The SIRPα-CD47 immune checkpoint in NK cells

Abstract

Here we report on the existence and functionality of the immune checkpoint signal regulatory protein α (SIRPα) in NK cells and describe how it can be modulated for cell therapy. NK cell SIRPα is up-regulated upon IL-2 stimulation, interacts with target cell CD47 in a threshold-dependent manner, and counters other stimulatory signals, including IL-2, CD16, or NKG2D. Elevated expression of CD47 protected K562 tumor cells and mouse and human MHC class I-deficient target cells against SIRPα+ primary NK cells, but not against SIRPα- NKL or NK92 cells. SIRPα deficiency or antibody blockade increased the killing capacity of NK cells. Overexpression of rhesus monkey CD47 in human MHC-deficient cells prevented cytotoxicity by rhesus NK cells in a xenogeneic setting. The SIRPα-CD47 axis was found to be highly species specific. Together, the results demonstrate that disruption of the SIRPα-CD47 immune checkpoint may augment NK cell antitumor responses and that elevated expression of CD47 may prevent NK cell-mediated killing of allogeneic and xenogeneic tissues.

Graphical abstract

Figure 1.

Cd47 overexpression protects MHC-deficient mouse iPSCs from killing by stimulated NK cells or macrophages in vivo. A 1:1 mixture of CFSE-labeled WT and either B2m^−/−Ciita^−/− or B2m^−/−Ciita^−/− Cd47 tg miPSCs was injected into the peritoneum of mice on a syngeneic C57BL/6 background. After 48 h, the ratio of recovered CFSE-positive miPSCs was determined (mean ± SD, triplicates in four animals per group). (A) Recipient WT C57BL/6 mice did not receive additional treatment. (B) A tg CD11b-DT receptor mouse on C57BL/6 background was used to selectively deplete macrophages. (C) The peritoneal cell populations in macrophage-depleted tg CD11b-DT receptor mice were restored by peritoneal cell transfer from WT C57BL/6 mice. (D) Macrophages were pharmacologically depleted in WT C57BL/6 mice using clodronate. (E) In macrophage-depleted mice, peritoneal NK cells were stimulated by peritoneal injections of mouse IL-2. (F) In macrophage-depleted mice, peritoneal NK cells were stimulated by injections of mouse IL-15. (G) In C57BL/6 mice, NK cells were depleted with an anti-NK1.1 depleting antibody. (H) In WT C57BL/6 mice, both macrophages and NK cells were depleted. (I) A 1:1 mixture of CFSE-labeled B2m^−/−Ciita^−/− and B2m^−/−Ciita^−/− Cd47 tg miPSCs was injected into WT C57BL/6 mice. wt, wild-type.

Figure S1.

Cd47 protects MHC-deficient miECs and CD47 protects HLA-deficient hiECs from killing by NK cells and macrophages in vitro. (A–D) WT, B2m^−/−Ciita^−/−, and B2m^−/−Ciita^−/− Cd47 tg miECs were challenged with syngeneic C57BL/6 NK cells (A), allogeneic BALB/c NK cells (B), syngeneic C57BL/6 macrophages (C), or allogeneic BALB/c macrophages (D). Where indicated, NK cells were stimulated with mouse IL-2 or IL-15. Graphs show mean ± SD and three independent replicates per group and time point; three different E:T ratios are shown. (E) B2m^−/−Ciita^−/− miECs were incubated with syngeneic C57BL/6 macrophages treated with an activating anti-Pirb antibody (mean ± SD, three independent replicates per group and time point, and three different E:T ratios are shown). (F and G) WT, B2M^−/−CIITA^−/−, and B2M^−/−CIITA^−/− CD47 tg hiECs were challenged with allogeneic human primary NK cells (F) or allogeneic human macrophages (G). Where indicated, NK cells were stimulated with IL-2 or IL-15. Where indicated, an agonist anti-LILRB1 antibody (clone GHI/75) was added to the assay. A specific blocking antibody against CD47 (clone B6.H12) or a blocking peptide against SIRPα was used in some assays. Graphs show mean ± SD and three independent replicates per group and time point; three different E:T ratios are shown. wt, wild-type.

