The immune checkpoint NKG2A/CD94 is a promising target for cancer immunotherapy, and its ligand major histocompatibility complex E (MHC-E) is frequently upregulated in cancer. NKG2A/CD94-mediated... Show moreThe immune checkpoint NKG2A/CD94 is a promising target for cancer immunotherapy, and its ligand major histocompatibility complex E (MHC-E) is frequently upregulated in cancer. NKG2A/CD94-mediated inhibition of lymphocytes depends on the presence of specific leader peptides in MHC-E, but when and where they are presented in situ is unknown. We apply a nanobody specific for the Qdm/Qa-1b complex, the NKG2A/CD94 ligand in mouse, and find that presentation of Qdm peptide depends on every member of the endoplasmic reticulum-resident peptide loading complex. With a turnover rate of 30 min, the Qdm peptide reflects antigen processing capacity in real time. Remarkably, Qdm/Qa-1b complexes require inflammatory signals for sur-face expression in situ, despite the broad presence of Qa-1b molecules in homeostasis. Furthermore, we identify LILRB1 as a functional inhibition receptor for MHC-E in steady state. These data provide a molecular understanding of NKG2A blockade in immunotherapy and assign MHC-E as a convergent ligand for multiple immune checkpoints. Show less
Sereti, I.; Verburgh, M.L.; Gifford, J.; A. lo; Boyd, A.; Verheij, E.; ... ; Vujkovic-Cvijin, I. 2023
Antiretroviral therapy (ART) has dramatically lengthened lifespan among people with HIV (PWH), but this population experiences heightened rates of inflammation-related comorbidities. HIV-associated... Show moreAntiretroviral therapy (ART) has dramatically lengthened lifespan among people with HIV (PWH), but this population experiences heightened rates of inflammation-related comorbidities. HIV-associated inflammation is linked with an altered microbiome; whether such alterations precede inflammation-related comorbidities or occur as their consequence remains unknown. We find that ART-treated PWH exhibit depletion of gut -resident bacteria that produce short-chain fatty acids (SCFAs)-crucial microbial metabolites with anti-inflammatory properties. Prior reports establish that fecal SCFA concentrations are not depleted in PWH. We find that gut-microbiota-mediated SCFA production capacity is better reflected in serum than in feces and that PWH exhibit reduced serum SCFA, which associates with inflammatory markers. Leveraging stool and serum samples collected prior to comorbidity onset, we find that HIV-specific microbiome alterations precede morbidity and mortality in ART-treated PWH. Among these microbiome alterations, reduced microbiome-mediated conversion of lactate to propionate precedes mortality in PWH. Thus, gut microbial fiber/lactate conversion to SCFAs may modulate HIV-associated comorbidity risk. Show less
The transcription factor DUX4 regulates a portion of the zygotic gene activation (ZGA) program in the early embryo. Many cancers express DUX4 but it is unknown whether this generates cells similar... Show moreThe transcription factor DUX4 regulates a portion of the zygotic gene activation (ZGA) program in the early embryo. Many cancers express DUX4 but it is unknown whether this generates cells similar to early embryonic stem cells. Here we identified cancer cell lines that express DUX4 and showed that DUX4 is transiently expressed in a small subset of the cells. DUX4 expression activates the DUX4-regulated ZGA transcriptional program, the subsequent 8C-like program, and markers of early embryonic lineages, while suppressing steady-state and interferon-induced MHC class I expression. Although DUX4 was expressed in a small number of cells under standard culture conditions, DNA damage or changes in growth conditions increased the fraction of cells expressing DUX4 and its downstream programs. Our demonstration that transient expression of endogenous DUX4 in cancer cells induces a metastable early embryonic stem cell program and sup-presses antigen presentation has implications for cancer growth, progression, and immune evasion. Show less
