Clinical characteristics of all MSI patients from different ICB-treated cohorts within DRUP.

5-OP-RU induces MAIT cell-mediated suppression of CD4+ T cell stimulation.

Type of antibiotics (ATB) administered to patients in the MMR-d/MSI-H cohort.

Clinical information and accession numbers for the Microbe-ICB cohort.

Immunoglobulin A (IgA) deficiency is the most common primary antibody deficiency and contributes to recurrent respiratory tract infections (rRTIs) in children, with prevalence rates in this group ranging from 1:4 to 1:65. Previous studies in healthy humans and animals suggest that the gut microbiota significantly influences IgA production. We investigated the role of the gut microbiota in IgA deficiency and its potential in promoting IgA induction in children with rRTIs. Children under 7 years of age with rRTIs were enrolled in a prospective cohort study. Serum and feces were collected for IgA level measurement using ELISA and to determine the composition of the intestinal microbiota by 16S rRNA sequencing. We included 82 children with rRTIs, 38

Metagenomics data distribution in the MMR-d/MSI-H cohort.

Clinical characteristics of the Microbe-ICB cohort.

Functional dysbiosis associates with ICB response in the Microbe-ICB cohort.

HMBPP-producing bacteria induce Vδ2 T cell-mediated anti-tumor activity.

HMBPP does not affect surface expression of tumor antigen presentation machinery, PD-L1, and BTN3A1/2/3.

HMBPP induces Vδ2 T cell anti-tumor activity.

Biopsy location of patients from the MMR-d/MSI-H cohort.

Bacterial glycans are key components in immune interactions. We lack insight into the diverse glycan landscape present in complex microbial communities since currentomics techniques do not capture this post-translational information. Here we employed C-type lectin receptors (CLRs), which are dedicated innate glycan-sensing receptors, as probes for bacterial cell sorting in combination with 16S rRNA gene sequencing to identify microbiota species with a specific CLR-reactive glycan profile. We established our experimental CLR-sequencing (CLR-seq) pipeline using soluble fluorescently-labeled human macrophage galactose C-type lectin (MGL, CD301) and langerin (CD207). Both receptors identified known langerin- or MGL-interacting Staphylococcus aureus strains in a synthetic microbial community even when present at low abundance. Subsequent application of CLR-seq on fecal microbiota samples from healthy donors identified specific langerin- and MGL-interacting bacterial species that were subsequently validated as monocultures. In summary, CLR-seq is a modular platform that allows identification of human microbiota species based on CLR-interacting glycans with easy expansion to other CLRs or microbiota samples from patients. Given that CLRs are densely expressed on dendrites of antigen-presenting cells, this pathway may play a role in cross-barrier recognition and sampling of the environment during homeostasis.

Metagenomics data distribution within the Microbe-ICB cohort.

Functional dysbiosis correlates with ICB response in the MMR-d/MSI-H cohort.

Information on patient-derived (tumor) organoid lines.

Studies have identified a link between specific microbiome-derived bacteria and immune checkpoint blockade (ICB) efficacy. However, these species lack consistency across studies, and their immunomodulatory mechanisms remain elusive. To understand the influence of the microbiome on ICB response, we studied its functional capacity. Using pan-cancer metagenomics data from ICB-treated patients, we showed that community-level metabolic pathways are stable across individuals, making them suitable for predicting ICB response. We identified several microbial metabolic processes significantly associated with response, including the methylerythritol 4-phosphate (MEP) pathway, which was associated with response and induced Vδ2 T cell-mediated antitumor responses in patient-derived tumor organoids. In contrast, riboflavin synthesis was associated with ICB resistance, and its intermediates induced mucosal-associated invariant T (MAIT) cell-mediated immune suppression. Moreover, gut metabolomics revealed that high riboflavin levels were linked to worse survival in patients with abundant intratumoral MAIT cells. Collectively, our results highlight the relevance of metabolite-mediated microbiome-immune cell cross-talk. SIGNIFICANCE:Microbial metabolic pathways are highly conserved across individuals and therefore offer an opportunity to link the microbiome to immunotherapy efficacy. We identified specific microbial metabolic pathways associated with response to ICB and provided mechanistic insights into the immunomodulatory influence of these pathways on antitumor immunity.

