The immune system constantly monitors the emergence of cancerous cells and eliminates them. CD8⁺ cytotoxic T lymphocytes (CTLs), which kill tumor cells and provide antitumor immunity, select their targets by recognizing tumor antigenic peptides presented by MHC class-I (MHC-I) molecules. Cancer cells circumvent immune surveillance using diverse strategies. A key mechanism of cancer immune evasion is downregulation of MHC-I and key proteins of the antigen processing and presentation machinery (APM). Even though impaired MHC-I expression in cancers is well-known, reversing the MHC-I defects remains the least advanced area of tumor immunology. The discoveries that NLRC5 is the key transcriptional activator of MHC-I and APM genes, and genetic lesions and epigenetic modifications of NLRC5 are the most common cause of MHC-I defects in cancers, have raised the hopes for restoring MHC-I expression. Here, we provide an overview of cancer immunity mediated by CD8⁺ T cells and the functions of NLRC5 in MHC-I antigen presentation pathways. We describe the impressive advances made in understanding the regulation of NLRC5 expression, the data supporting the antitumor functions of NLRC5 and a few reports that argue for a pro-tumorigenic role. Finally, we explore the possible avenues of exploiting NLRC5 for cancer immunotherapy.

Aggressive tumors evade cytotoxic T lymphocytes by suppressing MHC class-I (MHC-I) expression that also compromises tumor responsiveness to immunotherapy. MHC-I defects strongly correlate to defective expression of NLRC5, the transcriptional activator of MHC-I and antigen processing genes. In poorly immunogenic B16 melanoma cells, restoring NLRC5 expression induces MHC-I and elicits antitumor immunity, raising the possibility of using NLRC5 for tumor immunotherapy. As the clinical application of NLRC5 is constrained by its large size, we examined whether a smaller NLRC5-CIITA fusion protein, dubbed NLRC5-superactivator (NLRC5-SA) as it retains the ability to induce MHC-I, could be used for tumor growth control. We show that stable NLRC5-SA expression in mouse and human cancer cells upregulates MHC-I expression. B16 melanoma and EL4 lymphoma tumors expressing NLRC5-SA are controlled as efficiently as those expressing full-length NLRC5 (NLRC5-FL). Comparison of MHC-I-associated peptides (MAPs) eluted from EL4 cells expressing NLRC5-FL or NLRC5-SA and analyzed by mass spectrometry revealed that both NLRC5 constructs expanded the MAP repertoire, which showed considerable overlap but also included a substantial proportion of distinct peptides. Thus, we propose that NLRC5-SA, with its ability to increase tumor immunogenicity and promote tumor growth control, could overcome the limitations of NLRC5-FL for translational immunotherapy applications.

SOCS1 is a tumor suppressor in hepatocellular carcinoma (HCC). Recently, we showed that a loss of SOCS1 in hepatocytes promotes NRF2 activation. Here, we investigated how SOCS1 expression in HCC cells affected oxidative stress response and modulated the cellular proteome. Murine Hepa1-6 cells expressing SOCS1 (Hepa-SOCS1) or control vector (Hepa-Vector) were treated with cisplatin or tert-butyl hydroperoxide (t-BHP). The induction of NRF2 and its target genes, oxidative stress, lipid peroxidation, cell survival and cellular proteome profiles were evaluated. NRF2 induction was significantly reduced in Hepa-SOCS1 cells. The gene and protein expression of NRF2 targets were differentially induced in Hepa-Vector cells but markedly suppressed in Hepa-SOCS1 cells. Hepa-SOCS1 cells displayed an increased induction of reactive oxygen species but reduced lipid peroxidation. Nonetheless, Hepa-SOCS1 cells treated with cisplatin or t-BHP showed reduced survival. GCLC, poorly induced in Hepa-SOCS1 cells, showed a strong positive correlation with NFE2L2 and an inverse correlation with SOCS1 in the TCGA-LIHC transcriptomic data. A proteomic analysis of Hepa-Vector and Hepa-SOCS1 cells revealed that SOCS1 differentially modulated many proteins involved in diverse molecular pathways, including mitochondrial ROS generation and ROS detoxification, through peroxiredoxin and thioredoxin systems. Our findings indicate that maintaining sensitivity to oxidative stress is an important tumor suppression mechanism of SOCS1 in HCC.

