Longitudinal analysis of 20 Years of external quality assurance schemes for PCR/NAAT-based bacterial genome detection in diagnostic testing.

Background: Quality control (QC), quality assurance, and standardization are crucial for modern diagnostic testing in the field of medical microbiology. The need for efficient QC to ensure accurate laboratory results, treatment, and infection prevention has led to significant efforts in standardizing assay reagents and workflows. External quality assessment (EQA) schemes, like those offered by INSTAND, play a vital role in evaluating in-house and commercial routine diagnostic assays, regarded as mandatory by national and global guidelines. The recent impact of polymerase chain reaction/nucleic acid amplification technology (PCR/NAAT) assays in medical microbiology requires that high-performing assays be distinguished from inadequately performing ones, especially those made by inexperienced suppliers. Objectives: The study assesses the evolving diagnostic performance trends over 2 decades for the detection of EHEC/STEC, Borrelia (B.) burgdorferi, and MRSA/cMRSA. It explores the historical context of assay utilization, participant engagement, and rates of correct results in EQA schemes. The research seeks to identify patterns in assay preferences, participant proficiency, and the challenges encountered in detecting emerging variants or clinical strains. Results: The study highlights the decline in in-house PCR assay usage, the emergence of new diagnostic challenges, and educational aspects within EQA schemes. Specific examples, such as the inclusion, in certain EQA surveys, of EHEC strains carrying stx-2f or B. miyamotoi, highlight the role of EQAs in increasing awareness and diagnostic capabilities. Advancements in MRSA detection, especially through the adoption of commercial assays, demonstrate the impact that technology evolution has had on diagnostic performance. Conclusion: Achieving excellence in diagnostic molecular microbiology involves a multifaceted approach, including well-evaluated assays, careful instrumentation selection, and structured training programs. EQA schemes contribute significantly to this pursuit by providing insights into the evolving diagnostic landscape and identifying areas for improvement in the diagnostic workflow as well as in PCR/NAAT assay design.

Strain specific differences in vitamin D3 response: impact on gut homeostasis.

Vitamin D3 regulates a variety of biological processes irrespective of its well-known importance for calcium metabolism. Epidemiological and animal studies indicate a role in immune regulation, intestinal barrier function and microbiome diversity. Here, we analyzed the impact of different vitamin D3- containing diets on C57BL/6 and BALB/c mice, with a particular focus on gut homeostasis and also investigated effects on immune cells in vitro. Weak regulatory effects were detected on murine T cells. By trend, the active vitamin D3 metabolite 1,25-dihydroxyvitamin D3 suppressed IFN, GM-CSF and IL-10 cytokine secretion in T cells of C57BL/6 but not BALB/c mice, respectively. Using different vitamin D3-fortified diets, we found a tissue-specific enrichment of mainly CD11b+ myeloid cells but not T cells in both mouse strains e.g. in spleen and Peyer's Patches. Mucin Reg3γ and Batf expression, as well as important proteins for gut homeostasis, were significantly suppressed in the small intestine of C57BL76 but not BALB/c mice fed with a high-vitamin D3 containing diet. Differences between both mouse stains were not completely explained by differences in vitamin D3 receptor expression which was strongly expressed in epithelial cells of both strains. Finally, we analyzed gut microbiome and again an impact of vitamin D3 was detected in C57BL76 but not BALB/c. Our data suggest strain-specific differences in vitamin D3 responsiveness under steady state conditions which may have important implications when choosing a murine disease model to study vitamin D3 effects.

Bactericidal/permeability-increasing protein instructs dendritic cells to elicit Th22 cell response.

Neutrophil-derived bactericidal/permeability-increasing protein (BPI) is known for its bactericidal activity against gram-negative bacteria and neutralization of lipopolysaccharide. Here, we define BPI as a potent activator of murine dendritic cells (DCs). As shown in GM-CSF-cultured, bone-marrow-derived cells (BMDCs), BPI induces a distinct stimulation profile including IL-2, IL-6, and tumor necrosis factor expression. Conventional DCs also respond to BPI, while M-CSF-cultivated or peritoneal lavage macrophages do not. Subsequent to BPI stimulation of BMDCs, CD4+ T cells predominantly secrete IL-22 and, when naive, preferentially differentiate into T helper 22 (Th22) cells. Congruent with the tissue-protective properties of IL-22 and along with impaired IL-22 induction, disease severity is significantly increased during dextran sodium sulfate-induced colitis in BPI-deficient mice. Importantly, physiological diversification of intestinal microbiota fosters BPI-dependent IL-22 induction in CD4+ T cells derived from mesenteric lymph nodes. In conclusion, BPI is a potent activator of DCs and consecutive Th22 cell differentiation with substantial relevance in intestinal homeostasis.