Fusobacterium nucleatum is a Gram-negative oncobacterium that is associated with colorectal cancer. The molecular mechanisms utilized by F. nucleatum to promote colorectal tumor development have largely focused on adhesin-mediated binding to the tumor tissue and on the pro-inflammatory capacity of F. nucleatum. However, the exact manner in which F. nucleatum promotes inflammation in the tumor microenvironment and subsequent tumor promotion remains underexplored. Here, we show that both living F. nucleatum and sterile F. nucleatum-conditioned medium promote CXCL8 release from the intestinal adenocarcinoma HT-29 cell line. We determined that the observed pro-inflammatory effect was ALPK1-dependent in both HEK293 and HT-29 cells and that the released F. nucleatum molecule had characteristics that match those of the pro-inflammatory ALPK1 ligand ADP-heptose or related heptose phosphates. In addition, we determined that not only F. nucleatum promoted an ALPK1-dependent pro-inflammatory environment but also other Fusobacterium species such as F. varium, F. necrophorum and F. gonidiaformans generated similar effects, indicating that ADP-heptose or related heptose phosphate secretion is a conserved feature of the Fusobacterium genus. By performing transcriptional analysis of ADP-heptose stimulated HT-29 cells, we found several inflammatory and cancer-related pathways to be differentially regulated, including DNA mismatch repair genes and the immune inhibitory receptor PD-L1. Finally, we show that stimulation of HT-29 cells with F. nucleatum resulted in an ALPK1-dependent upregulation of PD-L1. These results aid in our understanding of the mechanisms by which F. nucleatum can affect tumor development and therapy and pave the way for future therapeutic approaches.

Currently, over 88 million people are estimated to have adopted a vegan or vegetarian diet. Cysteine is a semi-essential amino acid, which availability is largely dependent on dietary intake of meat, eggs and whole grains. Vegan/vegetarian diets are therefore inherently low in cysteine. Sufficient uptake of cysteine is crucial, as it serves as substrate for protein synthesis and can be converted to taurine and glutathione. We found earlier that intermolecular cystine bridges are essential for the barrier function of the intestinal mucus layer. Therefore, we now investigate the effect of low dietary cystine on the intestine. Mice (8/group) received a high fat diet with a normal or low cystine concentration for 2 weeks. We observed no changes in plasma methionine, cysteine, taurine or glutathione levels or bile acid conjugation after 2 weeks of low cystine feeding. In the colon, dietary cystine restriction results in an increase in goblet cell numbers, and a borderline significant increase mucus layer thickness. Gut microbiome composition and expression of stem cell markers did not change on the low cystine diet. Remarkably, stem cell markers, as well as the proliferation marker Ki67, were increased upon cystine restriction in the small intestine. In line with this, gene set enrichment analysis indicated enrichment of Wnt signaling in the small intestine of mice on the low cystine diet, indicative of increased epithelial proliferation. In conclusion, 2 weeks of cystine restriction did not result in apparent systemic effects, but the low cystine diet increased the proliferative capacity specifically of the small intestine and induced the number of goblet cells in the colon.

The intestinal microbiota plays a significant role in maintaining systemic and intestinal homeostasis, but can also influence diseases such as inflammatory bowel disease (IBD) and cancer. Certain bacterial species within the intestinal tract can chronically activate the immune system, leading to low-grade intestinal inflammation. As a result, plasma cells produce high levels of secretory antigen-specific immunoglobulin A (IgA), which coats the immunostimulatory bacteria. This IgA immune response against intestinal bacteria may be associated with the maintenance of homeostasis and health, as well as disease. Unraveling this dichotomy and identifying the immunostimulatory bacteria is crucial for understanding the relationship between the intestinal microbiota and the immune system, and their role in health and disease. IgA-SEQ technology has successfully identified immunostimulatory, IgA-coated bacteria from fecal material. However, the original technology is time-consuming and has limited downstream applications. In this study, we aimed to develop a next-generation, high-throughput, magnet-based sorting approach (ng-IgA-SEQ) to overcome the limitations of the original IgA-SEQ protocol. We show, in various settings of complexity ranging from simple bacterial mixtures to human fecal samples, that our magnetic 96-well plate-based ng-IgA-SEQ protocol is highly efficient at sorting and identifying IgA-coated bacteria in a high-throughput and time efficient manner. Furthermore, we performed a comparative analysis between different IgA-SEQ protocols, highlighting that the original FACS-based IgA-SEQ approach overlooks certain nuances of IgA-coated bacteria, due to the low yield of sorted bacteria. Additionally, magnetic-based ng-IgA-SEQ allows for novel downstream applications. Firstly, as a proof-of-concept, we performed metagenomic shotgun sequencing on 10 human fecal samples to identify IgA-coated bacterial strains and associated pathways and CAZymes. Secondly, we successfully isolated and cultured IgA-coated bacteria by performing the isolation protocol under anaerobic conditions. Our magnetic 96-well plate-based high-throughput next-generation IgA-SEQ technology efficiently identifies a great number of IgA-coated bacteria from fecal samples. This paves the way for analyzing large cohorts as well as novel downstream applications, including shotgun metagenomic sequencing, culturomics, and various functional assays. These downstream applications are essential to unravel the role of immunostimulatory bacteria in health and disease.