Combining gnotobioic, immunological and cytometric tools to understand T cell responses

“Non-pathogenic bacteria from the gut microbiota induce antigen-specific Th cell responses that are crucial for maintaining intes4nal health. The technical inability of uncoupling T cell induction from persistence of the microbe has precluded insights about the dynamics of such responses including their dose requirements and the formation of long-lived T cell memory.
To address this, we have genetically engineered a mutant strain of the prototypic gut commensal Limosilactobacillus reuteri to reversibly colonise germ-free mice. This transient intestinal exposure induces L. reuteri-specific Th17 cells that can be followed beyond bacterial clearance. Specific T cell receptors have been identified through scRNA-seq of sorted T cells and their cognate antigen through multiplexed in vitro library screenings. L. reuteri-specific T cells can thus be traced by adop4ve transfer of TCR-transgenic T cells or by peptide:MHC- mediated labelling of endogenous T cells.
This highly defined and controllable system has revealed new insights into the physiology of microbiota-specific T cells: (1) The T cell response is regulated by antigen dose in a non-linear fashion with different thresholds for T cell expansion, homing, and differentiation, respectively. (2) Microbiota-specific T cells form long-lived, antigen-independent, tissue- resident memory that persists for many weeks after bacterial clearance in the intestine but largely disappears from the circula4on. (3) Intestinal antigen challenge reactivates microbiota- specific memory T cells in situ to orchestrate a barrier protection response.
Overall, transient colonization of germ-free mice together with antigen-specific T cell reagents has revealed the regulation and function of memory T cells specific to the nonpathogenic gut microbiota. The tools developed herein are applicable to a wide range of microbes and T cell responses and may facilitate a beSer understanding of dysregulated immune responses in inflammatory bowel disease.”