System-level analysis of single-cell data is rapidly transforming the field of immunometabolism. Given the competitive demand for nutrients in immune microenvironments, there is a need to... Show moreSystem-level analysis of single-cell data is rapidly transforming the field of immunometabolism. Given the competitive demand for nutrients in immune microenvironments, there is a need to understand how and when immune cells access these nutrients. Here, we describe a new approach for single-cell analysis of nutrient uptake where we use in-cell biorthogonal labeling of a functionalized amino acid after transport into the cell. In this manner, the bona fide active uptake of glutamine via SLC1A5/ASCT2 could be quantified. We used this assay to interrogate the transport capacity of complex immune subpopulations, both in vitro and in vivo. Taken together, our findings provide an easy sensitive single-cell assay to assess which cells support their function via SLC1A5-mediated uptake. This is a significant addition to the single-cell metabolic toolbox required to decode the metabolic landscape of complex immune microenvironments. Show less
2-Hydroxyglutarate (2HG) is a byproduct of the tricarboxylic acid (TCA) cycle and is readily detected in the tissues of healthy individuals. 2HG is found in two enantiomeric forms: S-2HG and R-2HG... Show more2-Hydroxyglutarate (2HG) is a byproduct of the tricarboxylic acid (TCA) cycle and is readily detected in the tissues of healthy individuals. 2HG is found in two enantiomeric forms: S-2HG and R-2HG. Here, we investigate the differential roles of these two enantiomers in cluster of differentiation (CD)8+ T cell biology, where we find they have highly divergent effects on proliferation, differentiation, and T cell function. We show here an analysis of structural determinants that likely underlie these differential effects on specific α-ketoglutarate (αKG)-dependent enzymes. Treatment of CD8+ T cells with exogenous S-2HG, but not R-2HG, increased CD8+ T cell fitness in vivo and enhanced anti-tumor activity. These data show that S-2HG and R-2HG should be considered as two distinct and important actors in the regulation of T cell function. Show less
Inner ear disorders are among the most common congenital abnormalities; however, current tissue culture models lack the cell type diversity to study these disorders and normal otic development.... Show moreInner ear disorders are among the most common congenital abnormalities; however, current tissue culture models lack the cell type diversity to study these disorders and normal otic development. Here, we demon-strate the robustness of human pluripotent stem cell-derived inner ear organoids (IEOs) and evaluate cell type heterogeneity by single-cell transcriptomics. To validate our findings, we construct a single-cell atlas of hu-man fetal and adult inner ear tissue. Our study identifies various cell types in the IEOs including periotic mesenchyme, type I and type II vestibular hair cells, and developing vestibular and cochlear epithelium. Many genes linked to congenital inner ear dysfunction are confirmed to be expressed in these cell types. Additional cell-cell communication analysis within IEOs and fetal tissue highlights the role of endothelial cells on the developing sensory epithelium. These findings provide insights into this organoid model and its po-tential applications in studying inner ear development and disorders. Show less
Ferreira, A.V.; Kostidis, S.; Groh, L.A.; Koeken, V.A.C.M.; Bruno, M.; Baydemir, I.; ... ; Domínguez-Andrés, J. 2023
Itaconate is an immunomodulatory metabolite produced by immune cells under microbial stimulation and certain pro-inflammatory conditions and triggers antioxidant and anti-inflammatory responses.... Show moreItaconate is an immunomodulatory metabolite produced by immune cells under microbial stimulation and certain pro-inflammatory conditions and triggers antioxidant and anti-inflammatory responses. We show that dimethyl itaconate, a derivative of itaconate previously linked to suppression of inflammation and widely employed as an alternative to the endogenous metabolite, can induce long-term transcriptional, epigenomic, and metabolic changes, characteristic of trained immunity. Dimethyl itaconate alters glycolytic and mitochondrial energetic metabolism, ultimately leading to increased responsiveness to microbial ligand stimulation. Subsequently, mice treated with dimethyl itaconate present increased survival to infection with Staphylococcus aureus. Additionally, itaconate levels in human plasma correlate with enhanced ex vivo pro-inflammatory cytokine production. Collectively, these findings demonstrate that dimethyl itaconate displays short-term anti-inflammatory characteristics and the capacity to induce long-term trained immunity. This pro-and anti-inflammatory dichotomy of dimethyl itaconate is likely to induce complex immune responses and should be contemplated when considering itaconate derivatives in a therapeutic context. Show less
