Anergy is a physiological state induced by antigen in T lymphocytes that is characterized by impaired proliferative and cytokine responses to subsequent exposure to cognate antigen (33
). The induction of anergy plays a critical role in maintaining self-tolerance and its loss can result in autoimmunity. Understanding the mechanisms by which anergy is induced is important because it permits investigators to manipulate T cell responses. While anergy in conventional T cells is induced only in certain conditions, such as during situations of incomplete or chronic T cell stimulation (49
), anergy appears to be the physiological response of iNKT cells to cognate glycolipid antigens (21
). Expression of a semi-invariant TCR by iNKT cells permits the host to generate an effective response to glycolipid antigens. Because of their innate characteristics, the response of iNKT cells is fast and robust, with production of cytokines that are potentially toxic to the host. For example αGalCer can induce liver toxicity in mice and, in older animals, even death (51
). As such, the activity of iNKT cells requires tight regulation, to avoid exuberant inflammation and its associated pathology. Consequently, anergy might be a means to avoid such adverse outcomes of iNKT cell activation. While iNKT cell anergy might be beneficial to the host under normal conditions, it poses a problem with regard to the therapeutic activities of iNKT cells. Our finding that the PD-1:PD-L pathway plays a critical role in mediating αGalCer-induced iNKT cell anergy opens up the possibility to manipulate iNKT cell function during glycolipid therapy. Our findings are in general agreement with a recent study (52
), published after submission of the present manuscript, demonstrating a critical role for the PD-1:PD-L pathway in αGalCer-induced iNKT cell anergy.
Prior studies have provided evidence that PD-1:PD-L interactions contribute not only to the induction but also to maintenance of anergy in conventional T cells, at least in the systems tested (31
). In addition, blockade of PD-1:PD-L interactions was able to reverse the exhausted phenotype of CD8+
lymphocytes during chronic infections (44
). We found that blockade of PD-1:PD-L interactions at the time of αGalCer treatment was able to prevent induction of iNKT cell anergy, but had little effect on the anergic phenotype once it was established. This outcome was surprising, as anergic iNKT cells expressed high surface levels of PD-1 (). Thus, other intercellular interactions might play a role in maintaining iNKT cell anergy. Although CTLA-4 has been implicated in the induction of anergy in conventional CD4+
T cells (55
), it was not induced at significant levels on αGalCer-activated iNKT cells (Supplementary Fig. 2
). In addition, we found that αGalCer was able to induce iNKT cell anergy in mice with a combined deficiency in the ligands of CTLA-4 (B7-1 and B7-2) (Supplementary Fig. 5B
), arguing against a role for CTLA-4 in αGalCer-induced iNKT cell anergy.
Recent studies have shown that PD-L1 can bind with B7-1 and that this interaction delivers an inhibitory signal to T cells and inhibits proliferation (57
). Therefore, it is possible that the effects of PD-L1 on iNKT cell anergy were mediated, at least in part, through its interaction with B7-1. However, our finding that PD-1-deficient mice are resistant to induction of iNKT cell anergy by αGalCer argues for a critical role of PD-1. In addition, we found that iNKT cells from B7-1-deficient mice can be rendered anergic following treatment of these animals with αGalCer (Supplementary Fig. 5A
). Based on these findings we favor the notion that interactions between PD-1 and its ligands PD-L1 and PD-L2 are critical for the induction of iNKT cell anergy.
We found that antibodies directed against PD-L1 and PD-L2 were more effective than antibodies against PD-1 to prevent anergy induction ( and ). One likely reason is that the anti-PD-1 antibody employed is not as effective in blocking PD-1:PD-L interactions (36
). The effects of the anti-PD-1 antibody on anergy induction also depended on the parameter investigated, with few effects observed when iNKT cell anergy was analyzed by total proliferative and cytokine responses in spleen cell cultures ( and ) or by investigating iNKT cell expansion in vivo ( and ), but more profound effects were observed when investigating intracellular cytokine production by iNKT cells (), serum IFN-γ production (Supplementary Fig. 3A
) or intracellular IFN-γ production by NK cells (Supplementary Fig. 3B
Although our data clearly demonstrate a critical role for the PD-1:PD-L pathway in anergy induction, blockade of this interaction, or PD-1-deficiency, did not completely overcome iNKT cell hyporesponsiveness induced by αGalCer (, , and ). These findings suggest that additional mechanisms contribute to αGalCer-induced iNKT cell anergy.
In addition to αGalCer, several other conditions can result in the induction of iNKT cell hyporesponsiveness. Multiple bacterial microorganisms and microbial products induce a hyporesponsive phenotype in iNKT cells (39
). Although some bacteria contain glycolipid antigens that can activate iNKT cells, most bacteria activate these cells in an indirect manner, via microbial products that can activate toll-like receptors on DC (58
). DC activated in this manner elaborate cytokines such as IL-12 and IL-18 that can activate iNKT cells. In some, but not all cases, this mode of iNKT cell activation requires CD1d expression on the DC. Sulfatide, a ligand of a subset of CD1d-restricted T cells referred to as Type II NKT cells that express more diverse TCRs than iNKT cells, also induces iNKT cell anergy, in a manner that requires interactions with DC (40
). The phenotype of iNKT cells rendered hyporesponsive by bacterial microorganisms or sulfatide bears many similarities with iNKT cells that became anergic in response to αGalCer. It was therefore surprising that the PD-1:PD-L pathway did not appear to be critical for the induction of iNKT cell hyporesponsiveness by E. coli
bacteria or sulfatide (). Interestingly, we have previously shown that the induction of iNKT cell hyporesponsiveness by E. coli
or sulfatide requires IL-12 production whereas IL-12 is dispensable for αGalCer-induced iNKT cell anergy (39
). These findings suggest multiple pathways and mechanisms for the induction of peripheral iNKT cell tolerance.
iNKT cells hold substantial promise for the development of immunotherapies (45
). We have previously shown that iNKT cell anergy induced by αGalCer or bacterial microorganisms impairs the therapeutic activities of iNKT cells against metastatic cancers (21
). Here we have shown that blockade of PD-1:PD-L interactions at the time of αGalCer treatment prevents the induction of iNKT cell anergy, preserving the therapeutic activities of these cells against B16 tumors (). Likewise, iNKT cells in αGalCer-treated PD-1-deficient mice retained their anti-metastatic activities (). In addition to preventing the induction of iNKT cell anergy, we further found that PD-1:PD-L blockade enhanced the antimetastatic activities of αGalCer (). Therefore, our findings suggest that combined therapy with αGalCer and PD-1:PD-L blockade is superior to αGalCer therapy alone. Furthermore, because this protocol prevents the induction of iNKT cell anergy, it should be effective when employed repeatedly, such as during disease relapse.