Bacteria and bacteriophage consortia are associated with protective intestinal metabolites in patients receiving stem cell transplantation.

The microbiome is a predictor of clinical outcome in patients receiving allogeneic hematopoietic stem cell transplantation (allo-SCT). Microbiota-derived metabolites can modulate these outcomes. How bacteria, fungi and viruses contribute to the production of intestinal metabolites is still unclear. We combined amplicon sequencing, viral metagenomics and targeted metabolomics from stool samples of patients receiving allo-SCT (n = 78) and uncovered a microbiome signature of Lachnospiraceae and Oscillospiraceae and their associated bacteriophages, correlating with the production of immunomodulatory metabolites (IMMs). Moreover, we established the IMM risk index (IMM-RI), which was associated with improved survival and reduced relapse. A high abundance of short-chain fatty acid-biosynthesis pathways, specifically butyric acid via butyryl-coenzyme A (CoA):acetate CoA-transferase (BCoAT, which catalyzes EC 2.8.3.8) was detected in IMM-RI low-risk patients, and virome genome assembly identified two bacteriophages encoding BCoAT as an auxiliary metabolic gene. In conclusion, our study identifies a microbiome signature associated with protective IMMs and provides a rationale for considering metabolite-producing consortia and metabolite formulations as microbiome-based therapies.

Short-term effects of etifoxine on human gut microbiome in healthy men.

Background: Neurosteroids have recently gained in interest as a treatment strategy for affective disorders. Etifoxine is known for its dual mode of action, one of which is to stimulate endogenous neurosteroid synthesis. The gut microbiome has been studied in affective disorders, but it has not been investigated in the context of human etifoxine or neurosteroid interventions. Methods: We performed a crossover study with 36 healthy male volunteers who received etifoxine versus alprazolam and placebo in a balanced Williams design. Participants were randomized into six sequences and went through three 5-day treatments followed by wash-out phases of 9 days. Bacterial compositions in stool samples were determined by high-throughput 16S rRNA amplicon sequencing. Results: Gut microbiome analyses revealed no relevant effects between treatments with respect to alpha and beta diversity. Differential abundance analyses yielded etifoxine treatment as the only effect related to changes in microbial features with reductions of Faecalibacterium duncaniae, Roseburia hominis and Lactobacillus rogosae (i.e., Bacteroides galacturonicus). Conclusion: Here we report on the first human investigation of the gut microbiome with short-term etifoxine intervention. Differences in diversity and compositional structure of the microbiome were more likely due to between- subject effects rather than medication. However, five-day treatment with etifoxine reduced the abundance of a few bacterial species. These species are currently seen as beneficial components of a healthy intestinal microbiome. This reduction in abundances may be related to elevated endogenous neurosteroids.

Multimodal immune cell phenotyping in GI biopsies reveals microbiome-related T cell modulations in human GvHD.

Acute graft-versus-host disease (aGvHD) is a significant complication after allogeneic hematopoietic stem cell transplantation (aHSCT), but major factors determining disease severity are not well defined yet. By combining multiplexed tissue imaging and single-cell RNA sequencing on gastrointestinal biopsies from aHSCT-treated individuals with fecal microbiome analysis, we link high microbiome diversity and the abundance of short-chain fatty acid-producing bacteria to the sustenance of suppressive regulatory T cells (Tregs). Furthermore, aGvHD severity strongly associates with the clonal expansion of mainly CD8 T cells, which we find distributed over anatomically distant regions of the gut, persistent over time, and inversely correlated with the presence of suppressive Tregs. Overall, our study highlights the pathophysiological importance of expanded CD8 T cell clones in the progression of aGvHD toward more severe clinical manifestations and strongly supports the further development of microbiome interventions as GvHD treatment via repopulation of the gut Treg niche to suppress inflammation.

Restrictive Versus Permissive Use of Broad-spectrum Antibiotics in Patients Receiving Allogeneic Stem Cell Transplantation and With Early Fever Due to Cytokine Release Syndrome: Evidence for Beneficial Microbiota Protection Without Increase in Infectious Complications.