Figure 2.

The protective effect of Cd47 overexpression against NK cell and macrophage killing is mediated through Sirpα. (A) Sirpα expression on C57BL/6 macrophages and time course of Sirpα expression on C57BL/6 NK cells stimulated with mouse IL-2 (mean ± SD, four independent experiments per group, ANOVA). (B) Cd47 binding to C57BL/6 macrophages and time course of Cd47 binding to C57BL/6 NK cells stimulated with mouse IL-2 (mean ± SD, four independent experiments per group, ANOVA). (C) Sirpα expression on naive C57BL/6 NK cells and 48 h after in vivo stimulation with i.p. mouse IL-2 (mean ± SD, six independent experiments per group, Student’s t test). (D–I) A 1:1 mixture of CFSE-labeled WT and B2m^−/−Ciita^−/− Cd47 tg miPSCs was injected into the peritoneum of syngeneic C57BL/6 mice and after 48 h, and the ratio of recovered CFSE-positive miPSCs was determined (mean ± SD, triplicates in four animals per group). Animals after NK cell depletion received an anti-Cd47 blocking antibody (clone BE0270) with the miPSC injection (D). Animals after macrophage depletion received an anti-Cd47 blocking antibody with the miPSC injection (E) and additionally mouse IL-2 to activate NK cells in vivo (F). Animals after NK cell depletion received an anti-Sirpα blocking antibody (clone P84; G). Animals after macrophage depletion received an anti-Sirpα blocking antibody (H) and additionally mouse IL-2 to activate NK cells in vivo (I). (J) Sirpα expression on Sirpa^−/− macrophages and time course of Sirpα expression on Sirpa^−/− NK cells stimulated with mouse IL-2 (mean ± SD, four independent experiments per group, ANOVA). (K) Cd47 binding to Sirpa^−/− macrophages and time course of Cd47 binding to Sirpa^−/− NK cells stimulated with mouse IL-2 (mean ± SD, four independent experiments per group, ANOVA). (L–O) In some Sirpa^−/− mice, NK cells were depleted (L). In other Sirpa^−/− mice, macrophages were depleted, and NK cells were stimulated with mouse IL-2 (M); some animals in addition received a blocking antibody for Sirpα (N) or Cd47 (O). Graphs show mean ± SD and triplicates in four animals per group. (P) WT, B2m^−/−Ciita^−/−, and B2m^−/−Ciita^−/− Cd47 tg miECs were challenged with Sirpa^−/− macrophages (mean ± SD, three independent replicates per group and time point, and three different E:T ratios). (Q) B2m^−/−Ciita^−/− Cd47 tg miECs were challenged with mouse IL-2–stimulated C57BL/6 NK cells or Sirpa^−/− NK cells. In some groups, an anti-Cd47 or anti-Sirpα blocking antibody was used (mean ± SD, three independent replicates per group and time point, and three different E:T ratios). (R) IFNγ ELISpot assays with B2m^−/−Ciita^−/− Cd47 tg miEC target cells and either mouse IL-2–stimulated C57BL/6 or Sirpa^−/− NK cells. Yac-1 served as controls (boxes show 25th to 75th percentile with median, and whiskers show minimum to maximum; six independent samples per group, Student’s t test). wt, wild-type.

Figure S2.

Expression of SIRPα and CD47 binding of mouse and primary human NK cells. (A and B) The kinetics of Sirpα expression (A) and Cd47 binding (B) of C57BL/6 NK cells in the presence of mouse IL-2 was assessed by flow cytometry (representative histograms are shown of four independent experiments). (C and D) The kinetics of Sirpα expression (C) and Cd47 binding (D) of Sirpa^−/− NK cells in the presence of mouse IL-2 was assessed by flow cytometry (representative histograms are shown of four independent experiments). (E and F) The kinetics of SIRPα expression (E) and CD47 binding (F) of human primary NK cells in the presence of IL-2 was assessed by flow cytometry (representative histograms are shown of four independent experiments). (G) The expression of CD47 on five B2M^−/−CIITA^−/− CD47 tg hiEC clones was assessed by flow cytometry (representative histograms of four independent experiments are shown). wt, wild-type.

