Dennis L. Kasper’s research while affiliated with Harvard Medical School and other places

Therapeutic efficacy with durable responses has been demonstrated with several antibody drugs that block key immune checkpoint receptors, including PD-1, PD-L1, and CTLA-4. Despite the success of these drugs, a substantial proportion of patients do not benefit. Targeting multiple inhibitory pathways simultaneously to augment anti-tumor immunity has proven to be a promising approach. The emergence of Repulsive Guidance Molecule b (RGMb), a ligand for PD-L2, as a novel co-inhibitory pathway in T cells, together with its regulation by the gut microbiome, encouraged the discovery and development of fully human anti-RGMb antibodies. Here, we describe phage display-derived monoclonal antibodies (mAbs) 2C11 and 5C10 that bind human RGMb with high affinities of 1.4 nM and 0.72 nM, respectively. Both mAbs 2C11 and 5C10 potently inhibited RGMb interaction with PD-L2. MAb 2C11 effectively inhibited RGMb interaction with bone morphogenetic proteins 2 and 4 (BMP2–4), while leaving RGMb interaction with Neogenin 1 (Neo1) unaffected. Conversely, mAb 5C10 disrupted RGMb interaction with Neo1 while maintaining RGMb binding to BMP2–4. These findings map the 2C11 epitope at the membrane-distal N-terminal region of RGMb, which coincides with both PD-L2- and BMP2–4-binding sites. The PD-L2 binding interface is likely positioned between RGMb’s N-terminal BMP-binding and C-terminal Neo1-binding regions. The in vivo activity of mAb 2C11 in combination with anti-PD-1 or anti-PD-L1 was tested in MC38 and B16-OVA cancer models and demonstrated synergistic effects by significantly enhancing anti-tumor responses. These properties make mAb 2C11 a promising candidate for therapeutic use to overcome immune checkpoint inhibitor resistances, warranting further exploration in clinical settings.

N-acyl lipids are important mediators of several biological processes including immune function and stress response. To enhance the detection of N-acyl lipids with untargeted mass spectrometry-based metabolomics, we created a reference spectral library retrieving N-acyl lipid patterns from 2,700 public datasets, identifying 851 N-acyl lipids that were detected 356,542 times. 777 are not documented in lipid structural databases, with 18% of these derived from short-chain fatty acids and found in the digestive tract and other organs. Their levels varied with diet, microbial colonization, and in people living with diabetes. We used the library to link microbial N-acyl lipids, including histamine and polyamine conjugates, to HIV status and cognitive impairment. This resource will enhance the annotation of these compounds in future studies to further the understanding of their roles in health and disease and highlight the value of large-scale untargeted metabolomics data for metabolite discovery.

Background & Aims Gut bacterial sphingolipids, primarily produced by Bacteroidetes, have dual roles as bacterial virulence factors and regulators of the host mucosal immune system, including regulatory T cells and invariant natural killer T cells. Patients with inflammatory bowel disease display altered sphingolipids profiles in fecal samples. However, how bacterial sphingolipids modulate mucosal homeostasis and regulate intestinal inflammation remains unclear. Methods We used dextran sodium sulfate (DSS)-induced colitis in mice monocolonized with Bacteroides fragilis strains expressing or lacking sphingolipids to assess the influence of bacterial sphingolipids on intestinal inflammation using transcriptional, protein, and cellular analyses. Colonic explant and organoid were used to study the function of bacterial sphingolipids. Host mucosal immune cells and cytokines were profiled and characterized using flow cytometry, enzyme-linked immunosorbent assay, and Western blot, and cytokine function in vivo was investigated by monoclonal antibody injection. Results B fragilis sphingolipids exacerbated intestinal inflammation. Mice monocolonized with B fragilis lacking sphingolipids exhibited less severe DSS-induced colitis. This amelioration of colitis was associated with increased production of interleukin (IL)-22 by ILC3. Mice colonized with B fragilis lacking sphingolipids following DSS treatment showed enhanced epithelial STAT3 activity, intestinal cell proliferation, and antimicrobial peptide production. Protection against DSS colitis associated with B fragilis lacking sphingolipids was reversed on IL22 blockade. Furthermore, bacterial sphingolipids restricted epithelial IL18 production following DSS treatment and interfered with IL22 production by a subset of ILC3 cells expressing both IL18R and major histocompatibility complex class II. Conclusions B fragilis–derived sphingolipids exacerbate mucosal inflammation by impeding epithelial IL18 expression and concomitantly suppressing the production of IL22 by ILC3 cells.

Lipopolysaccharides (LPS) and lipooligosaccharides (LOS) are ubiquitous structures found on the outer membrane of gram-negative bacteria. Bacteroides fragilis , is a gram-negative anaerobe commonly inhabiting the human colon. The LOS of this organism is known to trigger a type I interferon response in dendritic cells. However, detailed structural analysis of this LOS has been largely elusive. Using top-down mass spectrometry, we have unraveled the comprehensive fine structure of B. fragilis LOS. Our analysis reveals that this LOS has a poly-galactose-rhamnose-KDO-lipid A architecture, which can be modified by hexuronic acid and ethanolamine via phosphodiester linkages. The lipid moiety typically includes three to five acyl chains of varying length on a glucosamine disaccharide. This investigation lays the groundwork for deeper immunological exploration of B. fragilis LOS and underscores the efficacy of top-down mass spectrometry in characterizing intact LOS/LPS structures and their modifications.