Potent T cell responses against infections and malignancies require a rapid yet tightly regulated production of toxic effector molecules. Their production level is defined by post-transcriptional... Show morePotent T cell responses against infections and malignancies require a rapid yet tightly regulated production of toxic effector molecules. Their production level is defined by post-transcriptional events at 3' untranslated regions (3' UTRs). RNA binding proteins (RBPs) are key regulators in this process. With an RNA aptamer-based capture assay, we identify >130 RBPs interacting with IFNG, TNF, and IL2 3' UTRs in human T cells. RBP-RNA interactions show plasticity upon T cell activation. Furthermore, we uncover the intricate and time-dependent regulation of cytokine production by RBPs: whereas HuR supports early cytokine produc-tion, ZFP36L1, ATXN2L, and ZC3HAV1 dampen and shorten the production duration, each at different time points. Strikingly, even though ZFP36L1 deletion does not rescue the dysfunctional phenotype, tu-mor-infiltrating T cells produce more cytokines and cytotoxic molecules, resulting in superior anti-tumoral T cell responses. Our findings thus show that identifying RBP-RNA interactions reveals key modulators of T cell responses in health and disease. Show less
Schimmel, J.; Munoz Subirana, N.; Kool, H.; Schendel, R. van; Vlies, S. van der; Kamp, J.A.; ... ; Tijsterman, M. 2023
Gene editing through repair of CRISPR-Cas9-induced chromosomal breaks offers a means to correct a wide range of genetic defects. Directing repair to produce desirable outcomes by modulating DNA... Show moreGene editing through repair of CRISPR-Cas9-induced chromosomal breaks offers a means to correct a wide range of genetic defects. Directing repair to produce desirable outcomes by modulating DNA repair path-ways holds considerable promise to increase the efficiency of genome engineering. Here, we show that inhibition of non-homologous end joining (NHEJ) or polymerase theta-mediated end joining (TMEJ) can be exploited to alter the mutational outcomes of CRISPR-Cas9. We show robust inhibition of TMEJ activity at CRISPR-Cas9-induced double-strand breaks (DSBs) using ART558, a potent polymerase theta (PolW) inhib-itor. Using targeted sequencing, we show that ART558 suppresses the formation of microhomology-driven deletions in favor of NHEJ-specific outcomes. Conversely, NHEJ deficiency triggers the formation of large kb-sized deletions, which we show are the products of mutagenic TMEJ. Finally, we show that combined chemical inhibition of TMEJ and NHEJ increases the efficiency of homology-driven repair (HDR)-mediated precise gene editing. Our work reports a robust strategy to improve the fidelity and safety of genome engi-neering. Show less
Hos, B.J.; Tondini, E.; Camps, M.G.M.; Rademaker, W.; Bulk, J. van den; Ruano, D.; ... ; Ossendorp, F. 2022
We report an approach to identify tumor-specific CD4+ T cell neo-epitopes of both mouse and human cancer cells by analysis of major histocompatibility complex (MHC) class II-eluted natural peptides... Show moreWe report an approach to identify tumor-specific CD4+ T cell neo-epitopes of both mouse and human cancer cells by analysis of major histocompatibility complex (MHC) class II-eluted natural peptides. MHC class II -presented peptide sequences are identified by introducing the MHC class II transactivator (CIITA) in tumor cells that were originally MHC class II negative. CIITA expression facilitates cell-surface expression of MHC class II molecules and the appropriate peptide-loading machinery. Peptide elution of purified MHC class II molecules and subsequent mass spectrometry reveals oncoviral-and neo-epitopes as well as shared epitopes. Immunological relevance of these epitopes is shown by natural presentation by dendritic cells and immunogenicity. Synthetic peptide vaccination induced functional CD4+ T cell responses, which helped tu-mor control in vivo. Thus, this CIITA transfection approach aids to identify relevant T helper epitopes pre-sented by any MHC class II allele that would be otherwise very difficult to predict and reveals important tar-gets for cancer immunotherapy. Show less