Figure 3.

CD47 is species specific, with no cross-reactivity between mouse and human. (A and B) IFN-γ ELISpot assays were performed with B2m^−/−Ciita^−/− and B2m^−/−Ciita^−/− Cd47 tg miPSCs as target cells and syngeneic C57BL/6 (A) or xenogeneic human primary NK cells as effector cells (B). Cd47 antibody blockade (clone BE0270) was used in some groups, and Yac-1 was used as a control (boxes show 25th to 75th percentile with median, and whiskers show minimum to maximum; 12 independent experiments per miPSC group and 6 for Yac-1 groups, ANOVA with Bonferroni’s post hoc test). (C and D) IFN-γ ELISpot assays were performed with B2M^−/−CIITA^−/− and B2M^−/−CIITA^−/− CD47 tg hiPSCs as target cells and allogeneic human primary NK cells (C) or xenogeneic C57BL/6 NK cells as effector cells (D). CD47 antibody blockade (clone B6.H12) was used in some groups, and K562 was used as a control (boxes show 25th to 75th percentile with median, and whiskers show minimum to maximum, 12 independent experiments per hiPSC group and 6 for K562 groups, ANOVA with Bonferroni’s post hoc test). (E–H) Fluc⁺ B2M^−/−CIITA^−/− and B2M^−/−CIITA^−/− CD47 tg hiPSCs were incubated with allogeneic human macrophages (E and F) or xenogeneic C57BL/6 macrophages (G and H), and the BLI signal was quantified (boxes show 25th to 75th percentile with median, and whiskers show minimum to maximum; n = 16 [control], 20 [macrophages], and 9 [Triton X-100] independent samples, ANOVA with Bonferroni’s post hoc test). CD47 antibody blockade was used in some groups. (I–L) Fluc⁺ B2m^−/−Ciita^−/− and B2m^−/−Ciita^−/− Cd47 tg miPSCs were incubated with syngeneic C57BL/6 macrophages (I and J) or xenogeneic human macrophages (K and L), and the BLI signal was quantified (boxes show 25th to 75th percentile with median, and whiskers show minimum to maximum; n = 16 [control], 20 [macrophages], and 9 [Triton X-100] independent samples, ANOVA with Bonferroni’s post hoc test). Cd47 antibody blockade was used in some groups.

Figure 4.

IL-2 alters the expression of inhibitory and stimulatory receptors on primary human NK cells. (A–C) CD3⁻CD7⁺CD56⁺ primary human NK cells were sorted to avoid contamination with myeloid cells. Representative plots with side scatter vs. CD3 (left column), CD7 (middle column), or CD56 (right column) are shown. Rows show the isotype controls (A) and CD3⁻CD7⁺CD56⁺ NK cells before (B) and after 5 d of IL-2 stimulation (C). (D) Whole transcriptome sequencing of CD3⁻CD7⁺CD56⁺ primary NK cells was performed without IL-2 and after 5 d of IL-2 stimulation (heatmap shows row z-scores for the normalized expression of genes at least twofold differentially expressed among groups; FDR < 0.1, n = 3 per group). (E) Changes in gene expression of inhibitory and stimulatory NK receptors during 5 d of IL-2 stimulation (heatmap shows row z-scores for the normalized expression of selected NK cell receptors). All depicted genes are significantly (FDR < 0.1) differentially expressed. Changes are at least twofold, except for SIGLEC7 (log2 fold change = 0.84). (F) Whole transcriptome sequencing of primary human NK cells and NK cell lines were conducted after 24 h of IL-2 stimulation (heatmap shows z-scores for normalized expression of genes that were at least twofold differentially expressed in any four NK cell line compared with primary NK cells). (G) Principal-component analysis showing the distance of primary NK cells to all NK cell lines and the clustering of NKL with its sublines NK-RL12 and NK-CT604. (H and I) Gene expression of stimulatory NK cell receptors (H) and inhibitory NK cell receptors (I; heatmaps show z-scores for normalized expression).

