Lalit. K. Beura

Laboratory-raised specific pathogen-free (SPF) mice have been indispensable for fundamental immunology research, yet their reliability in predicting human clinical outcomes has been questionable. A major factor contributing to this disconnect is the sanitized housing environment, which deprives laboratory mice of physiological microbial exposure critical for immune maturation. Various approaches have been developed to introduce microbes to SPF mice, aiming to mimic human-like microbial experiences and engender adult human-like immune traits. However, some of these methods, specifically the pet store mice cohousing approach suffer from significant variability in pathogen exposure driven by the uncontrolled nature of microbial exchange and is associated with heightened mortality. Here we present an alternative gavage-fomite (GaF) method that exposes mice to a similarly diverse array of pathogens and commensal as the pet store cohousing method while limiting mortality. GaF-treated mice exhibited consistent gut microbial composition, robust immune maturation characterized by mucosal T cell distribution, elevated serum inflammatory cytokines, and a splenic immune transcriptional signature closely aligned with that of adult humans. Furthermore, these mice demonstrated enhanced protection against a virulent bacterial challenge. The simplicity and effectiveness of the GaF method for generating “mice with natural microbiota,” may support broader use of these models in basic and translational immunological research across institutions.

Barrier mucosal tissues play an important role in the differentiation and function of resident T cells. Among the diverse cell populations within these tissues, epithelial cells are key drivers of T cell differentiation. However, our understanding of the epithelial-derived signals that shape T cell biology in mucosal environments remains limited, largely due to a lack of specific tools and the complexity of available model systems. Here, we describe a murine vaginal epithelial organoid-CD8 T cell co-culture system that models dynamic interactions between infected epithelium and virus-specific CD8 T cells. This protocol enables phenotypic and functional analysis of T cell responses within a physiologically relevant three-dimensional epithelial microenvironment. Using this system, we demonstrate the differentiation of effector CD8 T cells into tissue-resident memory T cells (TRM). This versatile in vitro culture system provides reductionist opportunities to investigate molecular details of epithelial cues behind CD8 T cell differentiation that govern protective immunity and immunopathology in barrier organs.

Barrier mucosal tissues play an important role in the differentiation and function of resident T cells. Among the diverse cell populations within these tissues, epithelial cells are key drivers of T cell differentiation. However, our understanding of the epithelial-derived signals that shape T cell biology in mucosal environments remains limited, largely due to a lack of specific tools and the complexity of available model systems. Here, we describe a murine vaginal epithelial organoid-CD8 T cell co-culture system that models dynamic interactions between infected epithelium and virus-specific CD8 T cells. This protocol enables phenotypic and functional analysis of T cell responses within a physiologically relevant three-dimensional epithelial microenvironment. Using this system, we demonstrate the differentiation of effector CD8 T cells into tissue-resident memory T cells (TRM). This versatile in vitro culture system provides reductionist opportunities to investigate molecular details of epithelial cues behind CD8 T cell differentiation that govern protective immunity and immunopathology in barrier organs.

Resident Memory T cells (TRM) play a vital role in regional immune defense in barrier organs. Although laboratory rodents have been extensively used to study fundamental TRM biology, poor isolation efficiency, sampling bias and low cell survival rates have limited our ability to conduct TRM-focused high-throughput assays. Here, we engineered a murine vaginal epithelial organoid (VEO)-CD8 T cell co-culture system that supports CD8 TRM differentiation in vitro . The three-dimensional VEOs established from murine adult stem cells resembled stratified squamous vaginal epithelium and induced gradual differentiation of activated CD8 T cells into epithelial TRM. These in vitro generated TRM were phenotypically and transcriptionally similar to in vivo TRM, and key tissue residency features were reinforced with a second cognate-antigen exposure during co-culture. TRM differentiation was not affected even when VEOs and CD8 T cells were separated by a semipermeable barrier, indicating soluble factors’ involvement. Pharmacological and genetic approaches showed that TGF-β signaling played a crucial role in their differentiation. We found that the VEOs in our model remained susceptible to viral infections and the CD8 T cells were amenable to genetic manipulation; both of which will allow detailed interrogation of antiviral CD8 T cell biology in a reductionist setting. In summary, we established a robust model which captures bonafide TRM differentiation that is scalable, open to iterative sampling, and can be subjected to high throughput assays that will rapidly add to our understanding of TRM.

Lymphocytic choriomeningitis virus (LCMV) is the prototypic arenavirus and a natural mouse pathogen. LCMV-Armstrong, an acutely resolved strain, and LCMV-clone 13, a mutant that establishes chronic infection, have provided contrasting infection models that continue to inform the fundamental biology of T cell differentiation, regulation of exhaustion, and response to checkpoint blockade. In this study, we report the isolation and characterization of LCMV-Minnesota (LCMV-MN), which was naturally transmitted to laboratory mice upon cohousing with pet shop mice and shares 80–95% amino acid homology with previously characterized LCMV strains. Infection of laboratory mice with purified LCMV-MN resulted in viral persistence that was intermediate between LCMV-Armstrong and -clone 13, with widely disseminated viral replication and viremia that was controlled within 15–30 d, unless CD4 T cells were depleted prior to infection. LCMV-MN–responding CD8+ T cells biased differentiation toward the recently described programmed death-1 (PD-1)+CXCR5+Tim-3lo stemlike CD8+ T cell population (also referred to as progenitor exhausted T cells) that effectuates responses to PD-1 blockade checkpoint inhibition, a therapy that rejuvenates responses against chronic infections and cancer. This subset resembled previously characterized PD-1+TCF1+ stemlike CD8+ T cells by transcriptional, phenotypic, and functional assays, yet was atypically abundant. LCMV-MN may provide a tool to better understand the breadth of immune responses in different settings of chronic Ag stimulation as well as the ontogeny of progenitor exhausted T cells and the regulation of responsiveness to PD-1 blockade.