory receptors which are specific for classical HLA class-I molecules. Peripheral blood and freshly prepared tumor cell suspensions (n = 60) as well as control samples (n = 32) were investigated for the distribution, phenotype, and functional relevance of CD158ab/KIR2DL1,-2/3 expressing NK-cells in glioblastoma (GBM) patients. We found that GBM were scarcely infiltrated by NK-cells that preferentially expressed CD158ab/KIR2DL1,-2/3 as inhibitory receptors, displayed reduced levels of the activating receptors CD335/NKp46, CD226/DNAM-1, CD159c/NKG2C, and showed diminished capacity to produce IFN-γ and perforin. Functional hypoactivity of GBM-derived NK-cells persisted despite IL-2 preactivation. Blockade with a specific KIR2DL-1,2/3 monoclonal antibody reversed NK-cell inhibition and significantly enhanced degranulation and IFN-γ production of IL-2 preactivated NK-cells in the presence of primary GBM cells and HLA-C expressing but not HLA class-I deficient K562 cells. Additional analysis revealed that significant amounts of IL-2 could be produced by tumor-derived CD4⁺ and CD8⁺CD45RA^- memory T-cells after combined anti-CD3/anti-CD28 stimulation. Our data indicate that both blockade of inhibitory KIR and IL-2 triggering of tumor-derived NK-cells are necessary to enhance NK-cell responsiveness in GBM.

Figure 1

Frequency, phenotype, and distribution of NK-cells within peripheral blood and tumor cell suspensions of GBM patients opposed to control individuals. (a) The frequencies of CD3^-CD56⁺ NK-cells within the CD45⁺ leucocyte and lymphocyte population, as well as the proportion of CD56^dim and CD56^bright NK-cells were calculated (b). The frequencies of CD159a/NKG2A⁺ and CD158ab/KIR2DL-1,-2/3 in CD56^dim and CD56^bright NK-cell subsets in peripheral blood (PBMC-GBM) and tumor cell suspensions of GBM patients (TIL-GBM) and control individuals (PBMC-Co) are shown. (c) In addition, the proportion of CD158ab⁺ and CD159a⁺ tumor-derived CD3^-CD56⁺ NK-cells, as well as the ratio of CD158ab to CD159a compared to peripheral blood of GBM patients or control individuals were determined. (d) Representative immunohistochemical staining of KIR2DL-1/-2/3-expressing cells with glioblastoma tissue. Mean values + /− SD are depicted. P values were calculated using the Kruskal–Wallis test with post-hoc Dunn´s multiple comparison analysis (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 2

Expression profile and functional activity of CD158ab/KIR2DL-1,-2/3⁺ NK-cells. (a) Expression profile of selected NK-cell receptors and activation markers on CD158ab⁺ CD3^-CD56⁺ NK-cells. The frequencies of CD158ab⁺ CD3^-CD56⁺ NK-cells co-expressing the natural cytotoxic receptor CD335/NKp46, the activating NK receptors CD226/DNAM-1 and CD159c/NKG2C, CD16/Fc γ RIII, CD25/IL2Rα and CD279/PD-1 in peripheral blood and tumor cell suspensions of GBM patients and control individuals were determined. (b) IFN-γ and perforin expression of CD3^-CD56⁺ NK-cells and CD158ab⁺ NK-cell subsets (n = 10): The proportion of IFN-γ and perforin producing cells within the entire CD3^-CD56⁺ NK-cell population and the CD158ab⁺ NK-cell subsets of peripheral blood and tumor cell suspensions of GBM patients and control individuals after stimulation with PMA/Ionomycin were determined. (c) IFN-γ expression of CD56^dim and CD56^bright as well as CD158ab⁺ CD56^dim and CD56^bright NK-cell subsets (n = 10): The proportion of IFN-γ cells within the CD56^dim and CD56^bright NK-cell population and CD158ab⁺ CD56^dim and CD56^bright NK-cell subsets of peripheral blood and tumor cell suspensions of GBM patients and control individuals after stimulation with PMA/Ionomycin were determined. Mean values + /− SD are shown. P values were calculated using the Kruskal–Wallis test with post-hoc Dunn´s multiple comparison analysis (*p < 0.05,**p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 3