Figure 5.

Threshold of CD47 expression for NK cell and macrophage inhibition. (A) SIRPα expression on macrophages and time course of SIRPα expression on highly selected CD3⁻CD7⁺CD56⁺ primary NK cells stimulated with IL-2 (mean ± SD, four independent experiments per group, ANOVA). (B) CD47 binding to macrophages and time course of CD47 binding to highly selected CD3⁻CD7⁺CD56⁺ primary NK cells stimulated with IL-2 (mean ± SD, four independent experiments per group, ANOVA). (C and D) From the pool of B2M^−/−CIITA^−/− CD47 tg hiECs, five clones with different levels of CD47 overexpression were selected. The CD47 expression on transduced cells was always higher than the basal expression level on WT hiECs but below or above the mean of the pool. CD47 was quantified by RT-PCR (C; mean ± SD, three independent experiments per group) and fluorescence (D; mean ± SD, four independent experiments per group). (E and F) The five B2M^−/−CIITA^−/− CD47 tg hiEC clones were challenged with IL-2–activated human primary NK cells (E) or human macrophages (F). Graphs show mean ± SD and three independent replicates per group and time point; three different E:T ratios are shown. (G) A 1:1 mixture of CFSE-labeled WT and one of the B2M^−/−CIITA^−/− CD47 tg hiEC clones was injected into the peritoneum of immunodeficient NSG mice. Additionally, either IL-2–activated primary NK cells or macrophages were coinjected. After 48 h, the ratio of recovered CFSE-positive hiECs was determined (mean ± SD, triplicates in four animals per group). wt, wild-type.

Figure S3.

Cytokine stimulation induces SIRPα expression on human NK cells. (A) Selected CD3⁻CD7⁺CD56⁺ primary NK cells showed a dose-dependent increase of SIRPα expression with IL-2 (representative histograms are shown of two independent experiments). (B) Unstimulated CD3⁻CD7⁺CD56⁺ primary NK cells did not express SIRPα but showed low-level expression of TIM-3 (representative histograms of two independent experiments are shown). (C and D) Five stimulatory cytokines for NK cells were assessed for their efficacy to induce SIRPα expression (C) and TIM-3 expression (B). While all five increased TIM-3, only IL-2, IL-15, and IFN-α increased NK cell SIRPα (representative histograms of two independent experiments are shown).

Figure S4.

Expression of SIRPα and CD47 binding of human NK cell lines. (A and B) The kinetics of SIRPα expression (A) and CD47 binding (B) of human NK cell lines after 24 h of IL-2 stimulation was assessed by flow cytometry (representative histograms are shown of four independent experiments, bar graphs show mean ± SD, four independent experiments per group, ANOVA).

Figure S5.

NK cell killing of hiECs in the presence and absence of CD47. (A–E) WT, B2M^−/−CIITA^−/−, and B2M^−/−CIITA^−/− CD47 tg hiECs were challenged with primary NK cells (A) or NK cells from a cell line (B–E) using in vitro impedance assays. Graphs show mean ± SD and three independent replicates per group and time point; three different E:T ratios are shown. wt, wild-type.

Figure 6.

Mechanistic interaction of CD47 with NK cell SIRPα. (A–E) B2M^−/−CIITA^−/− CD47 tg hiECs engaged with primary NK cells (A) or NK cells from a cell line (B–E) using in vitro impedance assays in the presence or absence of a specific blocking antibody against CD47 (clone B6.H12) or a blocking peptide against SIRPα. Graphs show mean ± SD and three independent replicates per group and time point; three different E:T ratios are shown.

Figure 7.

CD47 overexpression using plasmid vector transfection. (A) CD47 overexpression in B2M^−/−CIITA^−/− hiPSCs was achieved by plasmid vector transfection, and out of this B2M^−/−CIITA^−/− CD47 tg (plasmid) hiPSC pool, 28 clones were picked and expanded. Clone 21 showed the highest CD47 expression. (B) Clone 21 iPSCs were differentiated into hiECs, and the CD47 expression level was assessed by flow cytometry (mean ± SD, four independent experiments). (C and D) hiEC clone 21 was challenged with IL-2–activated human primary NK cells (C) or human macrophages (D). Graphs show mean ± SD and three independent replicates per group and time point; three different E:T ratios are shown.