Commensal bacteria of the Bacteroidetes phylum are the primary producers of sphingolipids in the gut lumen. These lipids serve dual roles as bacterial virulence factors and regulators of the host mucosal immune system, including regulatory T cells and invariant natural killer T cells (iNKT). Sphingolipid composition is significantly altered in fecal samples of patients with inflammatory bowel disease (IBD). However, the specific mechanisms by which bacterial sphingolipids modulate mucosal homeostasis and regulate intestinal inflammation remain unclear. In this study, we investigated the impact of bacterial sphingolipids on intestinal inflammation by mono-colonizing mice with Bacteroides fragilis strains that either express or lack sphingolipids during DSS-induced colitis. We discovered that B. fragilis sphingolipids exacerbate intestinal inflammation. Mice mono-colonized with B. fragilis lacking sphingolipids exhibited less severe DSS-induced colitis. This amelioration of colitis was associated with increased production of interleukin-22 (IL-22) by innate lymphoid cell type 3 (ILC3). Consistent with the inhibitory effect of sphingolipids on IL-22 production, mice colonized with B. fragilis lacking sphingolipids showed enhanced epithelial STAT3 activity, intestinal cell proliferation, and antimicrobial peptide production following DSS treatment compared to those colonized with B. fragilis producing sphingolipids. Additionally, colitis severity in mice colonized with B. fragilis lacking sphingolipids was exacerbated upon IL-22 blockade. Furthermore, our study reveals that bacterial sphingolipids restrict epithelial IL-18 production following DSS treatment and interfere with IL-22 production by a subset of ILC3 cells expressing both the interleukin-18 receptor (IL-18R) and major histocompatibility complex class II (MHC II). These findings indicate that B. fragilis-derived sphingolipids exacerbate mucosal inflammation by impeding epithelial IL-18 expression, resulting in compromised production of IL-22 by ILC3 cells. Work Model

Our bodies are inhabited by trillions of microorganisms. The host immune system constantly interacts with the microbiota in barrier organs, including the intestines. Over decades, numerous studies have shown that our mucosal immune system is dynamically shaped by a variety of microbiota-derived signals. Elucidating the mediators of these interactions is an important step for understanding how the microbiota is linked to mucosal immune homeostasis and gut-associated diseases. Interestingly, the efficacy of cancer immunotherapies that manipulate costimulatory and coinhibitory pathways has been correlated with the gut microbiota. Moreover, adverse effects of these therapies in the gut are linked to dysregulation of the intestinal immune system. These findings suggest that costimulatory pathways in the immune system might serve as a bridge between the host immune system and the gut microbiota. Here, we review mechanisms by which commensal microorganisms signal immune cells and their potential impact on costimulation. We highlight how costimulatory pathways modulate the mucosal immune system through not only classical antigen-presenting cells but also innate lymphocytes, which are highly enriched in barrier organs. Finally, we discuss the adverse effects of immune checkpoint inhibitors in the gut and the possible relationship with the gut microbiota.

Commensal bacteria of the Bacteroidetes phylum are the primary producers of sphingolipids in the gut lumen. These lipids serve dual roles as bacterial virulence factors and regulators of the host mucosal immune system, including regulatory T cells and invariant natural killer T cells (iNKT). Sphingolipid composition is significantly altered in fecal samples of patients with inflammatory bowel disease (IBD). However, the specific mechanisms by which bacterial sphingolipids modulate mucosal homeostasis and regulate intestinal inflammation remain unclear. In this study, we investigated the impact of bacterial sphingolipids on intestinal inflammation by mono-colonizing mice with Bacteroides fragilis strains that either express or lack sphingolipids during DSS-induced colitis. We discovered that B. fragilis sphingolipids exacerbate intestinal inflammation. Mice mono-colonized with B. fragilis lacking sphingolipids exhibited less severe DSS-induced colitis. This amelioration of colitis was associated with increased production of interleukin-22 (IL-22) by innate lymphoid cell type 3 (ILC3). Consistent with the inhibitory effect of sphingolipids on IL-22 production, mice colonized with B. fragilis lacking sphingolipids showed enhanced epithelial STAT3 activity, intestinal cell proliferation, and antimicrobial peptide production following DSS treatment compared to those colonized with B. fragilis producing sphingolipids. Additionally, colitis severity in mice colonized with B. fragilis lacking sphingolipids was exacerbated upon IL-22 blockade. Furthermore, our study reveals that bacterial sphingolipids restrict epithelial IL-18 production following DSS treatment and interfere with IL-22 production by a subset of ILC3 cells expressing both the interleukin-18 receptor (IL-18R) and major histocompatibility complex class II (MHC II). These findings indicate that B. fragilis -derived sphingolipids exacerbate mucosal inflammation by impeding epithelial IL-18 expression, resulting in compromised production of IL-22 by ILC3 cells. Highlights B. fragilis -derived sphingolipids exacerbate DSS-induced colitis in mono-colonized C57BL/6 mice. B. fragilis -derived sphingolipids constrain ILC3-derived IL-22, leading to reduced colonic epithelial cell proliferation and compromised barrier function. B. fragilis -derived sphingolipids restrict epithelial NLRC4 inflammasome activation and IL-18 secretion. B. fragilis -derived sphingolipids modulate IL-22 production by IL18R ⁺ MHC II ⁺ ILC3s.