Unlicensed NK cells target neuroblastoma following anti-GD2 antibody treatment (original) (raw)
Missing KIR ligand is associated with improved survival in patients with high-risk NB receiving anti-GD2 mAb. We previously reported that patients receiving ASCT with 3F8 had superior OS and progression-free survival (PFS) if they lacked HLA class I ligands for autologous inhibitory KIRs (25). To test whether ASCT is necessary for this benefit, we evaluated updated outcomes of 166 previously reported patients who received 3F8 following ASCT (25) and 76 new patients who received 3F8 following chemotherapy without ASCT. Patient characteristics for both cohorts are listed in Table 1. The median follow-up was 74 months from the institution of 3F8 immunotherapy, and the OS and PFS were similar in both patient groups (data not shown).
Patients were considered missing KIR ligand if they lacked one or more HLA class I ligand for their inhibitory KIRs. In contrast, patients with “all ligands present” possessed all HLA class I ligands for their inhibitory KIRs. 153 patients (63%) were missing KIR ligand, and the proportions of missing KIR ligand were comparable among patients who received chemotherapy without ASCT (62%) and those who received ASCT (64%). In both the non-ASCT and ASCT settings, missing KIR ligand was associated with increased OS and PFS (Figure 1, A and B), indicating that ASCT is not necessary for the “missing ligand” effect and suggesting that the effect may be more related to the mAb 3F8 or to NK-NB interaction in general. Among patients receiving 3F8, the median OS and PFS were 114 and 50 months, respectively, for patients missing KIR ligand compared with a median OS and PFS of 51 and 18 months, respectively, for patients with all ligands present (Figure 1C). Thus, compared with that of patients with all KIR ligands, patients lacking one or more class I ligands for autologous inhibitory KIRs had significantly longer OSs (hazard ratio [HR] = 0.57 [95% CI, 0.39–0.83], P = 0.003) and PFSs (HR = 0.58 [95% CI, 0.42–0.81], P = 0.001). In the multivariate analysis, which controlled for age, MYCN amplification, and bone metastases, the missing ligand effect was maintained for OS (HR = 0.48 [95% CI, 0.32–0.71], P < 0.001) and PFS (HR = 0.52 (95% CI, 0.36–0.74), P < 0.001) (Table 2), and there were no significant differences in disease status between the missing KIR ligand and all ligands present groups (P = 0.66; Supplemental Table 1; supplemental material available online with this article; doi:10.1172/JCI62749DS1). Although lack of any one class I ligand was associated with improved survival over that of those who possessed all KIR ligands, the highest OSs (HR = 0.38 [95% CI, 0.18–0.79], P = 0.01) and PFSs (HR = 0.28 [95% CI, 0.13–0.58], P = 0.001) were evident in patients who were HLA-Bw6/Bw6 and lacking the HLA-Bw4 ligand for autologous KIR3DL1 (Supplemental 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.
Licensed and unlicensed NK cells are activated in the presence of mAb 3F8. The improved outcome in patients with high-risk NB missing KIR ligand implies that unlicensed NK cells expressing NS-KIRs in these individuals become activated and contribute significantly to tumor control. To determine whether unlicensed NK cells expressing NS-KIRs can be mobilized by 3F8 for ADCC and tumor toxicity in vitro, we evaluated unlicensed and licensed NK cell activity from normal individuals in response to NB cell lines in the presence or absence of 3F8. Donors with KIR and HLA genotypes predictive of missing KIR ligand (n = 16) were selected for evaluation, allowing functional comparisons between NK populations expressing S-KIRs or NS-KIRs within each individual. Because donors were of a variety of KIR and HLA ligand backgrounds, licensed and unlicensed NK populations were defined by different KIR/self-HLA combinations (Supplemental Table 2). Four donors with all ligands present were also evaluated.
Multicolor flow cytometry provided single-cell assessment of NK cells exclusively expressing a single inhibitory KIR, permitting evaluation of unlicensed NK cells expressing NS-KIRs distinct from licensed NK cells expressing S-KIRs. Among individuals with missing KIR ligand, unlicensed NK cells exclusively expressing a single NS-KIR represented 3%–35% of the total NK repertoire (Supplemental Table 3). Among these same individuals, licensed NK cells exclusively expressing a single S-KIR represented 8%–31% of the total NK repertoire. In individuals with all ligands present, licensed NK cells exclusively expressing 1 S-KIR represented 19%–35% of the total NK repertoire. Thus, unlicensed and licensed NK cells expressing a single inhibitory KIR can represent substantial segments of the total NK repertoire.
