Susan Kloet

Trained immunity (TRIM) describes the long-term functional reprogramming of innate immune cells after an initial encounter with a microbial or endogenous stimulus, enabling cross-protection against unrelated pathogens. This memory has been studied almost exclusively as a single, pro-inflammatory state. Nevertheless, whether qualitatively distinct TRIM programs exist remains elusive. To answer this, we trained human PBMCs with stimuli representing different immunological contexts and defined four, distinct TRIM programs: antibacterial, antiparasitic/pro-allergic, antiviral, and pro-repair. Each TRIM is defined by a unique cytokine signature, distinct cellular requirements, and program-specific metabolic rewiring. Single-cell RNA sequencing and single-nucleus ATAC sequencing reveal program-specific transcriptional and chromatin states, with distinct transcription factor activities and gene regulatory networks underlying each program. As proof of concept, we demonstrate that in vivo induction of the antiviral TRIM program confers protection against lethal influenza A virus infection through transient hematopoietic stem and progenitor cell reprogramming and a type I interferon receptor-dependent mechanism. Finally, cross-comparison with patient transcriptomes shows that these four programs are differentially engaged across inflammatory and infectious diseases. These findings extend the concept of trained immunity beyond a single, pro-inflammatory response and establish a polarized framework of functionally specialized innate immune memory states, with relevance for infection, inflammation, and vaccine design.

Human immunoglobulin G4 (IgG4) shapes both protective and pathogenic immunity, influencing conditions ranging from allergy and autoimmunity to cancer. However, subclass-specific targeting of memory B cells remains challenging due to limited knowledge of their phenotypic and functional heterogeneity. Here, we show that IgG4 ⁺ memory B cells are overrepresented in a niche of FCER2 ⁺ BAFFR ⁺ memory B cells, can be classified in several subtypes, have a distinct transcription factor profile and are the only memory B cell subset to express sterile IGHE transcripts. While IgG4 ⁺ B cells display a unique transcriptional profile, only BAFFR, IL5Rb and the B cell receptor were upregulated on protein level on IgG4 ⁺ cells. IgG4 ⁺ B cells have normal repertoire diversity, but a distinct germline V-gene usage, suggesting IgG4 responses are driven by specific antigens. By labeling autoreactive B cells, we confirmed that MuSK myasthenia gravis (an archetypal IgG4-mediatedautoimmune disease) patients have a normal memory B cell profile and that autoreactive IgG4 ⁺ memory B cells are extremely rare. These results highlight the unique features of IgG4 ⁺ B cells, provide insight on potential subclass-specific therapeutic targets and point towards an antigen-driven IgG4 response within a largely non-autoreactive memory B cell pool.

Pathological USH2A mutations cause Usher syndrome type II, characterized by progressive retinitis pigmentosa and hearing and balance impairment. This study aims to investigate the cellular mechanisms underlying USH2A-related retinal degeneration using human induced pluripotent stem cell (hiPSC)-derived retinal organoids. The introduction of a homozygous nonsense mutation in the USH2A hotspot exon-13 resulted in normal photoreceptor development but loss of ciliary localization of usherin long form B and its interacting proteins, ADGRV1 and whirlin. Notably, single-cell RNA sequencing revealed unexpected significant transcriptional changes in Müller glial cells (MGCs), suggestive of disruptions in the translation, innate immune response, and endolysosomal system. These findings suggest that, while photoreceptor cells are mildly affected by the exon-13 USH2A mutation, MGCs exhibit major transcriptional changes, potentially contributing to the disease progression and therefore shedding light on potential alternative therapeutic targets.

Loss-of-function variants in PALB2 give rise to defects in DNA damage repair by homologous recombination (HR), increasing the risk of breast cancer in female carriers. However, genetic testing frequently reveals missense variants of uncertain significance (VUS) for which the impact on protein function and cancer risk are unclear. Here we assay 84% of all possible missense variants in 11 out of 13 PALB2 exons using site-saturation functional screens with PARP inhibitor sensitivity as a readout for HR. These exons encode the coiled-coil and WD40 domains, which we identify as the minimal regions required for HR. Furthermore, we reveal the functional impact of 6718 missense variants, classifying 3904 variants as functional (58%), 2422 as intermediate (36%), and 392 as damaging (6%). A burden-type analysis shows that damaging missense variants in PALB2 are associated with a significantly increased risk of breast cancer, similar to that observed for truncating variants. These results will be valuable for the classification of PALB2 missense VUS and clinical management of carriers.

