Joe N. Frost

TOX is a nuclear factor critical for thymic development of CD4⁺ thymocytes, natural killer and innate lymphoid cells. In post-thymic antigen-specific CD8⁺ T cells, TOX is highly expressed in settings of chronic antigen encounter such as cancer and chronic infection and required for the persistence of exhausted CD8⁺ T cells. The role of TOX in CD4⁺ T cells is less clear. Here, we show that TOX is critical for CD4⁺ type 1 helper T (T_H1) cell differentiation. Gain-of-function and loss-of-function studies show that TOX induces T_H1 cell-associated molecular programs that drive T_H1 cell-like phenotypes and interferon-γ production. TOX expression in CD4⁺ T cells from individuals with cancer was associated with increased cytotoxicity, antitumor immunity and improved responses to immunotherapy, as well as pathogenic responses in autoimmune and inflammatory diseases in mice and humans. Thus, TOX has opposing functions in CD4⁺ versus CD8⁺ T cells: while TOX is associated with CD8⁺ T cell exhaustion and generally with poor responsiveness to immunotherapy, in CD4⁺ T cells TOX drives T_H1 cell fate commitment and is associated with antitumor immunity and pathogenic autoimmune responses.

Iron is an irreplaceable co-factor for metabolism. Iron deficiency affects >1 billion people and decreased iron availability impairs immunity. Nevertheless, how iron deprivation impacts immune cell function remains poorly characterised. We interrogate how physiologically low iron availability affects CD8⁺ T cell metabolism and function, using multi-omic and metabolic labelling approaches. Iron limitation does not substantially alter initial post-activation increases in cell size and CD25 upregulation. However, low iron profoundly stalls proliferation (without influencing cell viability), alters histone methylation status, gene expression, and disrupts mitochondrial membrane potential. Glucose and glutamine metabolism in the TCA cycle is limited and partially reverses to a reductive trajectory. Previous studies identified mitochondria-derived aspartate as crucial for proliferation of transformed cells. Despite aberrant TCA cycling, aspartate is increased in stalled iron deficient CD8⁺ T cells but is not utilised for nucleotide synthesis, likely due to trapping within depolarised mitochondria. Exogenous aspartate markedly rescues expansion and some functions of severely iron-deficient CD8⁺ T cells. Overall, iron scarcity creates a mitochondrial-located metabolic bottleneck, which is bypassed by supplying inhibited biochemical processes with aspartate. These findings reveal molecular consequences of iron deficiency for CD8⁺ T cell function, providing mechanistic insight into the basis for immune impairment during iron deficiency.

Iron is a cofactor for hundreds of enzymes and biochemical processes that support cellular metabolism across the kingdoms of life. Because of this, the host and pathogen compete for iron as a vital resource. Moreover, research has shown that iron acquisition and iron trafficking have substantial effects on the immune system. This is especially important because iron-related disorders — both deficiency and overload — are common worldwide. In this Review, we describe how immune cells acquire and use iron, which branches of the immune system are most affected by iron and how changes in iron availability can affect infectious diseases, autoinflammatory disorders and antitumour immunity. We also discuss key unanswered questions and potential therapeutic opportunities to manipulate immunity by controlling iron trafficking.

Red blood cell development from erythroid progenitors requires profound reshaping of metabolism and gene expression. How these transcriptional and metabolic alterations are coupled is unclear. Nprl3 (an inhibitor of mTORC1) has remained in synteny with the α-globin genes for >500 million years, and harbours most of the a-globin enhancers. However, whether Nprl3 serves an erythroid role is unknown. We found that while haematopoietic progenitors require basal Nprl3 expression, erythroid Nprl3 expression is further boosted by the α-globin enhancers. This lineage-specific upregulation is required for sufficient erythropoiesis. Loss of Nprl3 affects erythroblast metabolism via elevating mTORC1 signalling, suppressing autophagy and disrupting glycolysis. Broadly consistent with these murine findings, human NPRL3-knockout erythroid progenitors produce fewer enucleated cells and demonstrate dysregulated mTORC1 signalling in response to nutrient availability and erythropoietin. Therefore, we propose that the anciently conserved linkage of NprI3, α-globin and their associated enhancers has coupled metabolic and developmental control of erythropoiesis.

SUMMARY: Regulatory T (Treg) cells contribute to solid organ cancer progression, except in colorectal cancer (CRC) despite being abundantly present. Here, we demonstrate that two distinct tumoral IL-10⁺ and IL-10⁻ Treg cell subsets exert opposing functions by counteracting and promoting CRC tumor growth, respectively. The tumor restraining activity of IL-10⁺ Treg cells was mediated by their suppression of effector CD4 T cell production of IL-17, which directly stimulates CRC tumor cell proliferation. Consistently, IL-10⁻ Treg cells were more abundant in both mouse and human CRC tumors than in tumor-adjacent normal tissues, whereas IL-10 ⁺ Treg cells exhibited the opposite distribution. Furthermore, relative abundance of IL-10⁺ and IL-10⁻ Treg cells correlated with better and worse disease prognoses in human CRC, respectively. This functional dichotomy between Treg cell subsets provides a rationale for therapeutic strategies to selectively target pro-tumoral Treg cells while preserving their anti-tumoral counterparts across barrier tissue cancers that harbor both subsets.

