Immune function is critically dependent on the nutrient microenvironment, of which fatty acids are central. Tools are lacking to quantify fatty acid uptake at the single-cell level, which has hindered understanding how primary immune cells use them in physiological settings. Here we show the synthesis and utilization of novel cyclopropenyl-fatty acids (cpFA) of different saturation and chain length, that can be detected by click chemistry. We validated that CpFA are compatible with downstream omics pipelines and thus could be used to determine phenotypic links to relative uptake. Using cpFA, we found that CD4+ regulatory T cells increase arachidonic acid uptake selectively in the intestine. RORγt+ Tregs possessed superior Cp-arachidonic acid uptake in the mesenteric lymph node, compared to the small intestine, suggesting cell-intrinsic and niche factors control arachidonic acid uptake. CpFA are therefore a key technological advancement in immunometabolism that can reveal FA preferences of immune cells in tissues at the single cell level. ### Competing Interest Statement The authors have declared no competing interest. European Research Council, https://ror.org/0472cxd90, 865175, 770769 Dutch Research Council, https://ror.org/04jsz6e67, iCNS Gravitation

Urbanization affects environmental exposures and lifestyle, shaping immune system variation and influencing disease susceptibility and vaccine responses. Here, we present systems analysis of immune profiles across the rural-urban gradient, comparing rural and urban Senegalese with urban Dutch individuals. By integrating single-cell phenotyping, metabolic profiling, and functional analysis, we reveal a trajectory of immune remodeling along the gradient. This includes enrichment of proinflammatory CD11c+ B cells associated with altered IgG Fc glycosylation, adaptive NK cells with reduced responsiveness to accessory cytokines, and CD161+CD4+T cells with enhanced cytokine production in rural settings. Metabolic perturbation studies demonstrated distinct dependencies on glycolysis, pentose phosphate pathway, and fatty acid synthesis for cellular cytokine responses across populations. We validate core rural-urban immune signatures in an independent Indonesian cohort, suggesting shared immunological adaptations to urbanization across ancestries and geographical areas. Our findings provide insights into rural-urban immune function in understudied populations.

Cytotoxic CD8+ T cells are essential mediators of immune responses against viral infections and tumors. Upon antigen encounter, antigen-specific CD8+ T cells undergo clonal expansion and produce effector cytokines, processes that require dynamic metabolic adaptation. However, profiling antigen-specific T cells at single-cell resolution remains technically challenging. We present a spectral flow cytometry-based workflow enabling metabolic profiling of antigen-specific CD8+ T cells identified via major histocompatibility complex (MHC) class I tetramers or CD137 upregulation. The approach integrates the analysis of metabolic protein expression to infer pathway activity, uptake of fluorescent probes to measure functional metabolism and metabolite utilization, and assays evaluating cellular energy metabolism. Applied to human and mouse samples, this method defined the metabolic profiles of cytomegalovirus-, SARS-CoV-2-, and tumor-specific CD8+ T cells across distinct activation states and tissues. By detailing each component of the workflow, we provide practical guidance for applying metabolic spectral flow cytometry to dissect disease mechanisms and therapeutic responses.

Parasitic helminths are well known master regulators of host immune responses. Yet, the underlying molecular principles remain largely enigmatic. Recent work from Bohnacker et al. reveals that glutamate dehydrogenase (GDH), a metabolic enzyme secreted by Heligmosomoides polygyrus, can suppress type 2 immunity by multimodal regulation of macrophage metabolism and epigenetics.

In the last couple of decades, cancer research has been shifting its focus to the immune system. Cancer cells, with their ability to adapt and evade immune responses, seem to accelerate the evolutionary pressure that has been put on our immune system during evolution. We thus try to aid these natural selection processes and assist our immune system to combat cancer. Here, we are discussing a study by Greg Delgoffe and colleagues that was published in Nature Immunology in December 2024, exploring a new approach to bring the dysfunctional immune cells back to life by blocking their lactate uptake.

IgA can form immune complexes (ICs) and activate myeloid cells via Fc alpha receptor-mediated signalling to secrete pro-inflammatory cytokines. It was previously described that of the two IgA subclasses (IgA1 and IgA2), IgA2 is more inflammatory than IgA1. However, the mechanisms underlying this differential pro-inflammatory potential remain poorly defined. Using anti-citrullinated protein IgA1 and IgA2 antibodies (ACPA) that are commonly found in rheumatoid arthritis (RA) patients and linked to chronic inflammation, we show here that, in macrophages, IgA2-ICs boost TLR-induced TNF and IL6 secretion, COX2 expression, and production of COX2-dependent lipid mediators to a higher level than IgA1-ICs. Metabolically, we found the amplification of TLR-induced cytokine production and COX2 induction by IgA2-ICs to be dependent on mitochondrial ATP synthesis, but not glycolysis. Finally, we found the potentiation of TLR-induced cytokine production by IgA-ICs to be COX2-dependent. Together this work points towards a key role for mitochondrial ATP synthesis in driving COX2 expression and subsequent IgA2-IC-dependent potentiation of TLR-induced cytokine production by macrophages. As such, our work provides new insights into the mechanisms underlying IgA2-induced inflammation in the context of RA. Thus, this may hold novel clues to be explored as therapeutic possibilities to target antibody-driven inflammation in chronic inflammatory diseases.

