Dysregulation of ILC3s unleashes progression and immunotherapy resistance in colon cancer.

Group 3 innate lymphoid cells (ILC3s) regulate immunity and inflammation, yet their role in cancer remains elusive. Here, we identify that colorectal cancer (CRC) manifests with altered ILC3s that are characterized by reduced frequencies, increased plasticity, and an imbalance with T cells. We evaluated the consequences of these changes in mice and determined that a dialog between ILC3s and T cells via major histocompatibility complex class II (MHCII) is necessary to support colonization with microbiota that subsequently induce type-1 immunity in the intestine and tumor microenvironment. As a result, mice lacking ILC3-specific MHCII develop invasive CRC and resistance to anti-PD-1 immunotherapy. Finally, humans with dysregulated intestinal ILC3s harbor microbiota that fail to induce type-1 immunity and immunotherapy responsiveness when transferred to mice. Collectively, these data define a protective role for ILC3s in cancer and indicate that their inherent disruption in CRC drives dysfunctional adaptive immunity, tumor progression, and immunotherapy resistance.

Manganese Increases the Sensitivity of the cGAS-STING Pathway for Double-Stranded DNA and Is Required for the Host Defense against DNA Viruses.

Manganese (Mn) is essential for many physiological processes, but its functions in innate immunity remain undefined. Here, we found that Mn2+ was required for the host defense against DNA viruses by increasing the sensitivity of the DNA sensor cGAS and its downstream adaptor protein STING. Mn2+ was released from membrane-enclosed organelles upon viral infection and accumulated in the cytosol where it bound directly to cGAS. Mn2+ enhanced the sensitivity of cGAS to double-stranded DNA (dsDNA) and its enzymatic activity, enabling cGAS to produce secondary messenger cGAMP in the presence of low concentrations of dsDNA that would otherwise be non-stimulatory. Mn2+ also enhanced STING activity by augmenting cGAMP-STING binding affinity. Mn-deficient mice showed diminished cytokine production and were more vulnerable to DNA viruses, and Mn-deficient STING-deficient mice showed no increased susceptibility. These findings indicate that Mn is critically involved and required for the host defense against DNA viruses.

Apoptotic Caspases Suppress Type I Interferon Production via the Cleavage of cGAS, MAVS, and IRF3.

Viral infection triggers host defenses through pattern-recognition receptor-mediated cytokine production, inflammasome activation, and apoptosis of the infected cells. Inflammasome-activated caspases are known to cleave cyclic GMP-AMP synthase (cGAS). Here, we found that apoptotic caspases are critically involved in regulating both DNA and RNA virus-triggered host defenses, in which activated caspase-3 cleaved cGAS, MAVS, and IRF3 to prevent cytokine overproduction. Caspase-3 was exclusively required in human cells, whereas caspase-7 was involved only in murine cells to inactivate cGAS, reflecting distinct regulatory mechanisms in different species. Caspase-mediated cGAS cleavage was enhanced in the presence of dsDNA. Alternative MAVS cleavage sites were used to ensure the inactivation of this critical protein. Elevated type I IFNs were detected in caspase-3-deficient cells without any infection. Casp3-/- mice consistently showed increased resistance to viral infection and experimental autoimmune encephalomyelitis. Our results demonstrate that apoptotic caspases control innate immunity and maintain immune homeostasis against viral infection.

Inflammasome Activation Triggers Caspase-1-Mediated Cleavage of cGAS to Regulate Responses to DNA Virus Infection.

Viral infection triggers host innate immune responses that result in the production of various cytokines including type I interferons (IFN), activation of inflammasomes, and programmed cell death of the infected cells. Tight control of inflammatory cytokine production is crucial for the triggering of an effective immune response that can resolve the infection without causing host pathology. In examining the inflammatory response of Asc-/- and Casp1-/- macrophages, we found that deficiency in these molecules resulted in increased IFN production upon DNA virus infection, but not RNA virus challenge. Investigation of the underlying mechanism revealed that upon canonical and non-canonical inflammasome activation, caspase-1 interacted with cyclic GMP-AMP (cGAMP) synthase (cGAS), cleaving it and dampening cGAS-STING-mediated IFN production. Deficiency in inflammasome signaling enhanced host resistance to DNA virus in vitro and in vivo, and this regulatory role extended to other inflammatory caspases. Thus, inflammasome activation dampens cGAS-dependent signaling, suggesting cross-regulation between intracellular DNA-sensing pathways.

NEMO-IKKß Are Essential for IRF3 and NF-κB Activation in the cGAS-STING Pathway.

Cytosolic dsDNA activates the cyclic GMP-AMP synthase (cGAS)-stimulator of IFN genes (STING) pathway to produce cytokines, including type I IFNs. The roles of many critical proteins, including NEMO, IKKß, and TBK1, in this pathway are unclear because of the lack of an appropriate system to study. In this article, we report that lower FBS concentrations in culture medium conferred high sensitivities to dsDNA in otherwise unresponsive cells, whereas higher FBS levels abrogated this sensitivity. Based on this finding, we demonstrated genetically that NEMO was critically involved in the cGAS-STING pathway. Cytosolic DNA activated TRIM32 and TRIM56 to synthesize ubiquitin chains that bound NEMO and subsequently activated IKKß. Activated IKKß, but not IKKα, was required for TBK1 and NF-κB activation. In contrast, TBK1 was reciprocally required for NF-κB activation, probably by directly phosphorylating IKKß. Thus, our findings identified a unique innate immune activation cascade in which TBK1-IKKß formed a positive feedback loop to assure robust cytokine production during cGAS-STING activation.

