Unlicensed NK cells target neuroblastoma following anti-GD2 antibody treatment

Abstract

Survival outcomes for patients with high-risk neuroblastoma (NB) have significantly improved with anti-disialoganglioside GD2 mAb therapy, which promotes NK cell activation through antibody-dependent cell-mediated cytotoxicity. NK cell activation requires an interaction between inhibitory killer cell immunoglobulin-like receptors (KIRs) and HLA class I ligands. NK cells lacking KIRs that are specific for self HLA are therefore "unlicensed" and hyporesponsive. mAb-treated NB patients lacking HLA class I ligands for their inhibitory KIRs have significantly higher survival rates, suggesting that NK cells expressing KIRs for non-self HLA are mediating tumor control in these individuals. We found that, in the presence of mAb, both licensed and unlicensed NK cells are highly activated in vitro. However, HLA class I expression on NB cell lines selectively inhibited licensed NK cell activity, permitting primarily unlicensed NK cells to mediate antibody-dependent cell-mediated cytotoxicity. These results indicate that unlicensed NK cells play a key antitumor role in patients undergoing mAb therapy via antibody-dependent cell-mediated cytotoxicity, thus explaining the potent "missing KIR ligand" benefit in patients with NB.

Figure 1. Missing KIR ligand is associated with improved OS and PFS in patients with high-risk NB treated with 3F8.

Among patients receiving 3F8, patients lacking class I ligands for autologous inhibitory KIRs (dotted lines) have higher OS and PFS compared with those of patients with all KIR ligands present (solid lines) following (A) chemotherapy alone (OS, HR = 0.53 [95% CI, 0.26–1.08], P = 0.077; PFS, HR = 0.49 [95% CI 0.28–0.88], P = 0.014) or (B) ASCT (OS, HR = 0.57 [95% CI, 0.37–0.88], P = 0.011; PFS, HR = 0.62 [95% CI, 0.41–0.94], P = 0.022), confirming that ASCT is not necessary for the missing ligand effect. (C) Among all patients receiving 3F8, patients missing KIR ligands have higher OS (HR = 0.57 [95% CI, 0.39–0.83], P = 0.003) and PFS (HR = 0.58 [95% CI, 0.42–0.81], P = 0.001) compared with those of patients with all KIR ligands.

Figure 2. 3F8 activates NK cells expressing S-KIRs, NS-KIRs, and NKG2A against NB targets.

In healthy individuals, CD107 degranulation was analyzed in the presence of NB target cells (LAN-1 cells), with and without mAb, among subsets of NK cells. (A) In individual no. 8, S-KIR spNK cells demonstrate strong activation in response to K562 compared with that of NS-KIR spNK cells. All NK populations show minimal activation in response to LAN-1 cells alone but are activated by the addition of 3F8 in response to LAN-1 cells. Data represent the average of 3 separate experiments. (B) Aggregate function of S-KIR and NS-KIR spNK cells from 16 healthy individuals. Addition of 3F8 to LAN-1 cells results in activation of S-KIR–positive, NS-KIR–positive, and KIR-negative NK cells (P < 0.0001), with S-KIR spNK cells more responsive (P = 0.004) and KIR-negative NK cells less responsive (P = 0.006) than NS-KIR spNK cells. (C) NKG2A expression contributes to NK response to LAN-1 cells in the presence of 3F8 among NK cells expressing S-KIR, and NS-KIR (P < 0.0001), and KIR-negative NK cells (P = 0.0004). NS-KIR–positive, NKG2A-negative NK cells are more responsive than KIR-negative, NKG2A-negative NK cells (P = 0.0008) and are equally responsive to KIR-negative, NKG2A-positive cells (P = 0.13). Symbols represent individual samples (mean ± SEM). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3. Licensed NK cells are selectively inhibited by HLA class I ligands induced on NB targets.

(A) Incubation with IFN-γ results in increased HLA class I antigen expression on LAN-1 and BE(2)N cells. HLA-Bw4 and HLA-E antigens are readily induced on BE(2)N but not LAN-1 cells. (B) CD107 degranulation and IFN-γ production in NKG2A-negative, KIR-positive NK cells in response to IFN-γ–treated NB targets in the presence of 3F8. When NK cells from individual no. 17 (HLA-C1/C1/Bw6/Bw6) are incubated with LAN-1 cells, mimicking effectors and NB cells in a patient lacking HLA-C2 and HLA-Bw4 ligands, both NS-KIR–positive (KIR2DL1 single positive [KIR2DL1sp] and KIR3DL1sp) and S-KIR–positive (KIR2DL3sp) NK cells are activated in the presence of 3F8, but only KIR2DL3 spNK cells are inhibited by IFN-γ–induced expression of self HLA on tumor target. When NK cells from individual no. 20 (HLA-C1/C2/Bw4/Bw4) are incubated with BE(2)N cells, mimicking effectors and NB cells in a patient with all ligands present, S-KIR–positive NK subsets (KIR2DL1sp, KIR2DL3sp, and KIR3DL1sp) are inhibited by IFN-γ–induced expression of cognate ligands on the tumor target. In both individuals, blocking antibodies to HLA class I fully restores response among S-KIR–positive NK cells. (C) Aggregate CD107 response among NK populations from 9 HLA-C1/C1 individuals to LAN-1 cells. HLA class I upregulation on the target inhibits S-KIR spNK cells, resulting in a comparatively stronger response from NS-KIR spNK cells (P = 0.003). Aggregate CD107 response among spNK cells from 3 HLA-C1/C2/Bw4 individuals to BE(2)N demonstrates that HLA class I upregulation on the target inhibits all self-specific NK subsets. Symbols represent individual samples (mean ± SEM). **P < 0.01,***P < 0.001.

Figure 4. IFN-γ released by activated NK cells induces upregulation of HLA class I on NB cells in vitro.

(A) HLA-Bw4 and HLA-A, HLA-B, and HLA-C expression on the LAN-1 and BE(2)N NB cell lines is shown following 72 hours in different culture conditions. Supernatants collected from PBMCs coincubated with LAN-1 and BE(2)N cells and 3F8, with or without GM-CSF, induced HLA class I expression on LAN-1 and BE(2)N cells, respectively; in comparison, supernatant collected from PBMCs incubated with or without GM-CSF did not induce HLA expression. (B) By ELISA, PBMCs alone or PBMCs with GM-CSF produced no or minimal IFN-γ. PBMCs activated by LAN-1 cells in the presence of 3F8 and GM-CSF released a substantial amount of IFN-γ compared with baseline and reached 60 pg/ml at 24 hours. (C) Titration assays demonstrate that HLA class I expression can be induced on LAN-1 and BE(2)N cells with 10 pg/ml IFN-γ and that expression increased in a dose-dependent manner.

Figure 5. Unlicensed NK cells dominate NB killing in the presence of 3F8.

3F8 activates both licensed and unlicensed NK cells through CD16. Engagement of inhibitory KIR with its cognate HLA class I ligand on NB cells inhibits licensed NK cell-mediated killing, leaving activation of unlicensed NK cells expressing KIR for non-self or missing ligands intact.