in-vitro-resident-memory-cd8-t-cell-differentiation-using-epithelial

<em>In Vitro</em> Resident Memory CD8 T Cell Differentiation Using Epithelial Organoid-T Cell Co-culture System

Barrier mucosal tissues play an important role in the differentiation and function of resident T cells. Among the diverse cell populations within these ...

Brown University

Central memory T (T) cells patrol lymph nodes and perform conventional memory responses on restimulation: proliferation, migration and differentiation into diverse T cell subsets while also self-renewing. Resident memory T (T) cells are parked within single organs, share properties with terminal effectors and contribute to rapid host protection. We observed that reactivated T cells rejoined the circulating pool. Epigenetic analyses revealed that T cells align closely with conventional memory T cell populations, bearing little resemblance to recently activated effectors. Fully differentiated T cells isolated from small intestine epithelium exhibited the potential to differentiate into T cells, effector memory T cells and T cells on recall. Ex-T cells, former intestinal T cells that rejoined the circulating pool, heritably maintained a predilection for homing back to their tissue of origin on subsequent reactivation and a heightened capacity to redifferentiate into T cells. Thus, T cells can rejoin the circulation but are advantaged to re-form local T when called on.

Developmental plasticity allows outside-in immune responses by resident memory T cells.

Numerous observations indicate that resident memory T cells (TRM) undergo unusually rapid attrition within the lung. Here we demonstrate that contraction of lung CD8+ T cell responses after influenza infection is contemporized with egress of CD69+/CD103+ CD8+ T cells to the draining mediastinal LN via the lymphatic vessels, which we term retrograde migration. Cells within the draining LN retained canonical markers of lung TRM, including CD103 and CD69, lacked Ly6C expression (also a feature of lung TRM), maintained granzyme B expression, and did not equilibrate among immunized parabiotic mice. Investigations of bystander infection or removal of the TCR from established memory cells revealed that the induction of the TRM phenotype was dependent on antigen recognition; however, maintenance was independent. Thus, local lung infection induces CD8+ T cells with a TRM phenotype that nevertheless undergo retrograde migration, yet remain durably committed to the residency program within the draining LN, where they provide longer-lived regional memory while chronicling previous upstream antigen experiences.

Retrograde migration supplies resident memory T cells to lung-draining LN after influenza infection.

The mouse () is the dominant organism used to investigate the mechanisms behind complex immunological responses because of their genetic similarity to humans and our ability to manipulate those genetics to understand downstream function. Indeed, our knowledge of immune system development, response to infection, and ways to therapeutically manipulate the immune response to combat disease were, in large part, delineated in the mouse. Despite the power of mouse-based immunology research, the translational efficacy of many new therapies from mouse to human is far from ideal. Recent data have highlighted how the naive, neonate-like immune system of specific pathogen-free mice differs dramatically in composition and function to mice living under barrier-free conditions (i.e., "dirty" mice). In this review, we discuss major findings to date and challenges faced when using dirty mice and specific areas of immunology research that may benefit from using animals with robust and varied microbial exposure.

New Insights into the Immune System Using Dirty Mice.

In metazoans, specific tasks are relegated to dedicated organs that are established early in development, occupy discrete locations and typically remain fixed in size. The adult immune system arises from a centralized haematopoietic niche that maintains self-renewing potential, and-upon maturation-becomes distributed throughout the body to monitor environmental perturbations, regulate tissue homeostasis and mediate organism-wide defence. Here we examine how immunity is integrated within adult mouse tissues, and address issues of durability, expansibility and contributions to organ cellularity. Focusing on antiviral T cell immunity, we observed durable maintenance of resident memory T cells up to 450 days after infection. Once established, resident T cells did not require the T cell receptor for survival or retention of a poised, effector-like state. Although resident memory indefinitely dominated most mucosal organs, surgical separation of parabiotic mice revealed a tissue-resident provenance for blood-borne effector memory T cells, and circulating memory slowly made substantial contributions to tissue immunity in some organs. After serial immunizations or cohousing with pet-shop mice, we found that in most tissues, tissue pliancy (the capacity of tissues to vary their proportion of immune cells) enables the accretion of tissue-resident memory, without axiomatic erosion of pre-existing antiviral T cell immunity. Extending these findings, we demonstrate that tissue residence and organ pliancy are generalizable aspects that underlie homeostasis of innate and adaptive immunity. The immune system grows commensurate with microbial experience, reaching up to 25% of visceral organ cellularity. Regardless of the location, many populations of white blood cells adopted a tissue-residency program within nonlymphoid organs. Thus, residence-rather than renewal or recirculation-typifies nonlymphoid immune surveillance, and organs serve as pliant storage reservoirs that can accommodate continuous expansion of the cellular immune system throughout life. Although haematopoiesis restores some elements of the immune system, nonlymphoid organs sustain an accrual of durable tissue-autonomous cellular immunity that results in progressive decentralization of organismal immune homeostasis.

