Investigating B cell central tolerance to a model commensal antigen

How the immune system establishes homeostasis with commensals is an area of intense study that is directly related to human health. B cells must balance the need for a diverse B cell receptor (BCR) repertoire while maintaining self-tolerance. Tolerance is enforced through multiple checkpoints, including deletion, receptor editing, and anergy. Importantly, anergized cells may become activated under specific circumstances, suggesting that these cells may help balance the need for robust pathogen reactivity with self-tolerance. Commensal microbes provide a complex challenge to this paradigm, as they harbor foreign antigens and yet must be contained without triggering overwhelming inflammation. Their persistence raises the question of how such antigens are distinguished from self. If, or how, microbial antigens could induce tolerance in host immunity is unknown. This proposal will test a new model in which a ubiquitous microbial antigen apparently functions as a tolerizing self-antigen for developing host B cells. To create the molecular and cellular tools to address these questions in a mechanistic and cause and effect fashion, we have generated three BCR-knock-in mice expressing LPS-specific BCR’s of either low- or high-affinity heavy chains (33HGL and 33Hhigh), or a high-affinity light chain (33Lhigh). These BCRs were derived from an expanded B cell clone that was responding to infection with Salmonella. B cells carrying these LPS-specific BCRs show clear evidence of clonal deletion and anergy. Intriguingly, lowering the BCR affinity for LPS reduces the stringency of negative selection. Critically, when bone marrow from SPF mice was transferred into lethally irradiated germ-free (GF) mice, we observed substantially reduced clonal deletion at each developmental stage. The marked reduction of self-tolerance in the absence of microbiota strongly suggests that these BCR knock-in B cells are tolerized to a microbial product, presumably LPS. Here we propose to formally prove and define the nature of this commensal-driven tolerance. In Aim 1 we will transfer GF bone marrow chimeras back to SPF conditions to define contexts in which tolerance is perturbed. In Aim 2 we will isolate specific commensal bacteria that react with the 33HL BCR, further defining tolerance mechanisms and creating tools for future study. Preliminary experiments also show that tolerized B cells indeed differentiate to antibody secreting cells in response to Salmonella infection. Thus, in the third Aim, we will define the nature and longevity of this B cell response, and determine whether they provide protection from challenge with a pathogenic strain of Salmonella. This will assess the functional consequences of tolerance to commensals on their capacity to respond to a cross-reactive infection. Developing and understanding this novel system, in which we track a single pathogen-specific B cell clonotype, to determine how it may be both tolerized by “commensal self” as well as provide protection during infection will provide new insights into how microbiota influences the B cell compartment. This in turn has implications for understanding intestinal inflammatory conditions, dysbiosis, and intestinal barrier breach such as with chemotherapy or infection. Project Number: 1R21AI185145-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: REBECCA ELSNER (+1 co-PI) | Institution: UNIVERSITY OF PITTSBURGH AT PITTSBURGH, PITTSBURGH, PA | Award Amount: $186,486 | Activity Code: R21 | Study Section: Adaptive Immunity Study Section[AI] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R21AI18514501A1