T cells are the most common immune cells in atherosclerotic plaques, and the function of T cells can be altered by fatty acids. Here, we show that pre -exposure of CD4 + T cells to oleic acid, an abundant fatty acid linked to cardiovascular events, upregulates core metabolic pathways and promotes differentiation into interleukin-9 (IL -9) -producing cells upon activation. RNA sequencing of non -activated T cells reveals that oleic acid upregulates genes encoding key enzymes responsible for cholesterol and fatty acid biosynthesis. Transcription footprint analysis links these expression changes to the differentiation toward T H 9 cells, a pro-atherogenic subset. Spectral flow cytometry shows that pre -exposure to oleic acid results in a skew toward IL -9 + -producing T cells upon activation. Importantly, pharmacological inhibition of either cholesterol or fatty acid biosynthesis abolishes this effect, suggesting a beneficial role for statins beyond cholesterol lowering. Taken together, oleic acid may affect inflammatory diseases like atherosclerosis by rewiring T cell metabolism.

The integration of spatial omics technologies can provide important insights into the biology of tissues. Here we combined mass spectrometry imaging-based metabolomics and imaging mass cytometry-based immunophenotyping on a single tissue section to reveal metabolic heterogeneity at single-cell resolution within tissues and its association with specific cell populations such as cancer cells or immune cells. This approach has the potential to greatly increase our understanding of tissue-level interplay between metabolic processes and their cellular components. The authors present a workflow integrating imaging mass cytometry and imaging mass spectrometry to deconvolute metabolic heterogeneity at the single-cell level.

Chronic infection with Schistosoma mansoni parasites is associated with reduced allergic sensitization in humans, while schistosome eggs protects against allergic airway inflammation (AAI) in mice. One of the main secretory/excretory molecules from schistosome eggs is the glycosylated T2-RNAse Omega-1 (ω1). We hypothesized that ω1 induces protection against AAI during infection. Peritoneal administration of ω1 prior to sensitization with Ovalbumin (OVA) reduced airway eosinophilia and pathology, and OVA-specific Th2 responses upon challenge, independent from changes in regulatory T cells. ω1 was taken up by monocyte-derived dendritic cells, mannose receptor (CD206)-positive conventional type 2 dendritic cells (CD206+ cDC2), and by recruited peritoneal macrophages. Additionally, ω1 impaired CCR7, F-actin, and costimulatory molecule expression on myeloid cells and cDC2 migration in and ex vivo, as evidenced by reduced OVA+ CD206+ cDC2 in the draining mediastinal lymph nodes (medLn) and retainment in the peritoneal cavity, while antigen processing and presentation in cDC2 were not affected by ω1 treatment. Importantly, RNAse mutant ω1 was unable to reduce AAI or affect DC migration, indicating that ω1 effects are dependent on its RNAse activity. Altogether, ω1 hampers migration of OVA+ cDC2 to the draining medLn in mice, elucidating how ω1 prevents allergic airway inflammation in the OVA/alum mouse model.

Cytomegalovirus (CMV) is one of the most common and relevant opportunistic pathogens in people who are immunocompromised, such as kidney transplant recipients (KTRs). The exact mechanisms underlying the disability of cytotoxic T cells to provide sufficient protection against CMV in people who are immunosuppressed have not been identified yet. Here, we performed in-depth metabolic profiling of CMV-specific CD8+ T cells in patients who are immunocompromised and show the development of metabolic dysregulation at the transcriptional, protein, and functional level of CMV-specific CD8+ T cells in KTRs with noncontrolled CMV infection. These dysregulations comprise impaired glycolysis and increased mitochondrial stress, which is associated with an intensified expression of the nicotinamide adenine dinucleotide nucleotidase (NADase) CD38. Inhibiting NADase activity of CD38 reinvigorated the metabolism and improved cytokine production of CMV-specific CD8+ T cells. These findings were corroborated in a mouse model of CMV infection under conditions of immunosuppression. Thus, dysregulated metabolic states of CD8+ T cells could be targeted by inhibiting CD38 to reverse hyporesponsiveness in individuals who fail to control chronic viral infection.