BACKGROUND: Intestinal microbiome contributes to the pathophysiology of acute gastrointestinal (GI) graft-versus-host disease (GvHD) and loss of microbiome diversity influences the outcome of patients after allogeneic stem cell transplantation (SCT). Systemic broad-spectrum antibiotics have been identified as a major cause of early intestinal dysbiosis. METHODS: In 2017, our transplant unit at the university hospital in Regensburg changed the antibiotic strategy from a permissive way with initiation of antibiotics in all patients with neutropenic fever independent of the underlying cause and risk to a restrictive use in cases with high likelihood of cytokine release syndrome (eg, after anti-thymocyte globulin [ATG] therapy). We analyzed clinical data and microbiome parameters obtained 7 days after allogeneic SCT from 188 patients with ATG therapy transplanted in 2015/2016 (permissive cohort, n = 101) and 2918/2019 (restrictive cohort, n = 87). RESULTS: Restrictive antibiotic treatment postponed the beginning of antibiotic administration from 1.4 ± 7.6 days prior to 1.7 ± 5.5 days after SCT (P = .01) and significantly reduced the duration of antibiotic administration by 5.8 days (P < .001) without increase in infectious complications. Furthermore, we observed beneficial effects of the restrictive strategy compared with the permissive way on microbiome diversity (urinary 3-indoxylsulfate, P = .01; Shannon and Simpson indices, P < .001) and species abundance 7 days post-transplant as well as a positive trend toward a reduced incidence of severe GI GvHD (P = .1). CONCLUSIONS: Our data indicate that microbiota protection can be achieved by a more careful selection of neutropenic patients qualifying for antibiotic treatment during allogeneic SCT without increased risk of infectious complications.

Is Autologous Fecal Microbiota Transfer after Exclusive Enteral Nutrition in Pediatric Crohn's Disease Patients Rational and Feasible? Data from a Feasibility Test.

BACKGROUND: Exclusive enteral nutrition (EEN) is a highly effective therapy for remission induction in pediatric Crohn's disease (CD), but relapse rates after return to a regular diet are high. Autologous fecal microbiota transfer (FMT) using stool collected during EEN-induced clinical remission might represent a novel approach to maintaining the benefits of EEN. METHODS: Pediatric CD patients provided fecal material at home, which was shipped at 4 °C to an FMT laboratory for FMT capsule generation and extensive pathogen safety screening. The microbial community composition of samples taken before and after shipment and after encapsulation was characterized using 16S rRNA amplicon sequencing. RESULTS: Seven pediatric patients provided fecal material for nine test runs after at least three weeks of nutritional therapy. FMT capsules were successfully generated in 6/8 deliveries, but stool weight and consistency varied widely. Transport and processing of fecal material into FMT capsules did not fundamentally change microbial composition, but microbial richness was <30 genera in 3/9 samples. Stool safety screening was positive for potential pathogens or drug resistance genes in 8/9 test runs. CONCLUSIONS: A high pathogen burden, low-diversity microbiota, and practical deficiencies of EEN-conditioned fecal material might render autologous capsule-FMT an unsuitable approach as maintenance therapy for pediatric CD patients.

Reliability of species detection in 16S microbiome analysis: Comparison of five widely used pipelines and recommendations for a more standardized approach.

The use of NGS-based testing of the bacterial microbiota is often impeded by inconsistent or non-reproducible results, especially when applying different analysis pipelines and reference databases. We investigated five frequently used software packages by submitting the same monobacterial datasets to them, representing the V1-2 and the V3-4 regions of the 16S-rRNA gene of 26 well characterized strains, which were sequenced by the Ion Torrent™ GeneStudio S5 system. The results obtained were divergent and calculations of relative abundance did not yield the expected 100%. We investigated these inconsistencies and were able to attribute them to failures either of the pipelines themselves or of the reference databases they rely on. On the basis of these findings, we recommend certain standards which should help to render microbiome testing more consistent and reproducible, and thus useful in clinical practice.

Reg3α concentrations at day of allogeneic stem cell transplantation predict outcome and correlate with early antibiotic use.