Hos, B.J.; Tondini, E.; Camps, M.G.M.; Rademaker, W.; Bulk, J. van den; Ruano, D.; ... ; Ossendorp, F. 2022
We report an approach to identify tumor-specific CD4+ T cell neo-epitopes of both mouse and human cancer cells by analysis of MHC class II-eluted natural peptides. MHC class II-presented peptide... Show moreWe report an approach to identify tumor-specific CD4+ T cell neo-epitopes of both mouse and human cancer cells by analysis of MHC class II-eluted natural peptides. MHC class II-presented peptide sequences are identified by introducing the MHC class II transactivator CIITA in tumor cells that were originally MHC class II-negative. CIITA expression facilitates cell-surface expression of MHC class II molecules and the appropriate peptide-loading machinery. Peptide elution of purified MHC class II molecules and subsequent mass spectrometry reveals many novel oncoviral-, shared and neo-epitopes. Immunological relevance of these epitopes is shown by natural presentation by dendritic cells and immunogenicity. Synthetic peptide vaccination induced functional CD4+ T cell responses which helped tumor control in vivo. Thus, this CIITA transfection approach aids to identify relevant T helper epitopes presented by any MHC class II allele that would be otherwise very difficult to predict and reveals important new targets for cancer immunotherapy. Show less
Pelgrom, L.R.; Patente, T.A.; Otto, F.; Nouwen, L.V.; Ozir-Fazalalikhan, A.; Ham, A.J. van der; ... ; Everts, B. 2022
How mechanistic target of rapamycin complex 1 (mTORC1), a key regulator of cellular metabolism, affects dendritic cell (DC) metabolism and T cell-priming capacity has primarily been investigated in... Show moreHow mechanistic target of rapamycin complex 1 (mTORC1), a key regulator of cellular metabolism, affects dendritic cell (DC) metabolism and T cell-priming capacity has primarily been investigated in vitro, but how mTORC1 regulates this in vivo remains poorly defined. Here, using mice deficient for mTORC1 component raptor in DCs, we find that loss of mTORC1 negatively affects glycolytic and fatty acid metabolism and maturation of conventional DCs, particularly cDC1s. Nonetheless, antigen-specific CD8(+) T cell responses to infection are not compromised and are even enhanced following skin immunization. This is associated with increased activation of Langerhans cells and a subpopulation of EpCAM-expressing cDC1s, of which the latter show an increased physical interaction with CD8(+) T cells in situ. Together, this work reveals that mTORC1 limits CD8(+) T cell priming in vivo by differentially orchestrating the metabolism and immunogenicity of distinct antigen-presenting cell subsets, which may have implications for clinical use of mTOR inhibitors. Show less
Huettner, I.; Krumm, S.A.; Serna, S.; Brzezicka, K.; Monaco, S.; Walpole, S.; ... ; IAVI African HIV Res Network 2022
The HIV-1 Envelope glycoprotein (Env) is the sole target for broadly neutralizing antibodies (bnAbs). Env is heavily glycosylated with host-derived N-glycans, and many bnAbs bind to, or are... Show moreThe HIV-1 Envelope glycoprotein (Env) is the sole target for broadly neutralizing antibodies (bnAbs). Env is heavily glycosylated with host-derived N-glycans, and many bnAbs bind to, or are dependent upon, Env glycans for neutralization. Although glycan-binding bnAbs are frequently detected in HIV-infected individuals, attempts to elicit them have been unsuccessful because of the poor immunogenicity of Env N-glycans. Here, we report cross-reactivity of glycan-binding bnAbs with self-and non-self N-glycans and glycoprotein antigens from different life-stages of Schistosoma mansoni. Using the IAVI Protocol C HIV infection cohort, we examine the relationship between S. mansoni seropositivity and development of bnAbs targeting glycandependent epitopes. We show that the unmutated common ancestor of the N332/V3-specific bnAb lineage PCDN76, isolated from an HIV-infected donor with S. mansoni seropositivity, binds to S. mansoni cercariae while lacking reactivity to gp120. Overall, these results present a strategy for elicitation of glycan-reactive bnAbs which could be exploited in HIV-1 vaccine development. Show less
