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. 2018 Jul;6(7):766-775.
doi: 10.1158/2326-6066.CIR-17-0498. Epub 2018 May 21.

Adaptive NK Cells Resist Regulatory T-cell Suppression Driven by IL37

Dhifaf Sarhan  1 , Keli L Hippen  2 , Amanda Lemire  2 , Skyler Hying  2 , Xianghua Luo  3   4 , Todd Lenvik  1 , Julie Curtsinger  4   5 , Zachary Davis  1 , Bin Zhang  1 , Sarah Cooley  1 , Frank Cichocki  1 , Bruce R Blazar  2 , Jeffrey S Miller  6
Affiliations

Adaptive NK Cells Resist Regulatory T-cell Suppression Driven by IL37

Dhifaf Sarhan et al. Cancer Immunol Res. 2018 Jul.

Abstract

Natural killer (NK) cells are capable of fighting viral infections and cancer. However, these responses are inhibited by immune suppressor cells in the tumor microenvironment. Tumor progression promotes the recruitment and generation of intratumoral regulatory T cells (Treg), associated with a poor prognosis in cancer patients. Here, we show that canonical NK cells are highly susceptible to Treg-mediated suppression, in contrast to highly resistant CD57+ FcεRγ-NKG2C+ adaptive (CD56+CD3-) NK cells that expand in cytomegalovirus exposed individuals. Specifically, Tregs suppressed canonical but not adaptive NK-cell proliferation, IFNγ production, degranulation, and cytotoxicity. Treg-mediated suppression was associated with canonical NK-cell downregulation of TIM3, a receptor that activates NK-cell IFNγ production upon ligand engagement, and upregulation of the NK-cell inhibitory receptors PD-1 and the IL1 receptor family member, IL1R8 (SIGIRR or TIR8). Treg production of the IL1R8 ligand, IL37, contributed to the phenotypic changes and diminished function in Treg-suppressed canonical NK cells. Blocking PD-1, IL1R8, or IL37 abrogated Treg suppression of canonical NK cells while maintaining NK-cell TIM3 expression. Our data uncover new mechanisms of Treg-mediated suppression of canonical NK cells and identify that adaptive NK cells are inherently resistant to Treg suppression. Strategies to enhance the frequency of adaptive NK cells in the tumor microenvironment or to blunt Treg suppression of canonical NK cells will enhance the efficacy of NK-cell cancer immunotherapy. Cancer Immunol Res; 6(7); 766-75. ©2018 AACR.

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Conflict of interest statement

Conflict of interest statement: Dr. JS Miller serves on the Scientific Advisory Board of Celgene, Fate Therapeutics, and GT Biopharma and has received research funds and/or clinical trials support from Fate Therapeutics and GT Biopharma. These relationships have been reviewed and managed by the University of Minnesota in accordance with its conflict of interest polices. These relationships had no role in funding this research, which has been funded by the NIH grants. Dr. BR Blazar declares a financial conflict with Tmunity and Kadmon Corp. These relationships have been reviewed and managed by the University of Minnesota in accordance with its conflict of interest polices. None of these relationships had any role in this research. The other authors have no conflict of interest to declare.