Intestinal microbiome diversity plays an important role in the pathophysiology of acute gastrointestinal (GI) graft-versus-host disease (GVHD) and influences the outcome of patients after allogeneic stem cell transplantation (ASCT). We analyzed clinical data and blood samples taken preconditioning and on the day of ASCT from 587 patients from 7 German centers of the Mount Sinai Acute GVHD International Consortium, dividing them into single-center test (n = 371) and multicenter validation (n = 216) cohorts. Regenerating islet-derived 3α (Reg3α) serum concentration of day 0 correlated with clinical data as well as urinary 3-indoxylsulfate (3-IS) and Clostridiales group XIVa, indicators of intestinal microbiome diversity. High Reg3α concentration at day 0 of ASCT was associated with higher 1-year transplant-related mortality (TRM) in both cohorts (P < .001). Cox regression analysis revealed high Reg3α at day 0 as an independent prognostic factor for 1-year TRM. Multivariable analysis showed an independent correlation of high Reg3α concentrations at day 0 with early systemic antibiotic (AB) treatment. Urinary 3-IS (P = .04) and Clostridiales group XIVa (P = .004) were lower in patients with high vs those with low day 0 Reg3α concentrations. In contrast, Reg3α concentrations before conditioning therapy correlated neither with TRM nor disease or treatment-related parameters. Reg3α, a known biomarker of acute GI GVHD correlates with intestinal dysbiosis, induced by early AB treatment in the period of pretransplant conditioning. Serum concentrations of Reg3α measured on the day of graft infusion are predictive of the risk for TRM of ASCT recipients.

Increased zinc levels facilitate phenotypic detection of ceftazidime-avibactam resistance in metallo-ß-lactamase-producing Gram-negative bacteria.

Ceftazidime-avibactam is one of the last resort antimicrobial agents for the treatment of carbapenem-resistant, Gram-negative bacteria. Metallo-ß-lactamase-producing bacteria are considered to be ceftazidime-avibactam resistant. Here, we evaluated a semi-automated antimicrobial susceptibility testing system regarding its capability to detect phenotypic ceftazidime-avibactam resistance in 176 carbapenem-resistant, metallo-ß-lactamase-producing Enterobacterales and Pseudomonas aeruginosa isolates. Nine clinical isolates displayed ceftazidime-avibactam susceptibility in the semi-automated system and six of these isolates were susceptible by broth microdilution, too. In all nine isolates, metallo-ß-lactamase-mediated hydrolytic activity was demonstrated with the EDTA-modified carbapenemase inactivation method. As zinc is known to be an important co-factor for metallo-ß-lactamase activity, test media of the semi-automated antimicrobial susceptibility testing system and broth microdilution were supplemented with zinc. Thereby, the detection of phenotypic resistance was improved in the semi-automated system and in broth microdilution. Currently, ceftazidime-avibactam is not approved as treatment option for infections by metallo-ß-lactamase-producing, Gram-negative bacteria. In infections caused by carbapenem-resistant Gram-negatives, we therefore recommend to rule out the presence of metallo-ß-lactamases with additional methods before initiating ceftazidime-avibactam treatment.

Infective Endocarditis: Predictive Factors for Diagnosis and Mortality in Surgically Treated Patients.

Background: Diagnosis of infective endocarditis (IE) often is challenging, and mortality is high in such patients. Our goal was to characterize common diagnostic tools to enable a rapid and accurate diagnosis and to correlate these tools with mortality outcomes. Methods: Because of the possibility of including perioperative diagnostics, only surgically treated patients with suspected left-sided IE were included in this retrospective, monocentric study. A clinical committee confirmed the diagnosis of IE. Results: 201 consecutive patients (age 64 ± 13 years, 74% male) were finally diagnosed with IE, and 14 patients turned out IE-negative. Preoperative tests with the highest sensitivity for IE were positive blood cultures (89.0%) and transesophageal echocardiography (87.5%). In receiver operating characteristics, vegetation size revealed high predictive power for IE (AUC 0.800, p < 0.001) with an optimal cut-off value of 11.5 mm. Systemic embolism was associated with mortality, and N-terminal prohormone of B-type natriuretic peptide (NT-proBNP) had predictive power for mortality. Conclusion: If diagnostic standard tools remain inconclusive, we suggest employing novel cut-off values to increase diagnostic accuracy and accelerate diagnosis. Patients with embolism or elevated NT-proBNP deserve a closer follow-up.

Downregulation of the vitamin D receptor expression during acute gastrointestinal graft versus host disease is associated with poor outcome after allogeneic stem cell transplantation.