Figure 8.

Redirected antibody-dependent cytotoxicity assay against P815. (A) CD3⁻CD7⁺CD56⁺ NK cells were stimulated with IL-2 for 72 h, and SIRPα expression was assessed by flow cytometry (representative histogram of two independent experiments). (B and C) CD3⁻CD7⁺CD56⁺ NK cells were added to Fluc⁺ target P815 cells (green bar), or NK cells were added together with activating antibodies against CD16 (B) or NKG2D (C). The effect of anti-SIRPα to offset the activating signals was assessed. An antibody against CD56 served as control (mean ± SD, three independent experiments per group, ANOVA with Bonferroni’s post hoc test).

Figure 9.

CD47 overexpression in K562 cells. (A) CD47 overexpression was achieved with lentiviral particles and the relative surface expression on K562 CD47 tg was assessed in flow cytometry (mean ± SD, three independent samples per group, Student’s t test). (B–D) CD3⁻CD7⁺CD56⁺ primary NK (B), NKL (C), or NK92 (D) cells were stimulated with IL-2 for 72 h before they were added to Fluc⁺ K562 or K562 CD47 tg, and target cell killing was assessed in BLI assays (mean ± SD, three independent samples per group, Student’s t test). (E) Fluc⁺ K562 cells were transplanted into NSG mice, which received adoptive transfer of IL-2–treated CD3⁻CD7⁺CD56⁺ primary NK cells. Cell survival was longitudinally monitored using BLI (five mice; each line represents one mouse). (F–G) Fluc⁺ K562 CD47 tg cells were transplanted into NSG mice, and cell survival was longitudinally monitored using BLI (five mice per graph; each line represents one mouse). IL-2–treated CD3⁻CD7⁺CD56⁺ primary NK cells were used as effector cells without blocking CD47 (F) or with CD47 antibody block and FcR block (G; clone B6.H12). (H and I) Fluc⁺ K562 CD47 tg cells were transplanted into NSG mice with (H) or without (I) CD47 antibody block but without transfer of human NK cells (three mice per graph; each line represents one mouse). (J) Fluc⁺ K562 CD47 tg cells were transplanted into NSG mice that received IL-2–treated CD3⁻CD7⁺CD56⁺ primary NK cells (five mice; each line represents one mouse). Free Fab fragments of the CD47 antibody (clone B6.H12; H) were used to block CD47 interactions. (K) IL-2–treated NK92 effector cells were transferred in conjunction with the CD47 blocking antibody in mice transplanted with Fluc⁺ K562 CD47 tg cells (five mice; each line represents one mouse).

Figure 10.

Inhibition of rhesus monkey NK cells and macrophages by B2M^−/−CIITA^−/− hiECs overexpressing rhesus CD47. (A) WT, B2M^−/−CIITA^−/−, and B2M^−/−CIITA^−/− CD47 tg hiECs were challenged with rhesus NK cells using in vitro impedance assays. Rhesus NK cells were used either unstimulated or stimulated with rhesus IL-2. Graphs show mean ± SD and three independent replicates per group and time point; three different E:T ratios are shown. (B) The rhesus CD47 tg was expressed in B2M^−/−CIITA^−/− rhCD47 hiECs, and expression by fluorescence is compared with primary rhesus ECs (representative histogram of two independent experiments). (C) B2M^−/−CIITA^−/− rhCD47 tg hiECs were challenged with unstimulated or rhesus IL-2–stimulated rhesus NK cells. Graphs show mean ± SD and three independent replicates per group and time point; three different E:T ratios are shown. (D) WT, B2M^−/−CIITA^−/−, and B2M^−/−CIITA^−/− rhCD47 tg hiECs were challenged with rhesus macrophages. Graphs show mean ± SD and three independent replicates per group and time point; three different E:T ratios are shown. wt, wild-type.