Sahin, M.; Remy, M.M.; Fallet, B.; Sommerstein, R.; Florova, M.; Langner, A.; ... ; Pinschewer, D.D. 2022
Across the animal kingdom, multivalency discriminates antibodies from all other immunoglobulin superfamily members. The evolutionary forces conserving multivalency above other structural hallmarks... Show moreAcross the animal kingdom, multivalency discriminates antibodies from all other immunoglobulin superfamily members. The evolutionary forces conserving multivalency above other structural hallmarks of antibodies remain, however, incompletely defined.Here, we engineer monovalent either Fc-competent or -deficient antibody formats to investigate mechanisms of protection of neutralizing antibodies (nAbs) and non-neutralizing antibodies (nnAbs) in virus-infected mice. Antibody bivalency enables the tethering of virions to the infected cell surface, inhibits the release of virions in cell culture, and suppresses viral loads in vivo independently of Fc gamma receptor (Fc gamma R) interactions. In return, monovalent antibody formats either do not inhibit virion release and fail to protect in vivo or their protective efficacy is largely Fc gamma R dependent. Protection in mice correlates with virus-release-inhibiting activity of nAb and nnAb rather than with their neutralizing capacity.These observations provide mechanistic insights into the evolutionary conservation of antibody bivalency and help refining correlates of nnAb protection for vaccine development. Show less
Autotaxin (ATX; ENPP2) produces lysophosphatidic acid (LPA) that regulates multiple biological functions via cognate G protein-coupled receptors LPAR1-6. ATX/LPA promotes tumor cell migration and... Show moreAutotaxin (ATX; ENPP2) produces lysophosphatidic acid (LPA) that regulates multiple biological functions via cognate G protein-coupled receptors LPAR1-6. ATX/LPA promotes tumor cell migration and metastasis via LPAR1 and T cell motility via LPAR2, yet its actions in the tumor immune microenvironment remain unclear. Here, we show that ATX secreted by melanoma cells is chemorepulsive for tumor-infiltrating lymphocytes (TILs) and circulating CD8(+) T cells ex vivo, with ATX functioning as an LPA-producing chaperone. Mechanistically, T cell repulsion predominantly involves G alpha(12/13)-coupled LPAR6. Upon anti-cancer vaccination of tumor-bearing mice, ATX does not affect the induction of systemic T cell responses but, importantly, suppresses tumor infiltration of cytotoxic CD8(+) T cells and thereby impairs tumor regression. Moreover, single-cell data from melanoma tumors are consistent with intratumoral ATX acting as a T cell repellent. These findings highlight an unexpected role for the pro-metastatic ATX-LPAR axis in suppressing CD8(+) T cell infiltration to impede anti-tumor immunity, suggesting new therapeutic opportunities. Show less
Katsuki, Y.; Abe, M.; Park, S.Y.; Wu, W.W.; Yabe, H.; Yabe, M.; ... ; Takata, M. 2021
SLX4/FANCP is a key Fanconi anemia (FA) protein and a DNA repair scaffold for incision around a DNA inter strand crosslink (ICL) by its partner XPF nuclease. The tandem UBZ4 ubiquitin-binding... Show moreSLX4/FANCP is a key Fanconi anemia (FA) protein and a DNA repair scaffold for incision around a DNA inter strand crosslink (ICL) by its partner XPF nuclease. The tandem UBZ4 ubiquitin-binding domains of SLX4 are critical for the recruitment of SLX4 to damage sites, likely by binding to K63-linked polyubiquitin chains. However, the identity of the ubiquitin E3 ligase that mediates SLX4 recruitment remains unknown. Using small interfering RNA (siRNA) screening with a GFP-tagged N-terminal half of SLX4 (termed SLX4-N), we identify the RNF168 E3 ligase as a critical factor for mitomycin C (MMC)-induced SLX4 foci formation. RNF168 and GFP-SLX4-N colocalize in MMC-induced ubiquitin foci. Accumulation of SLX4-N at psoralen-laser ICL tracks or of endogenous SLX4 at Digoxigenin-psoralen/UVA ICL is dependent on RNF168. Finally, we find that RNF168 is epistatic with SLX4 in promoting MMC tolerance. We conclude that RNF168 is a critical component of the signal transduction that recruits SLX4 to ICL damage. Show less