Tumors evade cytotoxic T lymphocyte (CTL)-mediated killing by downregulating MHC-I, which mainly results from the loss of its transcriptional activator NLRC5. Expressing full-length NLRC5 (NLRC5-FL) or a shorter NLRC5-CIITA fusion protein termed NLRC5 super-activator (NLRC5-SA) in cancer cells upregulates MHC-I expression and promotes antitumor immunity. To distinguish the role of NLRC5 expressed within tumor cells and antigen presenting cells, we studied B16-F10 melanoma expressing NLRC5-FL (B16-N-FL) or NLRC5-SA (B16-N-SA) in Nlrc5+/+ and Nlrc5−/− mice. Both tumors were efficiently controlled in both Nlrc5+/+ and Nlrc5−/− hosts with abundant immune cell infiltration, enriched for activated and differentiated CD8+ and CD4+ T cells, NK, NKT and iNKT cells. B16-N-FL and B16-N-SA tumors showed increased collagen deposition and vascularization, with upregulation of CCL4 and CXCL9 chemokine genes in B16-N-SA tumors. Depletion of either CD8+ T cells or NK1.1+ cells increased the growth of B16-N-FL and B16-N-SA tumors in Nlrc5+/+ mice, and that of B16-N-SA tumors in Nlrc5-/- hosts. Proteomes of B16-N-FL and B16-N-SA cells showed downmodulation of dominant tumor antigens and upregulation of ubiquitination and protein processing pathway proteins. Differentially expressed proteins shared between B16-N-FL and B16-N-SA cells showed enrichment in phagosome and autophagy pathways. We conclude that tumor cell-intrinsic NLRC5 expression is critical for efficient tumor antigen presentation and adaptive and innate immune cell activation, and establishment of an immune-supportive tumor microenvironment to permit immune cell infiltration and their effector functions and achieve tumor control, and that all these effects can be accomplished in MHC-I-low immune evasive tumors by delivering NLRC5-SA.

Introduction: Tumors downregulate NLRC5 expression to reduce MHC-I and evade CD8+ T cell–mediated killing. Introducing full-length NLRC5 (N5FL) or NLRC5-CIITA chimeric protein (N5CA) can restore tumor immunogenicity. Here, we elucidated the underlying mechanisms and also evaluated the requirement of NLRC5 expression in antigen-presenting cells (APCs) of the host for tumor control. Materials and methods: Nlrc5−/− and Nlrc5+/+ mice were implanted with B16-F10 melanoma expressing N5FL or NC5A. Tumor growth, activation of lymphoid cells, their infiltration into tumors and the requirement for tumor control were assessed. The proteomes of B16-F10 cells expressing N5FL or N5CA were analyzed. Results: NLRC5-expressing tumors were efficiently controlled in both Nlrc5+/+ and Nlrc5−/− mice, characterized by infiltration of CD8+ and CD4+ cells, natural killer (NK) cells, NK T (NKT) cells and invariant NKT (iNKT) cells, increased tumor vascularization and upregulation of Ccl4 and Cxcl9 genes. Both CD8+ and NK1.1+ cells contribute to NLRC5-dependent tumor control. NLRC5 expression in the Tumor Cancer Genome Atlas–skin cutaneous melanoma correlated with NK cell markers and nonclassical MHC-Ib expression. Proteomes of NLRC5-expressing tumor cells showed downmodulation of dominant tumor antigens, but displayed significant changes in the lysosome and the proteasome pathways, upregulated the protein processing in endoplasmic reticulum pathway, and downregulated the autophagy pathway, all of which could impact processing and presentation of subdominant tumor antigens. Conclusion: NLRC5 expression in APCs is dispensable for NLRC5-dependent antitumor immunity. NLRC5-dependent tumor control requires both CD8+ and NK1.1+ cells. Tumor-intrinsic NLRC5 promotes vasculature that is permissive to infiltration by antitumor immune cells. In addition to upregulating MHC-I, NLRC5 may enhance alternative antigen-processing pathways and attenuate autophagic degradation of MHC-I, thereby augmenting tumor immunogenicity.

Background: IL-15 is a promising cytokine for cancer immunotherapy. IL-15 promotes differentiation and homeostasis of innate and adaptive immune cells, and their cytolytic effector functions. The IL-15 receptor is composed of IL-15Rα, IL-15Rβ and the common γ_c chains. IL-15 is also trans-presented as IL-15Rα:IL-15 complex to IL-15Rβ:γ_c on neighboring cells. IL-15Rα is dispensable for early immune response to infections and in autoimmune diabetes. The role of IL-15Rα in antitumor immune responses remains unclear. Methods: In WT, Il15^−/− and Il15ra^−/− mice, we studied the growth of syngeneic tumor cell lines and tumor immune surveillance against endogenous fibrosarcoma induced by methylcholanthrene (MCA). Immune gene signature and total proteome analysis were performed on MCA-induced tumors. Results: Lack of IL-15 or IL-15Rα did not enhance the growth of implanted tumor cell lines, despite reduced immune cell infiltration. MCA-induced tumor incidence was reduced in mice lacking IL-15Rα but not IL-15, although both are required for efficient tumor immunoediting. Il15^−/− and Il15ra^−/− tumors showed reduced Ifng expression but displayed differential modulation of Ifng-responsive genes. Proteome profiles of Il15^−/− tumors, but not tumor-derived cell lines, showed significant reduction in antigen presentation pathways. B16-F10 melanoma cells expressing NLRC5, the IFNγ-induced transcriptional activator of tumor antigen presentation, still required IL-15 but not IL-15Rα for efficient tumor control. Conclusions: Our findings show that IL-15 plays a negligible role in immunosurveillance against spontaneous tumor development, whereas IL-15Rα restrains immunosurveillance. Neither IL-15 nor IL-15Rα have a significant impact on implanted tumor models, although IL-15 facilitates efficient control of highly immunogenic tumors for which IL-15Rα is dispensable.