The double‐edged sword of IFN‐γ‐dependent immune‐based therapies

Immunomodulatory therapies that enhance immune recognition of tumors are emerging as a successful approach for the treatment of poorly immunogenic hematological cancers such as non-Hodgkin's B-cell lymphoma (B-NHL). A key player in these therapies is the pro-inflammatory cytokine interferon gamma (IFNγ). This effector molecule, often referred to as a ‘master regulator’ of anti-tumor immunity is produced by several types of immune cells including natural killer (NK) cells, NK T cells (NKT), CD4+ T cells, CD8+ T cells and B cells.1 This cytokine has been shown to have a critical protective role in therapeutic anti-tumor responses enabling long-term survival. However, despite this, immunotherapy-induced IFNγ can also drive acquired tumor resistance to treatment, leading to tumor escape and poor prognosis.2 In this issue Rearden et al.3 describe the use of the Eμ-myc murine B-NHL mouse model as an immunotherapeutic setting to directly investigate the effects of vaccine-induced IFNγ on host immunity versus lymphoma cell immunogenicity, and the acquisition of therapeutic resistance. Eμ-myc transgenic mice develop B-cell lymphomas as a result of constitutive expression of the oncogenic transcription factor c-myc, and therefore, this model closely represents clinical features of human non-Hodgkin's lymphoma (B-NHL).4 The group had previously reported transient control of B-NHL in mice with a therapeutic vaccine approach that targeted the NKT cells.5 Here, a single vaccination of irradiated tumor cells loaded with α-galactosylceramide (α-GalCer) that targets the immune adjuvant properties of NKT cells provoked rapid production of IFNγ from NKT and NK cells in the Eμ-myc model. This resulted in inhibition of tumor growth and prolonged survival in some mice.5 However, several questions remained on whether IFNγ was mediating a direct effect on the tumor cells and/or was regulating host immunity following therapeutic cell-based vaccination. Deciphering the cellular targets of IFNγ is critically important to the success of IFNγ-dependent immunotherapies in order to predict patient responses, especially in cancers where this cytokine may also drive acquired tumor resistance and relapse in the long term. In this study, a repeat therapeutic vaccination with irradiated, α-GalCer-loaded Eμ-myc tumor cells 7 days later failed to improve survival beyond that of the single vaccination. The loss of the therapeutic effect following secondary vaccination was attributed to decreased production of IFNγ and reduced downstream activation of NK cells and CD8+ T cells (Figure 1). This data also suggested that the presence of the tumor could also reduce the hosts’ response to the therapy as Eμ-myc tumors that ‘escaped’ suppression from vaccine-induced immunity grew more rapidly and demonstrated complete resistance to therapeutic vaccination in naive immunocompetent hosts. To determine the importance of tumor IFNγ signaling in driving this acquired resistance, the authors utilized Eμ-myc tumors with a dysfunctional, dominant negative IFNγ receptor (dnIFNγR) to show that with each repeated vaccination, both wild-type and dnIFNγR tumors displayed increasing resistance to the vaccine therapy with similar levels of tumor burden as unvaccinated mice (Figure 1). Furthermore, a similar in vivo response of wild-type tumors to vaccination was observed regardless of exposure or non-exposure to recombinant IFNγ prior to transfer into immunocompetent mice. Collectively, these data supported the notion that acquired vaccine-induced tumor resistance did not depend upon direct tumor IFNγ signaling. Previous studies have reported that IFNy does not have a direct effect on apoptosis and growth of Eμ-myc lymphoma cells.6 Hence the authors speculated that the production of IFNγ in the B-NHL mouse model was acting through host cell signaling to enhance therapeutic efficacy. Upregulation of MHCI and costimulatory molecule CD86 on Eμ-myc tumors following IFNγ-induced vaccine monotherapy was shown to be dependent on host IFNγ production and diminished in IFNγR−/− recipient mice. Interestingly, MHCI and CD86 were also upregulated in dnIFNγR tumors demonstrating that the initial vaccine-induced effects were independent of tumor IFNy signaling and, more so, due to host cell-mediated sensitization of Eμ-myc tumors to CD8+ T-cell targeting following vaccination (Figure 1). Over time, both MHCI and CD1d surface