Summary T cells are the most common immune cells in atherosclerotic plaques and the function of T cells can be altered by fatty acids. Here, we show that pre-exposure of CD4 + T cells to oleic acid, an abundant fatty acid linked to cardiovascular events, results in a preferential differentiation into pro-inflammatory subsets upon activation by upregulating core metabolic pathways. RNA-sequencing of non-activated CD4 + T cells revealed that oleic acid upregulates genes encoding enzymes responsible for cholesterol and fatty acid biosynthesis. Transcription footprint analysis linked this rewiring to the differentiation of pro-inflammatory subsets. Indeed, spectral flow cytometry showed that pre-exposure to oleic acid results in a skew toward IL-9, IL-17A, IL-5 and IL-13 producing T cells upon activation. Importantly, inhibition of either cholesterol or fatty acid biosynthesis abolishes this effect, suggesting a beneficial role for statins beyond cholesterol lowering. Taken together, fatty acids may affect inflammatory diseases by influencing T cell metabolism.

Dendritic cell (DC) activation and function are underpinned by profound changes in cellular metabolism. Several studies indicate that the ability of DCs to promote tolerance is dependent on catabolic metabolism. Yet the contribution of AMP-activated kinase (AMPK), a central energy sensor promoting catabolism, to DC tolerogenicity remains unknown. Here, we show that AMPK activation renders human monocyte-derived DCs tolerogenic as evidenced by an enhanced ability to drive differentiation of regulatory T cells, a process dependent on increased RALDH activity. This is accompanied by several metabolic changes, including increased breakdown of glycerophospholipids, enhanced mitochondrial fission-dependent fatty acid oxidation, and upregulated glucose catabolism. This metabolic rewiring is functionally important as we found interference with these metabolic processes to reduce to various degrees AMPK-induced RALDH activity as well as the tolerogenic capacity of moDCs. Altogether, our findings reveal a key role for AMPK signaling in shaping DC tolerogenicity and suggest AMPK as a target to direct DC-driven tolerogenic responses in therapeutic settings.

The parasitic helminth Schistosoma mansoni is a potent inducer of type 2 immune responses by stimulating dendritic cells (DCs) to prime T helper 2 (Th2) responses. We previously found that S. mansoni soluble egg antigens (SEA) promote the synthesis of Prostaglandin E2 (PGE2) by DCs through ERK-dependent signaling via Dectin-1 and Dectin-2 that subsequently induces OX40L expression, licensing them for Th2 priming, yet the ligands present in SEA involved in driving this response and whether specific targeting of PGE2 synthesis by DCs could affect Th2 polarization are unknown. We here show that the ability of SEA to bind Dectin-2 and drive ERK phosphorylation, PGE2 synthesis, OX40L expression, and Th2 polarization is impaired upon cleavage of high-mannose glycans by Endoglycosidase H treatment. This identifies high-mannose glycans present on glycoproteins in SEA as important drivers of this signaling axis. Moreover, we find that OX40L expression and Th2 induction are abrogated when microsomal prostaglandin E synthase-1 (mPGES) is selectively inhibited, but not when a general COX-1/2 inhibitor is used. This shows that the de novo synthesis of PGE2 is vital for the Th2 priming function of SEA-stimulated DCs as well as points to the potential existence of other COX-dependent lipid mediators that antagonize PGE2-driven Th2 polarization. Lastly, specific PGE2 inhibition following immunization with S. mansoni eggs dampened the egg-specific Th cell response. In summary, our findings provide new insights in the molecular mechanisms underpinning Th2 induction by S. mansoni and identify druggable targets for potential control of helminth driven-Th2 responses.

Cellular metabolism is a key determinant of immune cell function. Here we found that CD14 + monocytes from Sub-Saharan Africans produce higher levels of IL-10 following TLR-4 stimulation and are bioenergetically distinct from monocytes from Europeans. Through metabolomic profiling, we identified the higher IL-10 production to be driven by increased baseline production of NADPH oxidase-dependent reactive oxygen species, supported by enhanced pentose phosphate pathway activity. Together, these data indicate that NADPH oxidase-derived ROS is a metabolic checkpoint in monocytes that governs their inflammatory profile and uncovers a metabolic basis for immunological differences across geographically distinct populations.

