When treated with anti-GD2 mAb, patients with NB who lack one or more HLA ligand for their inhibitory KIRs respond better to treatment, have lower rates of relapse, and survive longer compared with patients who possess all HLA KIR ligands. These results are independent of stem cell transplantation and suggest that the missing ligand effect is a result of either the 3F8 mAb therapy and/or the interaction between NK cells and the NB target. The response and survival advantages associated with missing KIR ligand genotypes, denoting individuals who lack HLA ligands for his/her autologous inhibitory KIR, suggested to us that the unlicensed NK cells expressing KIRs for non–self-HLA ligands in these individuals may be more effective in tumor eradication than the licensed NK cells expressing KIRs for self HLA. We considered 2 possible mechanisms underlying these clinical findings: activation among the NS-KIR–specific NK population by 3F8 and/or selective inhibition of the S-KIR–specific population. For NB treated with 3F8, both mechanisms appear to be important.
In vitro, licensed NK cells expressing S-KIRs and unlicensed NK cells expressing NS-KIRs are both activated by NB targets in the presence of 3F8, but only the licensed cells are inhibited by cytokine-induced expression of self-HLA molecules on tumor targets. While NKG2A expression contributes to the ADCC response, NKG2A-bearing NK cells are also silenced by HLA-E expression on tumor cells (27
). Taken together, the data suggest that upon encountering target cells with HLA class I expression, unlicensed NK cells bearing receptors for non-self HLA are the primary mediators of ADCC. Several studies examining tumor samples from patients with NB and mouse models indicate that upregulation of HLA class I occurs in vivo. All patients in this study received chemotherapy, GM-CSF, and anti-GD2 mAb therapy, and most patients received isotretinoin. It has been reported that tumor specimens from patients with NB treated with chemotherapy upregulate class I expression as a result of partial differentiation (29
); retinoid derivatives induce cellular differentiation and HLA class I expression on NB cells (30
); and, in NB-bearing mice treated with anti-GD2 mAb, recurrent tumors exhibit greater than 5-fold higher MHC class I expression (31
). We could not demonstrate in vitro that agents used to treat NB could directly upregulate HLA class I expression on certain NB cell lines, suggesting that in vivo conditions may have contributed to previous findings or that the cell lines we used may not respond with HLA class I upregulation to these agents (30
). We show clearly, however, that PBMCs responding to NB in the presence of mAb produce cytokines, in particular IFN-γ, at concentrations sufficient to induce HLA class I expression on NB cells. Other cytokines, such as tumor necrosis factor, can further enhance HLA class I expression, including HLA-Bw4 (data not shown), and it is possible that these cytokines are also released in ADCC-dependent lymphocyte activation. These in vitro findings offer insight to in vivo conditions, in which recruitment of NK cells to the tumor microenvironment may result in effector/target interactions, leading to significant local cytokine concentrations. Taken together, the data indicate that it is highly likely that NK activation in vivo by 3F8-dependent ADCC leads to cytokine release, inducing HLA class I expression on the NB target, inhibiting licensed NK cells, and facilitating the striking missing KIR ligand effect (Figure ).
Unlicensed NK cells dominate NB killing in the presence of 3F8.
The in vitro studies presented in this report represented activity among NK cells expressing a single inhibitory KIR. Examination of the NK repertoire among 20 individuals revealed that the single-positive unlicensed and licensed NK populations, while varying in size from individual to individual, can each represent a significant proportion of the total NK repertoire. The repertoire also includes smaller populations of NK cells that express two or more inhibitory receptors, often resulting in higher levels of responsiveness (19
). However, interaction between just one receptor and its HLA class I ligand is sufficient to inhibit these highly responsive NK cells (19
). Thus, licensed NK cells expressing S-KIRs, whether expressing one or more than one inhibitory KIR, will be inactivated by the presence of self-HLA class I on the tumor target.
These findings are highly relevant to patients with other malignancies treated with mAbs, such as rituximab, whose mechanisms of action involve NK-mediated ADCC (37
). Higher HLA class I expression on CD20-positive lymphoma cells is associated with attenuation of rituximab-induced NK-mediated ADCC (38
). However, correlation of KIR/HLA genotyping among patients receiving rituximab and other mAb therapies has not been systematically explored for the missing ligand effect.
Our data support the incorporation of KIR and HLA genotypes as a prognostic marker in patients with high-risk NB receiving antibody immunotherapy. Indeed, KIR/HLA genotypes are as strong a predictor of response and survival, if not better than several conventional NB biomarkers (25
). An analogous study including only patients with NB, particularly those who do not receive antibody treatment, should be performed to confirm whether mAb is necessary for this effect.
Since licensed NK cells expressing S-KIRs have higher ADCC capacity in general, rescuing licensed NK activity from class I inhibition is desirable and could increase response in all patients, including those with all KIR ligands present. Use of newly developed clinical antibodies directed to the inhibitory KIRs would prevent their engagement with HLA cognate ligands and potentially restore licensed NK function (39
). In addition, the use of exogenous NK cells in the treatment of patients with NB may be potentially useful if the patient lacks class I ligands for the donor inhibitory KIRs. Augmentation of innate immunity through adoptive transfer of allogeneic NK cells or the use of agents that increase endogenous NK cell number and activity, such as IL-2, lenalidomide, and anti-CD137 antibody, may all improve NB control, particularly in the presence of 3F8 (40
Unlicensed NK cells expressing NS-KIRs have a clinically significant impact in the control of acute myelogenous leukemia in allogeneic hematopoietic stem cell transplantation (23
), and it is clear from this study that their impact extends to other tumor targets. While transplantation and mAb therapy are hardly normal physiologic conditions, they both take advantage of the important pool of unlicensed NK cells, previously thought to be hyporesponsive and therefore potentially less clinically relevant. In mice, unlicensed NK cells play a dominant role in clearing murine cytomegalovirus (45
), in which the proinflammatory cytokine environment of viral infection leads to higher function among unlicensed cells. These cells can be recalled upon viral rechallenge to eradicate infected cells more efficiently than licensed NK cells, whose cytotoxic and proliferative function is diminished in the presence of MHC class I expression (45
). It is possible that activity among unlicensed NK cells in patients with NB may also be augmented by the proinflammatory state associated with tumor burden. The existence of unlicensed NK cells that have been presumed to be hyporesponsive in an otherwise parsimoniously constructed human immune system has been a paradox. These findings contribute to our understanding of the critical role of the unlicensed NK population in tumor control.