KIR2DL-1,-2/3 blockade reverses the functional hypoactivity of GBM-derived NK-cells. (a) Functional analysis of blood- and GBM-derived NK-cells: NK-cell responsiveness was assessed by cell surface mobilization of CD107a and IFN-γ production following K562 stimulation of resting or IL-2 preactivated PBMC and TIL at an effector to target ratio of 10:1 for 4.5 h. Representative dot plots are shown illustrating the capacity of blood and tumor-derived CD3^-CD56⁺ NK-cells to express CD107a, IFN-γ or both CD107a and IFN-γ after stimulation with K562-0 cells or culture medium alone (none). (b,c) The activity and cytolytic potential of glioblastoma-derived NK-cells after KIR2DL-1,-2/3 blockade were assessed. (b) Blocking experiments revealed that Lirilumab significantly inhibited the binding of CD158a and CD158b mAb to blood- and tumor-derived NK-cells at a concentration of 30 μg/ml (****p < 0.0001). (c) IL-2 preactivated PBMC and TIL from different glioblastoma patients (n = 5) were pre-incubated with a human IgG4 isotype control mAb or Lirilumab (30 μg/ml), cocultured with culture medium (none), IFN-γ pretreated HLA-class I deficient K562 cells (K562-0) and HLA-C expressing K562 cells (K562-HLA-C) at an effector to target ratio of 10:1 for 4.5 h and then analyzed by flow cytometry. The frequencies of CD107a⁺, IFN-γ⁺and CD107a⁺IFN-γ⁺CD3^-CD56⁺ NK-cells were determined. Mean values + /− SD are depicted. P values were calculated using the two-tailed Mann–Whitney test (*p < 0.05, **p < 0.01).

Figure 4

KIR2DL-1,-2/3 blockade improves the NK-cell stimulatory properties of HLA class-I expressing GBM cells. (a) Primary GBM cell cultures were prepared and evaluated for the expression of HLA class-I molecules (HLA-ABC) by flow cytometry (n = 5). (b) Representative immunohistochemical staining of HLA class I-expressing cells within glioblastoma tissue. (c) Expression of HLA class-I molecules on unstimulated (grey histograms) or IFN-γ pretreated (red histograms) K562-HLA-C and primary GBM cells (GBM1-3). (d) IL-2 preactivated PBMC from glioblastoma patients (n = 3) were pre-incubated with a human IgG4 isotype control mAb or Lirilumab (30 μg/ml), cocultured with IFN-γ pretreated HLA-C expressing K562 cells (K562-HLA-C) or primary GBM cells at an effector to target ratio of 10:1 for 4.5 h and then analyzed by flow cytometry. The frequencies of CD107a⁺, IFN-γ⁺ and CD107a⁺IFN-γ⁺ CD3^-CD56⁺ NK-cells were determined. Mean values + /− SD are depicted. P values were calculated using the two-tailed Mann–Whitney test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 5

Functional analysis of GBM-derived CD4⁺ and CD8⁺ memory T-cells. (a) The frequency of CD4⁺ and CD8⁺ CD45RA^- memory T-cells in PBMC and TIL of GBM patients were determined by flow cytometry (n = 10). Mean values + /− SD are depicted. P values were calculated using the two-tailed Mann–Whitney test (****p < 0.0001). (b) IL-2 and (c) IFN-γ production of CD4⁺ and CD8⁺ CD45RA^- memory T-cells after stimulation with culture medium (none), immobilized anti-CD3 mAb, immobilized anti-CD3/anti-CD28 mAb or PMA/Ionomycin for 6 h in the presence of Brefeldin A and Monsenin (n = 10). Mean values + /− SD from five different GBM patients are depicted. P values were calculated using the Kruskal–Wallis test with post-hoc Dunn´s multiple comparison analysis (**p < 0.01, ***p < 0.001, ****p < 0.0001).