Dendritic cells (DCs) play a crucial role in promoting tolerance through priming of regulatory T cells (Treg). Several studies indicate DC tolerogenicity is dependent on catabolic metabolism. However, the role of AMP-activated Kinase (AMPK), a key energy and nutrient sensor driving catabolic metabolism, in this process is unclear. We found that human retinoic acid-induced tolerogenic CD103 + DCs (RA-DCs) display increased AMPK signaling. Interestingly, RA-DCs, but not vitamin-D3- or dexamethasone-induced tolerogenic DCs, required AMPK for Treg induction. Mechanistically, AMPK underpinned RA-driven tolerogenicity by promoting RALDH activity in a FoxO3-dependent manner. Correspondingly, mice deficient for AMPK in DCs (CD11c ΔAMPKα1 ) harbored reduced frequencies of intestinal CD103 + CD11b + DCs with impaired RALDH activity. Importantly, upon infection with parasitic worm Schistosoma mansoni , that elicits strong Th2 and Treg responses, CD11c ΔAMPKα1 mice showed a defect in Treg accumulation and concomitantly, displayed an impaired ability to control Type 2 immunity-driven granulomatous inflammation against the parasite eggs. Together, our findings identify AMPK as a key regulator of tolerance by CD103 + DCs. Summary Dendritic cells (DCs) are critical for inducing tolerance. However, how metabolic cues control their tolerogenicity is still poorly understood. Patente et al demonstrate that AMPK is crucial for Treg induction by retinoic acid-primed tolerogenic CD103 + DCs.

Vaccination is one of medicine’s greatest achievements; however, its full potential is hampered by considerable variation in efficacy across populations and geographical regions. For example, attenuated malaria vaccines in high-income countries confer almost 100% protection, whereas in low-income regions these same vaccines achieve only 20–50% protection. This trend is also observed for other vaccines, such as bacillus Calmette–Guérin (BCG), rotavirus and yellow fever vaccines, in terms of either immunogenicity or efficacy. Multiple environmental factors affect vaccine responses, including pathogen exposure, microbiota composition and dietary nutrients. However, there has been variable success with interventions that target these individual factors, highlighting the need for a better understanding of their downstream immunological mechanisms to develop new ways of modulating vaccine responses. Here, we review the immunological factors that underlie geographical variation in vaccine responses. Through the identification of causal pathways that link environmental influences to vaccine responsiveness, it might become possible to devise modulatory compounds that can complement vaccines for better outcomes in regions where they are needed most.

Obesity-associated metabolic inflammation drives the development of insulin resistance and type 2 diabetes, notably through modulating innate and adaptive immune cells in metabolic organs. The nutrient sensor liver kinase B1 (LKB1) has recently been shown to control cellular metabolism and T cell priming functions of DCs. Here, we report that hepatic DCs from high-fat diet–fed (HFD-fed) obese mice display increased LKB1 phosphorylation and that LKB1 deficiency in DCs (CD11cΔLKB1) worsened HFD-driven hepatic steatosis and impaired glucose homeostasis. Loss of LKB1 in DCs was associated with increased expression of Th17-polarizing cytokines and accumulation of hepatic IL-17A+ Th cells in HFD-fed mice. Importantly, IL-17A neutralization rescued metabolic perturbations in HFD-fed CD11cΔLKB1 mice. Mechanistically, deficiency of the canonical LKB1 target AMPK in HFD-fed CD11cΔAMPKα1 mice recapitulated neither the hepatic Th17 phenotype nor the disrupted metabolic homeostasis, suggesting the involvement of other and/or additional LKB1 downstream effectors. We indeed provide evidence that the control of Th17 responses by DCs via LKB1 is actually dependent on both AMPKα1 and salt-inducible kinase signaling. Altogether, our data reveal a key role for LKB1 signaling in DCs in protection against obesity-induced metabolic dysfunctions by limiting hepatic Th17 responses.

Supplementary Methods, figures and tables

Cells pre-treated for 3h with 20 µM nelfinavir.

Type 2 immune responses characterise both helminth infections and atopic disease such as allergy or asthma, but a better understanding of the mechanisms that regulate these responses is key to improving therapeutic and vaccination strategies. Immuno-metabolic studies over the last two decades have suggested T cell activation broadly requires rapid increases in glycolysis and oxidative phosphorylation. In contrast, we show that CD4 + T helper 2 (Th2) cells activated in vivo , using models of helminth infection, do not acquire a glycolytic metabolism. Instead, we show that Th2 cells solely increase their oxidative metabolism, associated with increased fatty acid uptake. Rather than contributing directly to effector function, our data reveal that Th2 cells switch to fatty acid oxidation downstream of PD-1 signalling to promote expression of the IL-33 receptor (ST2). These data provide insight into the spatial regulation of T cell metabolism, and suggest that PD-1 blockade therapies may be effective in Th2 disorders.