expression were reduced on both IFNγ-sensitive and IFNγ-insensitive tumor cells following repeated vaccination, further suggesting that alteration of the tumor was a result of an ‘immunoediting’ process that was independent of direct tumor IFNγ signaling. The importance of host IFNγ signaling in NKT cell-targeted vaccination was highlighted in experiments utilizing IFNγR−/− mice. In the unvaccinated setting, Eμ-myc tumors established in these mice which have high systemic levels of IFNγ and transferred to naive immunocompetent mice grew more slowly compared with tumors originating in wild-type mice suggesting that the importance of IFNγ in immune surveillance of tumors by host cells. These transferred Eμ-myc tumors from IFNγR−/− mice also remained sensitive to vaccination in naive immunocompetent mice suggesting that the lack of surveillance by the IFNγR−/− host cells increases the immunogenicity of the tumor, but at the same time the presence of high systemic IFNγ does not act on the tumor directly to induce resistance. Despite the increased IFNγ levels, as well as upregulation of tumor MHCI and CD86 expression post vaccination, the NKT cell-based therapy failed in IFNγR−/− mice due to reduced activation of host effector cells. This strongly suggests that intact host IFNγ signaling is critical for therapeutic vaccine efficacy. Future work to investigate the effect of other cell-based vaccine approaches and their requirement of intact host IFNγ signaling would be informative, especially in other tumor settings. Furthermore, determining how IFNy-independent mechanisms may be driving acquired immune resistance to therapy in NHL and other hematological cancers may enable development of more effective therapies in the future with predicted patient outcomes leading to improved responses. Overall, the study by Rearden et al.3 provides valuable insight into the critical role of IFNγ in controlling B-cell lymphomas in a therapeutic vaccination setting. Their work highlighted that intact IFNγ signaling within the host is more critical for the successful treatment of B-NHL compared with tumor cell sensitivity to IFNγ. Finally, direct tumor IFNγ-signaling alone does not drive acquired tumor resistance to vaccination, suggesting that additional tumor intrinsic immunoediting pathways may be directly or indirectly instigating tumor escape. For example, increased systemic IFNγ by effector cells has been shown to activate checkpoint inhibitor pathways such as PD-1/PDL-1 expression in hematological cancers including Hodgkin's lymphoma, B-NHL, multiple myeloma and myeloid malignancies.7 IFNγ can also activate indoleamine dioxygenase, an enzyme known to regulate the immune response by indirectly causing effector T cell anergy and differentiation of FoxP3+ Tregs.8 It has been implicated in acute myeloid leukemia following activation of NK cells.9 Thus, activation of these pathways may, in part, explain why a second NKT cell-targeted vaccination fails to prolong anti-tumor immunity and prevent relapse of B-cell lymphoma (Figure 1). Taken altogether, these data have implications for potentially improving the therapeutic outcomes of IFNγ-dependent immune therapies. Further investigation into the mechanisms of acquired tumor resistance may better direct combination approaches using NKT-based vaccination and immunotherapy for significantly improving anti-tumor responses. The authors declare no conflict of interest. Effects of single versus repeat therapeutic cell-based vaccination on treatment of B-NHL. (a) A single vaccination of irradiated wild-type (wtIFNγR) or dysfunctional, dominant negative (dnIFNγR) IFNγ receptor Eμ-myc tumor cells loaded with α-GalCer resulted in enhanced frequency and activation of NKT cells resulting in increased production of IFNγ and subsequent activation of NK and CD8+ T cells. This led to inhibition of tumor growth and long-term survival in some mice, although immune resistance was observed in both WT and dnIFNγR Eμ-myc settings. (b) Repeat vaccination failed to upregulate tumor MHC1 and CD1d surface expression, and resulted in NKT anergy with no observed increase in IFNγ in both WT and dnIFNγR tumor settings. Hence in this model, vaccine-induced IFNγ exerts much of its anti-tumor activity via regulation of host immunity and was independent of direct tumor IFNγ signaling. Acquired resistance may be caused by IFN-γ-induced activation of other tumor immunoediting pathways such as PD-1/PDL-1 or indoleamine dioxygenase.