The vitamin D receptor (VDR) is critical in regulating intestinal homeostasis and emerging evidence demonstrates that VDR deficiency is a critical factor in inflammatory bowel disease pathology. However, no clinical data exist regarding the intestinal expression of VDR in patients after allogeneic haematopoietic stem cell transplantation (HSCT). Analyzing intestinal biopsies from 90 patients undergoing HSCT with mortality follow-up, we demonstrated that patients with severe acute gastrointestinal graft versus host disease (GI-GvHD) showed significant downregulation of VDR gene expression compared to mild or no acute GI-GvHD patients (p = 0.007). Reduced VDR expression was already detectable at acute GI-GvHD onset compared to GvHD-free patients (p = 0.01). These results were confirmed by immunohistochemistry (IHC) where patients with severe acute GI-GvHD showed fewer VDR+ cells (p = 0.03) and a reduced VDR staining score (p = 0.02) as compared to mild or no acute GI-GvHD patients. Accordingly, low VDR gene expression was associated with a higher cumulative incidence of treatment-related mortality (TRM) (p = 1.6x10-6) but not with relapse-related mortality (RRM). A multivariate Cox regression analysis identified low VDR as an independent risk factor for TRM (p = 0.001, hazard ratio 4.14, 95% CI 1.78-9.63). Furthermore, VDR gene expression significantly correlated with anti-microbial peptides (AMPs) gene expression (DEFA5: r = 0.637, p = 7x10-5, DEFA6: r 0 0.546, p = 0.001). In conclusion, our findings suggest an essential role of the VDR in the pathogenesis of gut GvHD and the prognosis of patients undergoing HSCT.

Contribution of Symptomatic, Herbal Treatment Options to Antibiotic Stewardship and Microbiotic Health.

Epithelial surfaces in humans are home to symbiotic microbes (i.e., microbiota) that influence the defensive function against pathogens, depending on the health of the microbiota. Healthy microbiota contribute to the well-being of their host, in general (e.g., via the gut-brain axis), and their respective anatomical site, in particular (e.g., oral, urogenital, skin, or respiratory microbiota). Despite efforts towards a more responsible use of antibiotics, they are often prescribed for uncomplicated, self-limiting infections and can have a substantial negative impact on the gut microbiota. Treatment alternatives, such as non-steroidal anti-inflammatory drugs, may also influence the microbiota; thus, they can have lasting adverse effects. Herbal drugs offer a generally safe treatment option for uncomplicated infections of the urinary or respiratory tract. Additionally, their microbiota preserving properties allow for a more appropriate therapy of uncomplicated infections, without contributing to an increase in antibiotic resistance or disturbing the gut microbiota. Here, herbal treatments may be a more appropriate therapy, with a generally favorable safety profile.

Targeted escape of SARS-CoV-2 in vitro from monoclonal antibody S309, the precursor of sotrovimab.

Class 1 and 2 monoclonal antibodies inhibit SARS-CoV-2 entry by blocking the interaction of the viral receptor-binding domain with angiotensin-converting enzyme 2 (ACE2), while class 3 antibodies target a highly conserved epitope outside the ACE2 binding site. We aimed to investigate the plasticity of the spike protein by propagating wild-type SARS-CoV-2 in the presence of class 3 antibody S309. After 12 weeks, we obtained a viral strain that was completely resistant to inhibition by S309, due to successively evolving amino acid exchanges R346S and P337L located in the paratope of S309. The antibody lost affinity to receptor-binding domains carrying P337L or both amino acid exchanges, while ACE2 binding was not affected. The resistant strain replicated efficiently in human CaCo-2 cells and was more susceptible to inhibition of fusion than the original strain. Overall, SARS-CoV-2 escaped inhibition by class 3 antibody S309 through a slow, but targeted evolution enabling immune escape and altering cell entry. This immune-driven enhancement of infectivity and pathogenicity could play an important role in the future evolution of SARS-CoV-2, which is under increasing immunological pressure from vaccination and previous infections.

Ecological Effects of Daily Antiseptic Treatment on Microbial Composition of Saliva-Grown Microcosm Biofilms and Selection of Resistant Phenotypes.