In each individual, we compared CD107 mobilization as a marker of cytotoxic response among NS-KIR–positive and S-KIR–positive NK cells following stimulation with the HLA class I–negative K562 target cell line or with HLA-genotyped NB cell lines (Supplemental Table 4) in the presence or absence of mAb 3F8. Among all individuals, both NS-KIR and S-KIR NK cell populations had dim CD56 expression, indicating their equivalent terminal differentiation and potential for cytotoxic response (27, 28). Furthermore, there was no difference in CD16 expression among the NK populations (data not shown).
As expected, in an HLA-C1/C1, HLA-Bw4/Bw6 individual, licensed NK cells exclusively expressing the self-specific KIR2DL3 or KIR3DL1 receptor were responsive to K562 cells, while unlicensed NK cells expressing the non-self-specific KIR2DL1 receptor were hyporesponsive (Figure 2A). Although NB cell lines have low to no cell surface expression of HLA class I molecules (7, 8) (Figure 3A), rendering them potentially susceptible to NK cytotoxicity, all NK cells, regardless of their licensed status, responded poorly to stimulation with the NB cell line LAN-1 (Figure 2A). In the presence of the anti-GD2 mAb 3F8, however, licensed single-positive NK (spNK) cells for S-KIRs were highly activated when stimulated (Figure 2A). Unexpectedly, the unlicensed NS-KIR spNK cells also demonstrated higher activation in the presence of 3F8, compared with that of the K562 control. KIR-negative NK cells were also activated but to a lesser degree. These results were confirmed with the IMR-32 cell line and 4 other NB cell lines (data not shown), demonstrating that multiple NK subsets are recruited by 3F8 to mediate ADCC against NB target cells. Cytotoxicity assays confirmed that addition of 3F8 was critical for NK-mediated NB lysis (Supplemental 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.
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.
Aggregate analysis of NK response to the NB cell line, LAN-1, from 16 individuals demonstrated that NK cells expressing S-KIRs or NS-KIRs showed strong CD107 mobilization mediated by 3F8 (P < 0.0001) (Figure 2B). A higher percentage of S-KIR spNK cells compared with NS-KIR spNK cells exhibited CD107 mobilization (P = 0.004), confirming that licensed NK cells are more activated for ADCC (18, 19). The aggregate analysis also showed response among KIR-negative NK cells, albeit to a lower level than that in the NS-KIR–positive population (P = 0.006).
Based on the results in the KIR-negative NK cells, we surmised that CD94/NKG2A may contribute to the NK cell response to LAN-1 cells in the presence of 3F8. Indeed, NKG2A expression contributed to response among all NK cell populations, including the KIR-negative cells (Figure 2C). Among individuals with missing KIR ligand, NKG2A-positive KIR-negative NK cells represented 13%–51% of the total NK repertoire, and there was no preferential coexpression of NKG2A among NS-KIR or S-KIR populations (data not shown).
HLA class I expression on tumor cells selectively inhibits licensed NK cells, sparing ADCC by unlicensed NK cells. In vitro, NK cells exclusively expressing S-KIRs or NS-KIRs are activated by 3F8 for ADCC against NB, although comparatively S-KIR–positive NK cells have higher response. In vivo, however, higher survival among patients missing KIR ligands for autologous inhibitory KIRs suggests not only that unlicensed NK cells expressing NS-KIRs are major contributors to tumor control but also that licensed NK cells expressing S-KIRs in patients with all ligands present are inhibited or silenced. Others have shown that HLA class I molecules, including the nonclassical HLA-E, are upregulated on NB cells following exposure to chemotherapy, isotretinoin, and anti-GD2 antibodies (29–32), therapeutic agents that the patients with high-risk NB in this study received. We therefore hypothesized that upregulation of self-HLA class I ligands on NB cells specifically inhibits licensed NK cells expressing inhibitory receptors for self-HLA ligands while sparing unlicensed NK cells lacking self-specific inhibitory receptors.
To isolate the role of KIR and HLA in NK-NB interactions, we evaluated NKG2A-negative NK cells for CD107 mobilization and IFN-γ production in response to NB cell lines and 3F8 with induced self-HLA expression. HLA class I expression on NB cells was induced following culture for 72 hours with IFN-γ (33). Reproducing autologous NK-NB interactions in a patient missing KIR ligand, NK cells from an HLA-C1/C1/Bw6/Bw6 individual were incubated with the target LAN-1 cells. Although the HLA genotype of LAN-1 cells is HLA-C1/C1/Bw4/Bw6, IFN-γ–induced HLA class I expression did not include HLA-Bw4 (Figure 3A), therefore mimicking an HLA-C1/C1/Bw6/Bw6 tumor target. While both S-KIR–positive and NS-KIR–positive NK cells were activated by LAN-1 cells with 3F8, upregulation of HLA expression on the target cell led to selective inhibition of the self-KIR2DL3 spNK cells (Figure 3B). In contrast, the non-self-specific KIR2DL1 and KIR3DL1 spNK cells maintained their level of activation in the presence of the class I–expressing tumor target. Reproducing autologous NK-NB interactions in an individual with all ligands present, NK cells from an HLA-C1/C2/Bw4/Bw4 individual were incubated with the HLA-C1/C2/Bw4/Bw4 cell line BE(2)N. IFN-γ induced HLA class I expression, including HLA-Bw4, on the BE(2)N target cell line (Figure 3A). Licensed NK cells exclusively expressing KIR2DL1, KIR2DL3, or KIR3DL1 were activated by BE(2)N cells in the presence of 3F8, and all were inhibited by induced HLA class I expression on the target cell (Figure 3B). Confirming that the inhibition was due to interaction of cognate HLA class I molecules on the target cells with self-specific KIRs on the licensed NK cells, addition of anti–HLA class I blocking antibodies to the effector/target mix fully restored activity among the S-KIR NK cells, while leaving activity among the NS-KIR NK cells unchanged (Figure 3B). Selective inhibition of licensed NK cells by induced cognate HLA class I ligands was verified with the NB-1691 and IMR-32 cell lines (data not shown).