Manganese salts function as potent adjuvants.

Aluminum-containing adjuvants have been used for nearly 100 years to enhance immune responses in billions of doses of vaccines. To date, only a few adjuvants have been approved for use in humans, among which aluminum-containing adjuvants are the only ones widely used. However, the medical need for potent and safe adjuvants is currently continuously increasing, especially those triggering cellular immune responses for cytotoxic T lymphocyte activation, which are urgently needed for the development of efficient virus and cancer vaccines. Manganese is an essential micronutrient required for diverse biological activities, but its functions in immunity remain undefined. We previously reported that Mn2+ is important in the host defense against cytosolic dsDNA by facilitating cGAS-STING activation and that Mn2+ alone directly activates cGAS independent of dsDNA, leading to an unconventional catalytic synthesis of 2'3'-cGAMP. Herein, we found that Mn2+ strongly promoted immune responses by facilitating antigen uptake, presentation, and germinal center formation via both cGAS-STING and NLRP3 activation. Accordingly, a colloidal manganese salt (Mn jelly, MnJ) was formulated to act not only as an immune potentiator but also as a delivery system to stimulate humoral and cellular immune responses, inducing antibody production and CD4+/CD8+ T-cell proliferation and activation by either intramuscular or intranasal immunization. When administered intranasally, MnJ also worked as a mucosal adjuvant, inducing high levels of secretory IgA. MnJ showed good adjuvant effects for all tested antigens, including T cell-dependent and T cell-independent antigens, such as bacterial capsular polysaccharides, thus indicating that it is a promising adjuvant candidate.

Metalloimmunology: The metal ion-controlled immunity.

Metals are essential components in all forms of life required for the function of nearly half of all enzymes and are critically involved in virtually all fundamental biological processes. Especially, the transition metals iron (Fe), zinc (Zn), manganese (Mn), nickel (Ni), copper (Cu) and cobalt (Co) are crucial micronutrients known to play vital roles in metabolism as well due to their unique redox properties. Metals carry out three major functions within metalloproteins: to provide structural support, to serve as enzymatic cofactors, and to mediate electron transportation. Metal ions are also involved in the immune system from metal allergies to nutritional immunity. Within the past decade, much attention has been drawn to the roles of metal ions in the immune system, since increasing evidence has mounted to suggest that metals are critically implicated in regulating both the innate immune sensing of and the host defense against invading pathogens. The importance of ions in immunity is also evidenced by the identification of various immunodeficiencies in patients with mutations in ion channels and transporters. In addition, cancer immunotherapy has recently been conclusively demonstrated to be effective and important for future tumor treatment, although only a small percentage of cancer patients respond to immunotherapy because of inadequate immune activation. Importantly, metal ion-activated immunotherapy is becoming an effective and potential way in tumor therapy for better clinical application. Nevertheless, we are still in a primary stage of discovering the diverse immunological functions of ions and mechanistically understanding the roles of these ions in immune regulation. This review summarizes recent advances in the understanding of metal-controlled immunity. Particular emphasis is put on the mechanisms of innate immune stimulation and T cell activation by the essential metal ions like calcium (Ca2+), zinc (Zn2+), manganese (Mn2+), iron (Fe2+/Fe3+), and potassium (K+), followed by a few unessential metals, in order to draw a general diagram of metalloimmunology.

Manganese is critical for antitumor immune responses via cGAS-STING and improves the efficacy of clinical immunotherapy.

CD8+ T cell-mediated cancer clearance is often suppressed by the interaction between inhibitory molecules like PD-1 and PD-L1, an interaction acts like brakes to prevent T cell overreaction under normal conditions but is exploited by tumor cells to escape the immune surveillance. Immune checkpoint inhibitors have revolutionized cancer therapeutics by removing such brakes. Unfortunately, only a minority of cancer patients respond to immunotherapies presumably due to inadequate immunity. Antitumor immunity depends on the activation of the cGAS-STING pathway, as STING-deficient mice fail to stimulate tumor-infiltrating dendritic cells (DCs) to activate CD8+ T cells. STING agonists also enhance natural killer (NK) cells to mediate the clearance of CD8+ T cell-resistant tumors. Therefore STING agonists have been intensively sought after. We previously discovered that manganese (Mn) is indispensable for the host defense against cytosolic dsDNA by activating cGAS-STING. Here we report that Mn is also essential in innate immune sensing of tumors and enhances adaptive immune responses against tumors. Mn-insufficient mice had significantly enhanced tumor growth and metastasis, with greatly reduced tumor-infiltrating CD8+ T cells. Mechanically, Mn2+ promoted DC and macrophage maturation and tumor-specific antigen presentation, augmented CD8+ T cell differentiation, activation and NK cell activation, and increased memory CD8+ T cells. Combining Mn2+ with immune checkpoint inhibition synergistically boosted antitumor efficacies and reduced the anti-PD-1 antibody dosage required in mice. Importantly, a completed phase 1 clinical trial with the combined regimen of Mn2+ and anti-PD-1 antibody showed promising efficacy, exhibiting type I IFN induction, manageable safety and revived responses to immunotherapy in most patients with advanced metastatic solid tumors. We propose that this combination strategy warrants further clinical translation.