Expansible residence decentralizes immune homeostasis.

Memory CD8+ T cells populate non-lymphoid tissues (NLTs) following pathogen infection, but little is known about the establishment of endogenous tumor-specific tissue-resident memory T cells (T) during cancer immunotherapy. Using a transplantable mouse model of prostate carcinoma, here we report that tumor challenge leads to expansion of naïve neoantigen-specific CD8+ T cells and formation of a small population of non-recirculating T in several NLTs. Primary tumor destruction by irreversible electroporation (IRE), followed by anti-CTLA-4 immune checkpoint inhibitor (ICI), promotes robust expansion of tumor-specific CD8+ T cells in blood, tumor, and NLTs. Parabiosis studies confirm that T establishment following dual therapy is associated with tumor remission in a subset of cases and protection from subsequent tumor challenge. Addition of anti-PD-1 following dual IRE + anti-CTLA-4 treatment blocks tumor growth in non-responsive cases. This work indicates that focal tumor destruction using IRE combined with ICI is a potent in situ tumor vaccination strategy that generates protective tumor-specific T.

Irreversible electroporation augments checkpoint immunotherapy in prostate cancer and promotes tumor antigen-specific tissue-resident memory CD8+ T cells.

Tissue resident memory T cells (T) are enriched in non-lymphoid tissues and represent a formidable barrier against invading pathogens and tumors. T are armed with deployment ready effector molecules which combined with their frontline location allows them to be early organizing centers of our immune defense. Despite their autonomous nature, T rely on careful collaboration with other immune and non-immune cells located within the barrier organ to exert their superior protective role. Here, we highlight recent studies focusing on cellular interactions that regulate T establishment and function. A deeper understanding of these processes is instrumental in designing new means to target T for desirable outcomes in infectious diseases, cancers and autoimmunity.

Cellular interactions in resident memory T cell establishment and function.

The coronavirus disease 2019 (COVID-19) pandemic has created unprecedented challenges worldwide. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19 and has a complex interaction with the immune system, including growing evidence of sex-specific differences in the immune response. Sex-disaggregated analyses of epidemiological data indicate that males experience more severe symptoms and suffer higher mortality from COVID-19 than females. Many behavioural risk factors and biological factors may contribute to the different immune response. This review examines the immune response to SARS-CoV-2 infection in the context of sex, with emphasis on potential biological mechanisms explaining differences in clinical outcomes. Understanding sex differences in the pathophysiology of SARS-CoV-2 infection will help promote the development of specific strategies to manage the disease.

The immune response to COVID-19: Does sex matter?

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)CXCR5Tim-3 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-1TCF1 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.

Novel Lymphocytic Choriomeningitis Virus Strain Sustains Abundant Exhausted Progenitor CD8 T Cells without Systemic Viremia.

Adaptive immunity is didactically partitioned into humoral and cell-mediated effector mechanisms, which may imply that each arm is separate and does not function together. Here, we report that the activation of CD8+ resident memory T cells (T) in nonlymphoid tissues triggers vascular permeability, which facilitates rapid distribution of serum antibodies into local tissues. T reactivation was associated with transcriptional upregulation of antiviral signaling pathways as well as Fc receptors and components of the complement cascade. Effects were local, but evidence is presented that T in brain and reproductive mucosa are both competent to induce rapid antibody exudation. T reactivation in the mouse female genital tract increased local concentrations of virus-specific neutralizing antibodies, including anti-vesicular stomatitis virus, and passively transferred anti-HIV antibodies. We showed that this response was sufficient to increase the efficacy of ex vivo vesicular stomatitis virus neutralization. These results indicate that CD8+ T antigen recognition can enhance local humoral immunity.

Tissue-resident memory T cells trigger rapid exudation and local antibody accumulation.

Immigration to a highly industrialized nation has been associated with metabolic disease and simultaneous shifts in microbiota composition, but the underlying mechanisms are challenging to test in human studies. Here, we conducted a pilot study to assess the differential effects of human gut microbiota collected from the United States (US) and rural Thailand on the murine gut mucosa and immune system. Colonization of germ-free mice with microbiota from US individuals resulted in an increased accumulation of innate-like CD8 T cells in the small intestine lamina propria and intra-epithelial compartments when compared to colonization with microbiota from Thai individuals. Both TCRγδ and CD8αα T cells showed a marked increase in mice receiving Western microbiota and, interestingly, this phenotype was also associated with an increase in intestinal mucus thickness. Serendipitously, an accidentally infected group of mice corroborated this association between elevated inflammatory response and increased mucus thickness. These results suggest that Western-associated human gut microbes contribute to a pro-inflammatory immune response.