During acute malaria, most individuals mount robust inflammatory responses that limit parasite burden. However, long-lived sterilizing anti-malarial memory responses are not efficiently induced,... Show moreDuring acute malaria, most individuals mount robust inflammatory responses that limit parasite burden. However, long-lived sterilizing anti-malarial memory responses are not efficiently induced, even following repeated Plasmodium exposures. Using multiple Plasmodium species, genetically modified parasites, and combinations of host genetic and pharmacologic approaches, we find that the deposition of the malarial pigment hemozoin directly limits the abundance and capacity of conventional type 1 dendritic cells to prime helper T cell responses. Hemozoin-induced dendritic cell dysfunction results in aberrant Plasmodium-specific CD4 T follicular helper cell differentiation, which constrains memory B cell and long-lived plasma cell formation. Mechanistically, we identify that dendritic cell-intrinsic NLRP3 inflammasome activation reduces conventional type 1 dendritic cell abundance, phagocytosis, and T cell priming functions in vivo. These data identify biological consequences of hemozoin deposition during malaria and highlight the capacity of the malarial pigment to program immune evasion during the earliest events following an initial Plasmodium exposure. Show less
Cabello-Lobato, M.J.; Gonzalez-Garrido, C.; Cano-Linares, M.I.; Wong, R.P.; Yanez-Vilchez, A.; Morillo-Huesca, M.; ... ; Prado, F. 2021
The minichromosome maintenance (MCM) helicase physically interacts with the recombination proteins Rad51 and Rad52 from yeast to human cells. We show, in Saccharomyces cerevisiae, that these... Show moreThe minichromosome maintenance (MCM) helicase physically interacts with the recombination proteins Rad51 and Rad52 from yeast to human cells. We show, in Saccharomyces cerevisiae, that these interactions occur within a nuclease-insoluble scaffold enriched in replication/repair factors. Rad51 accumulates in a MCM- and DNA-binding-independent manner and interacts with MCMhelicases located outside of the replication origins and forks. MCM, Rad51, and Rad52 accumulate in this scaffold in G1 and are released during the S phase. In the presence of replication-blocking lesions, Cdc7 prevents their release from the scaffold, thus maintaining the interactions. We identify a rad51 mutant that is impaired in its ability to bind to MCM but not to the scaffold. This mutant is proficient in recombination but partially defective in single-stranded DNA (ssDNA) gap filling and replication fork progression through damaged DNA. Therefore, cells accumulate MCM/Rad51/Rad52 complexes at specific nuclear scaffolds in G1 to assist stressed forks through non-recombinogenic functions. Show less
Responding to different dynamic levels of stress is critical for mammalian survival. Disruption of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) signaling is proposed to underlie... Show moreResponding to different dynamic levels of stress is critical for mammalian survival. Disruption of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) signaling is proposed to underlie hypothalamic-pituitary-adrenal (HPA) axis dysregulation observed in stress-related psychiatric disorders. In this study, we show that FK506-binding protein 51 (FKBP5) plays a critical role in fine-tuning MR:GR balance in the hippocampus. Biotinylated-oligonucleotide immunoprecipitation in primary hippocampal neurons reveals that MR binding, rather than GR binding, to the Fkbp5 gene regulates FKBP5 expression during baseline activity of glucocorticoids. Notably, FKBP5 andMR exhibit similar hippocampal expression patterns in mice and humans, which are distinct from that of the GR. Pharmacological inhibition and region- and cell type-specific receptor deletion in mice further demonstrate that lack of MR decreases hippocampal Fkbp5 levels and dampens the stress-induced increase in glucocorticoid levels. Overall, our findings demonstrate that MR-dependent changes in baseline Fkbp5 expression modify GR sensitivity to glucocorticoids, providing insight into mechanisms of stress homeostasis. Show less