Antiseptics are widely used in dental practice and included in numerous over-the-counter oral care products. However, the effects of routine antiseptic use on microbial composition of oral biofilms and on the emergence of resistant phenotypes remain unclear. Microcosm biofilms were inoculated from saliva samples of four donors and cultured in the Amsterdam Active Attachment biofilm model for 3 days. Then, they were treated two times daily with chlorhexidine digluconate (CHX) or cetylpyridinium chloride (CPC) for a period of 7 days. Ecological changes upon these multiple antiseptic treatments were evaluated by semiconductor-based sequencing of bacterial 16S rRNA genes and identification of amplicon sequence variants (ASVs). Furthermore, culture-based approaches were used for colony-forming units (CFU) assay, identification of antiseptic-resistant phenotypes using an agar dilution method, and evaluation of their antibiotic susceptibilities. Both CHX and CPC showed only slight effects on CFU and could not inhibit biofilm growth despite the two times daily treatment for 7 days. Both antiseptics showed significant ecological effects on the microbial compositions of the surviving microbiota, whereby CHX led to enrichment of rather caries-associated saccharolytic taxa and CPC led to enrichment of rather gingivitis-associated proteolytic taxa. Antiseptic-resistant phenotypes were isolated on antiseptic-containing agar plates, which also exhibited phenotypic resistance to various antibiotics. Our results highlight the need for further research into potential detrimental effects of antiseptics on the microbial composition of oral biofilms and on the spread of antimicrobial resistance in the context of their frequent use in oral healthcare.

Low Intestinal IL22 Associates With Increased Transplant-Related Mortality After Allogeneic Stem Cell Transplantation.

The role of IL-22 in adult patients undergoing allogeneic stem cell transplantation (SCT) is of major interest since animal studies showed a protective and regenerative effect of IL-22 in graft versus host disease (GvHD). However, no clinical data exist on the tissue expression. Here we demonstrate that patients not suffering from transplant-related mortality (TRM) show significantly upregulated IL22 expression during histological and clinical GI-GvHD (p = 0.048 and p = 0.022, respectively). In contrast, in GvHD patients suffering from TRM, IL22 was significantly lower (p = 0.007). Accordingly, lower IL22 was associated with a higher probability of TRM in survival analysis (p = 0.005). In a multivariable competing risk Cox regression analysis, low IL22 was identified as an independent risk factor for TRM (p = 0.007, hazard ratio 2.72, 95% CI 1.32 to 5.61). The expression of IL22 seemed to be microbiota dependent as broad-spectrum antibiotics significantly diminished IL22 expression (p = 0.019). Furthermore, IL22 expression significantly correlated with G-protein coupled receptor (GPR)43 (r = 0.263, p = 0.015) and GPR41 expression (r = 0.284, p = 0.009). In conclusion, our findings reveal an essential role of IL-22 for the prognosis of patients undergoing allogeneic SCT.

The Pre-Analytical CEN/TS Standard for Microbiome Diagnostics-How Can Research and Development Benefit?

Recently, CEN/TS 17626:2021, the European pre-analytical standard for human specimens intended for microbiome DNA analysis, was published. Although this standard relates to diagnostic procedures for microbiome analysis and is relevant for in vitro diagnostic (IVD) manufacturers and diagnostic laboratories, it also has implications for research and development (R&D). We present here why standards are needed in biomedical research, what pre-analytical standards can accomplish, and which elements of the pre-analytical workflow they cover. The benefits of standardization for the generation of FAIR (findable, accessible, interoperable, reusable) data and to support innovation are briefly discussed.

Microbiota-dependent activation of the myeloid calcineurin-NFAT pathway inhibits B7H3- and B7H4-dependent anti-tumor immunity in colorectal cancer.

Bacterial sensing by intestinal tumor cells contributes to tumor growth through cell-intrinsic activation of the calcineurin-NFAT axis, but the role of this pathway in other intestinal cells remains unclear. Here, we found that myeloid-specific deletion of calcineurin in mice activated protective CD8+ T cell responses and inhibited colorectal cancer (CRC) growth. Microbial sensing by myeloid cells promoted calcineurin- and NFAT-dependent interleukin 6 (IL-6) release, expression of the co-inhibitory molecules B7H3 and B7H4 by tumor cells, and inhibition of CD8+ T cell-dependent anti-tumor immunity. Accordingly, targeting members of this pathway activated protective CD8+ T cell responses and inhibited primary and metastatic CRC growth. B7H3 and B7H4 were expressed by the majority of human primary CRCs and metastases, which was associated with low numbers of tumor-infiltrating CD8+ T cells and poor survival. Therefore, a microbiota-, calcineurin-, and B7H3/B7H4-dependent pathway controls anti-tumor immunity, revealing additional targets for immune checkpoint inhibition in microsatellite-stable CRC.