Clostridioides difficile infection (CDI) is a toxin-mediated gastro-intestinal disease. Yet, C. difficile is a phylogenetically diverse species that includes many non-toxigenic strains. In general, these are understudied, despite having significant potential impact for our understanding of the colonization process and as therapeutic modalities. Here, we present an in-depth characterization - including the complete genome sequence - of the non-toxigenic C. difficile strain L-NTCD03. This strain belongs to PCR ribotype 416, clade 4 and multilocus sequence type 39. It is resistant to multiple antimicrobials, but not those used for treatment of CDI. We validate the relevance of the cfr(B) gene from this strain in antimicrobial resistance. We find the L-NTCD03 strain to be non-toxic in various assays. Altogether, L-NTCD03 is a promising candidate for developing into a live biotherapeutic product.

Pathological USH2A mutations cause Usher Syndrome type II, characterized by progres-sive retinitis pigmentosa and hearing and balance impairment. This study aims to inves-tigate the cellular mechanisms underlying USH2A-related retinal degeneration using hu-man induced pluripotent stem cell (hiPSC)-derived retinal organoids. The introduction of a homozygous nonsense mutation in the USH2A hotspot exon-13 resulted in normal photoreceptor development, but loss of ciliary localization of usherin long form B and its interacting proteins, ADGRV1 and whirlin. Notably, single-cell RNA sequencing revealed unexpected significant changes in Müller glial cells (MGCs), with disruptions in the translation, innate immune response, and endolysosomal system. These findings suggest that, while photoreceptor cells are mildly affected by the exon-13 USH2A mutation, MGCs exhibit major dysfunction, potentially contributing to the disease progression and there-fore shedding light on potential alternative therapeutic targets.

BRCA1 (breast cancer-associated protein 1) plays a central role in homologous recombination (HR) through interactions with multiple proteins across its various domains. The C-terminal BRCT domains bind HR regulators such as ABRAXAS1, CtIP, and BRIP1, each contributing to distinct, sometimes opposing, functions. While pathogenic mutations frequently cluster within the canonical BRCT phospho-binding pocket, the broader mutational landscape and its functional consequences remain poorly understood. Here, we used a site-saturation mutagenesis library of the BRCT domains to test >4,000 single-residue variants for their ability to bind ABRAXAS1 and CtIP. Using a yeast two-hybrid screen, we systematically assessed these interactions and validated key findings in mammalian cells. The resulting interaction map identified previously uncharacterized residues critical for partner binding and demonstrated their detrimental impact on HR. Importantly, we identified separation-of-function mutations that selectively disrupt individual protein interactions, enabling a more detailed analysis of each partner’s contribution to HR. Functional assays suggested that disruption of CtIP binding had the most pronounced impact on HR. Furthermore, integration of our data with clinical variant data revealed a strong correlation between loss of protein binding and pathogenicity, highlighting the potential utility of our interaction map for clinical variant interpretation.

DUX4 regulates the expression of genic and nongenic elements and modulates chromatin accessibility during zygotic genome activation in cleavage stage embryos. Its misexpression in skeletal muscle causes facioscapulohumeral dystrophy (FSHD). By leveraging full-length RNA isoform sequencing with short-read RNA sequencing of DUX4-inducible myoblasts, we elucidate an isoform-resolved transcriptome featuring numerous unannotated isoforms from known loci and novel intergenic loci. While DUX4 activates similar programs in early embryos and FSHD muscle, the isoform usage of known DUX4 targets is notably distinct between the two contexts. DUX4 also activates hundreds of previously unannotated intergenic loci dominated by repetitive elements. The transcriptional and epigenetic profiles of these loci in myogenic and embryonic contexts indicate that the usage of DUX4-binding sites at these intergenic loci is influenced by the cellular environment. These findings demonstrate that DUX4 induces context-specific transcriptomic programs, enriching our understanding of DUX4-induced muscle pathology.

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.

Ruminococcus gnavus is a gut bacterium found in > 90% of healthy individuals, but its increased abundance is also associated with chronic inflammatory diseases, particularly Crohn’s disease. Nevertheless, its global distribution and intraspecies genomic variation remain understudied. By surveying 12,791 gut metagenomes, we recapitulated known associations with metabolic diseases and inflammatory bowel disease. We uncovered a higher prevalence and abundance of R. gnavus in Westernized populations and observed bacterial relative abundances up to 83% in newborns. Next, we built a resource of R. gnavus isolates (N = 45) from healthy individuals and Crohn’s disease patients and generated complete R. gnavus genomes using PacBio circular consensus sequencing. Analysis of these genomes and publicly available high-quality draft genomes (N = 333 genomes) revealed multiple clades which separated Crohn’s-derived isolates from healthy-derived isolates. Presumed R. gnavus virulence factors could not explain this separation. Bacterial genome-wide association study revealed that Crohn’s-derived isolates were enriched in genes related to mobile elements and mucin foraging. Together, we present a large R. gnavus resource that will be available to the scientific community and provide novel biological insights into the global distribution and genomic variation of R. gnavus.