In the aggregate analysis of unlicensed and licensed NK response to LAN-1 cells from 9 individuals homozygous for HLA-C1 and therefore lacking KIR ligand HLA-C2, HLA class I upregulation on the NB target cell led to selective inhibition of CD107 mobilization among self-specific KIR2DL2/3+ cells and, consequently, a stronger relative response in the non-self-specific KIR2DL1+ cells (P = 0.003, Figure 3C). Among NKG2A-negative cells, NS-KIR–positive NK cells were more responsive than KIR-negative NK cells (P = 0.001). Similar results were obtained with intracellular IFN-γ response (data not shown). Aggregate analysis of NK activity among 3 individuals with all ligands present was consistent, with inhibition of all S-KIR–bearing NK subsets by NB target cells expressing the cognate HLA class I ligands (P < 0.0001, Figure 3C).
When NKG2A-positive cells were evaluated, NKG2A coexpression enhanced ADCC response of all NK cell subsets, but it did not prevent HLA class I–mediated inhibition of licensed cells (Supplemental Figure 3). Moreover, NKG2A-positive cells were also inhibited by HLA-E expression on the BE(2)N target cell, as evidenced by suppression of activity among the NKG2A-positive KIR-negative subset (Supplemental Figure 3).
Cytokines released by activated NK cells induce HLA class I expression on NB cell lines. Induced HLA expression has been described in NB tumors isolated from patients receiving treatment (29). To determine whether upregulation of HLA class I molecules on NB target cell lines is directly induced by agents commonly used in the treatment of NB, the NB cell lines LAN-1 and BE(2)N were incubated with GM-CSF, isotretinoin, or with different chemotherapy agents. No increase in HLA class I expression on the NB cell lines was detected following treatment with these agents (data not shown). We then investigated whether cytokines released from NK cells activated by 3F8-dependent ADCC could induce HLA class I expression on NB cells. PBMCs and NB targets were coincubated in the presence of 3F8, with or without GM-CSF, which enhances 3F8-dependent ADCC (34). Supernatant obtained following 24 hours of coculture was collected and used as a culture medium for the NB cell lines LAN-1 and BE(2)N (Figure 4). Supernatant from PBMCs alone did not affect HLA class I expression on NB cell lines, and the addition of GM-CSF to PBMCs resulted in supernatant that minimally increased HLA class I expression on the NB target. Coincubation of 3F8 with PBMCs and NB cells, however, produced a supernatant capable of inducing HLA class I expression on LAN-1 and BE(2)N cells (Figure 4A) to levels nearly equivalent to the level attained following the direct addition of IFN-γ (Figure 3A). HLA-Bw4 expression was also specifically induced, albeit to a lower level than that induced in the other class I molecules.
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.
We surmised that release of cytokines by tumor-activated PBMCs in the presence of mAb was responsible for the supernatant-mediated upregulation of HLA class I on the tumor target. Using ELISA, we determined the concentrations of IFN-γ released in the different supernatants. PBMCs alone did not produce detectable IFN-γ, and the addition of GM-CSF resulted in the release of a minimal amount of IFN-γ at 24 hours (Figure 4B). However, PBMCs activated by NB target cells in the presence of 3F8 released a substantial amount of IFN-γ, peaking at 12 hours, and the addition of GM-CSF to 3F8 further enhanced IFN-γ production, reaching 60 pg/ml by 24 hours (Figure 4B). Using titration assays, we determined that HLA expression on LAN-1 and BE(2)N cells could be induced with as little as 10 pg/ml IFN-γ (Figure 4C). We therefore conclude that concentrations of IFN-γ released by tumor-activated PBMCs in the presence of 3F8 and GM-CSF are adequate to induce HLA expression on the tumor.