Figures

Figure 1
Figure 1. Canonical but not adaptive NK-cell function is suppressed by Tregs
NK cells were cocultured with IL2 (50 IU/ml) and autologous HLA-DR+ cells (APCs, 1:1) ± allogeneic Tregs at different Treg:NK ratios (1:1–1:8) for 6 days. CD3−CD56+CD57+FCεRγ+NKG2C− gated canonical and CD3−CD56+CD57+FCεRγ−NKG2C+ gated adaptive NK-cell proliferation (as determined by Ki67 activation) was evaluated by flow cytometry. A) Scatter plots of Ki67 against CD56 for one representative donor and cumulative data from 7 donors shown as mean ± SEM. B) NK cells were cultured with IL2 (50 IU/ml) and APCs ± Tregs at different Treg:NK ratios (1:2–1:8) for 6 days and then stimulated with anti-CD16 antibody (1 μg/ml), IL12 (5 ng/ml), and IL18 (50 ng/ml) 6 hours prior to analysis. Canonical and adaptive NK-cell degranulation (CD107a) and IFNγ production were evaluated by flow cytometry. Results from 4 independent experiments, and pooled data (n = 8–11) are shown as mean ± SEM and statistical analyses were performed using a paired t test.
Figure 2
Figure 2. Adaptive NK cells express high levels of TIM3 and low levels of PD-1 and IL1R8
NK cells were cocultured with IL2 (50 IU/ml) and APCs ± Tregs and evaluated for TIM3, PD-1, and IL1R8 expression before culture and 6 days post-culture. A) Representative histogram plots of receptor expression on gated subsets of NK cells prior to coculture, after culture in the absence of Tregs, or after culture with Tregs. B) Pooled data (n = 6) are shown as mean ± SEM. Data are shown from 3 independent experiments statistical analysis was performed using a paired t test.
Figure 3
Figure 3. Canonical and adaptive NK-cell respond differently to Treg-mediated suppression
NK cells were cocultured with APCs + Tregs for 6 days in the presence of IL2 (50 IU/ml) and stimulated with anti-CD16, IL12, and IL18 6 hours prior to analysis. A) The gating strategy is shown for different NK cells subsets. B) Histogram plots showing the expression of TIM3 and PD-1 from one representative donor. C) IFNγ production and correlation analyses were done by evaluating IFNγ production of different NK-cell subsets (n = 5 donors) and the expression of TIM3 and PD-1 following 6 days of coculture with IL2, APCs and Tregs after terminal stimulation with anti-CD16 antibody, IL12, and IL18. Pooled data (n = 5) are shown from two independent experiments as mean ± SEM and statistical analyses were performed using paired t test and Mixed effects model.
Figure 4
Figure 4. Blocking IL1R8 and PD-1 abrogates Treg-mediated suppression of canonical NK cells
A) Canonical and B) adaptive NK cells cultured with IL2 (50 IU/ml) and APCs ± Tregs (Treg:NK, 1:2) in the presence of blocking antibodies against IL1R8, PD-1, TIM3, or control IgG for 6 days. NK-cell cultures were then stimulated with anti-CD16 antibody, and IL12, and IL18 for 6 hours prior analysis of IFNγ production and proliferation (Ki67). Cumulative data (n = 7) are shown from three independent experiments as mean ± SEM. C) Canonical NK-cell TIM3 (upper panel) and PD-1 (lower panel) expression were analyzed following culture with APCs ± Tregs and in the presence of blocking antibodies against IL37, IL1R8, and PD-1 or IgG. D) Canonical NK cells in culture with APCs were treated with recombinant IL37 (3 μg/ml) or PD-L1 Fc chimera protein (10 μg/ml) and evaluated for the expression of TIM3 and PD-1 expression after 6 days of culture. Cumulative data (n = 6–8) are shown from three independent experiments as mean ± SEM and statistical analyses were performed using pairwise comparison mixed-effects model.
Figure 5
Figure 5. Induction of TIM3 on canonical NK cells restores function in cocultures with Tregs
Purified NK cells were cocultured with APCs (1:1) overnight and stimulated with IL12 (5 ng/ml) and IL18 (50 ng/ml) to increase TIM3 or left unstimulated. Following overnight stimulation, cells were washed and cultured with IL2 (50 IU/ml) ± Tregs (Treg:NK, 1:2) for 6 days in the presence of anti-TIM3 or control IgG. (A) Representative histogram of TIM3 expression is shown before and after coculture. B) NK-cell function was evaluated following stimulation with anti-CD16 antibody and IL2 and IL18 6 hours prior staining. C) Following coculture, NK cells were stimulated with soluble recombinant Gal9 (50 nM) for 20 min. prior to analysis for the phosphorylation of NF-κB (pNF-κB) and Akt (pAkt). Representative and cumulative data are shown from 2 independent experiments. Cumulative data (n = 6–12) are shown as mean ± SEM and statistical analyses were performed using pairwise comparisons and mixed effect models.
Figure 6
Figure 6. Adaptive NK cells resist immunosuppression in the tumor microenvironment
FACS-sorted NK cells were cultured with IL2 (50 IU/ml) and APCs ± Tregs (Treg:NK, 1:2) for 6 days. A) Canonical and B) adaptive NK cells were separately co-incubated with dye-labeled tumor cell lines, K562, THP-1, DU-145 at a 3:1 effector to target cell ratio, and NK-cell cytotoxicity was evaluated over 48-hours by live imaging. Cumulative data (n = 6) of quantified relative killing are shown from two independent experiments as mean ± SEM and statistical analyses were performed using mixed effect models to compare slopes between groups.
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