Natural killer (NK) cells are innate cytotoxic lymphocytes with adaptive immune features, including antigen specificity, clonal expansion and memory. As such, NK cells share many transcriptional and epigenetic programs with their adaptive CD8⁺ T cell siblings. Various signals ranging from antigen, co-stimulation and proinflammatory cytokines are required for optimal NK cell responses in mice and humans during virus infection; however, the integration of these signals remains unclear. In this study, we identified that the transcription factor IRF4 integrates signals to coordinate the NK cell response during mouse cytomegalovirus infection. Loss of IRF4 was detrimental to the expansion and differentiation of virus-specific NK cells. This defect was partially attributed to the inability of IRF4-deficient NK cells to uptake nutrients required for survival and memory generation. Altogether, these data suggest that IRF4 is a signal integrator that acts as a secondary metabolic checkpoint to orchestrate the adaptive response of NK cells during viral infection.

Malaria and iron deficiency are major global health problems with extensive epidemiological overlap. Iron deficiency-induced anaemia can protect the host from malaria by limiting parasite growth. On the other hand, iron deficiency can significantly disrupt immune cell function. However, the impact of host cell iron scarcity beyond anaemia remains elusive in malaria. To address this, we employed a transgenic mouse model carrying a mutation in the transferrin receptor ( Tfrc ^Y20H/Y20H ), which limits the ability of cells to internalise iron from plasma. At homeostasis Tfrc ^Y20H/Y20H mice appear healthy and are not anaemic. However, Tfrc ^Y20H/Y20H mice infected with Plasmodium chabaudi chabaudi AS showed significantly higher peak parasitaemia and body weight loss. We found that Tfrc ^Y20H/Y20H mice displayed a similar trajectory of malaria-induced anaemia as wild-type mice, and elevated circulating iron did not increase peak parasitaemia. Instead, P. chabaudi infected Tfrc ^Y20H/Y20H mice had an impaired innate and adaptive immune response, marked by decreased cell proliferation and cytokine production. Moreover, we demonstrated that these immune cell impairments were cell-intrinsic, as ex vivo iron supplementation fully recovered CD4 T cell and B cell function. Despite the inhibited immune response and increased parasitaemia, Tfrc ^Y20H/Y20H mice displayed mitigated liver damage, characterised by decreased parasite sequestration in the liver and an attenuated hepatic immune response. Together, these results show that host cell iron scarcity inhibits the immune response but prevents excessive hepatic tissue damage during malaria infection. These divergent effects shed light on the role of iron in the complex balance between protection and pathology in malaria.

Low plasma iron (hypoferremia) induced by hepcidin is a conserved inflammatory response that protects against infections but inhibits erythropoiesis. How hypoferremia influences leukocytogenesis is unclear. Using proteomic data, we predicted that neutrophil production would be profoundly more iron-demanding than generation of other white blood cell types. Accordingly in mice, hepcidin-mediated hypoferremia substantially reduced numbers of granulocytes but not monocytes, lymphocytes, or dendritic cells. Neutrophil rebound after anti-Gr-1–induced neutropenia was blunted during hypoferremia but was rescued by supplemental iron. Similarly, hypoferremia markedly inhibited pharmacologically stimulated granulopoiesis mediated by granulocyte colony-stimulating factor and inflammation-induced accumulation of neutrophils in the spleen and peritoneal cavity. Furthermore, hypoferremia specifically altered neutrophil effector functions, suppressing antibacterial mechanisms but enhancing mitochondrial reactive oxygen species–dependent NETosis associated with chronic inflammation. Notably, antagonizing endogenous hepcidin during acute inflammation enhanced production of neutrophils. We propose plasma iron modulates the profile of innate immunity by controlling monocyte-to-neutrophil ratio and neutrophil activity in a therapeutically targetable system.

Summary: Vaccination programmes are critically important to suppress the burden of infectious diseases, saving countless lives globally, as emphasised by the current COVID-19 pandemic. Effective adaptive immune responses are complex processes subject to multiple influences. Recent genetic, pre-clinical, and clinical studies have converged to show that availability of iron is a key factor regulating the development of T and B cell responses to infection and immunisation. Lymphocytes obtain iron from circulating transferrin. The amount of iron bound to transferrin is dependent on dietary iron availability and is decreased during inflammation via upregulation of the iron-regulatory hormone, hepcidin. As iron deficiency and chronic inflammatory states are both globally prevalent health problems, the potential impact of low iron availability on immune responses is significant. We describe the evidence supporting the importance of iron in immunity, highlight important unknowns, and discuss how therapeutic interventions to modulate iron availability might be implementable in the context of vaccination and infectious disease.

Cyclic GMP-AMP (cGAMP) is an immunostimulatory second messenger produced by cGAS that activates STING. Soluble cGAMP acts as an adjuvant when administered with antigens. cGAMP is also incorporated into enveloped virus particles during budding. We hypothesised that inclusion of the adjuvant cGAMP within viral vaccine vectors would promote adaptive immunity against vector antigens. We immunised mice with virus-like particles (VLPs) containing the HIV-1 Gag protein and VSV-G. Inclusion of cGAMP within these VLPs augmented splenic VLP-specific CD4 and CD8 T cell responses. It also increased VLP- and VSV-G-specific serum antibody titres and enhancedin vitrovirus neutralisation. The superior antibody response was accompanied by increased numbers of T follicular helper cells in draining lymph nodes. Vaccination with cGAMP-loaded VLPs containing haemagglutinin induced high titres of influenza A virus neutralising antibodies and conferred protection following subsequent influenza A virus challenge. Together, these results show that incorporating cGAMP into VLPs enhances their immunogenicity, making cGAMP-VLPs an attractive platform for novel vaccination strategies.Short summary: cGAMP is an innate immune signalling molecule that can be transmitted between cells by inclusion in enveloped virions. This study demonstrates enhanced immunogenicity of HIV-derived virus-like particles containing cGAMP. Viral vectors loaded with cGAMP may thus be potent vaccines.