Industrialized human gut microbiota increases CD8+ T cells and mucus thickness in humanized mouse gut.

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 . 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  generated TRM were phenotypically and transcriptionally similar to  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.

Epithelial organoid supports resident memory CD8 T cell differentiation.

Antibiotics cause collateral damage to resident microbes that is associated with various health risks. To date, studies have largely focused on the impacts of antibiotics on large intestinal and fecal microbiota. Here, we employ a gastrointestinal (GI) tract-wide integrated multiomic approach to show that amoxicillin (AMX) treatment reduces bacterial abundance, bile salt hydrolase activity, and unconjugated bile acids in the small intestine (SI). Losses of fatty acids (FAs) and increases in acylcarnitines in the large intestine (LI) correspond with spatially distinct expansions of Proteobacteria. Parasutterella excrementihominis engage in FA biosynthesis in the SI, while multiple Klebsiella species employ FA oxidation during expansion in the LI. We subsequently demonstrate that restoration of unconjugated bile acids can mitigate losses of commensals in the LI while also inhibiting the expansion of Proteobacteria during AMX treatment. These results suggest that the depletion of bile acids and lipids may contribute to AMX-induced dysbiosis in the lower GI tract.

Spatial analysis of murine microbiota and bile acid metabolism during amoxicillin treatment.

Resident memory T cells (TRMs) play a vital role in regional immune defense. Although laboratory rodents have been extensively used to study fundamental TRM biology, poor isolation efficiency and low cell survival rates have limited the implementation of TRM-focused high-throughput assays. Here, we engineer a murine vaginal epithelial organoid (VEO)-CD8 T cell co-culture system that supports CD8 TRM differentiation. These in-vitro-generated TRMs are phenotypically and transcriptionally similar to in vivo TRMs. Pharmacological and genetic approaches showed that transforming growth factor β (TGF-β) signaling plays a crucial role in their differentiation. The VEOs in our model are susceptible to viral infections and the CD8 T cells are amenable to genetic manipulation, both of which will allow a detailed interrogation of antiviral CD8 T cell biology. Altogether we have established a robust in vitro TRM differentiation system that is scalable and can be subjected to high-throughput assays that will rapidly add to our understanding of TRMs.

Epithelial organoid supports resident memory CD8 T cell differentiation.

The optimal vaginal microbiome is a Lactobacillus-dominant community. Apart from Lactobacillus iners, the presence of Lactobacillus species is associated with reduced vaginal inflammation and reduced levels of pro-inflammatory cytokines. Loss of Lactobacillus-dominance is associated with inflammatory conditions, such as bacterial vaginosis (BV). We have identified that Lactobacillus crispatus, a key vaginal bacterial species, produces a family of β-carboline compounds with anti-inflammatory activity. These compounds suppress nuclear factor κB (NF-κB) and interferon (IFN) signaling downstream of multiple pattern recognition receptors in primary human cells and significantly dampen type I IFN receptor (IFNAR) activation in monocytes. Topical application of an anti-inflammatory β-carboline compound, perlolyrine, was sufficient to significantly reduce vaginal inflammation in a mouse model of genital herpes infection. These compounds are enriched in cervicovaginal lavage (CVL) of healthy people compared with people with BV. This study identifies a family of compounds by which vaginal lactobacilli mediate host immune homeostasis and highlights a potential therapeutic avenue for vaginal inflammation.

Vaginal lactobacilli produce anti-inflammatory β-carboline compounds.

Monocyte-derived TGF-β shapes memory CD8+ T cells.

Inducing robust resident memory T cell (TRM) establishment in mucosal tissues could enhance immunotherapy and vaccine efficacy. However, the factors influencing TRM formation are not fully understood. We present a protocol to coculture murine effector CD8 T cells with epithelial organoids, which serve as a reductionist model for investigating TRM differentiation in vitro. We describe steps for naive CD8 T cell activation, coculture of T cells with vaginal epithelial organoids (VEOs), and analysis of T cell phenotypes via flow cytometry. For complete details on the use and execution of this protocol, please refer to Ulibarri et al..

Protocol for the coculture of murine vaginal epithelial organoids and T cells to induce resident memory CD8 T cell differentiation.

Naturalized, wilded, wildling, and dirty/pet store mouse models represent a spectrum of approaches designed to make laboratory mice more immunologically and physiologically similar to wild or human contexts by increasing their exposure to naturally occurring microbes and pathogens. In this study, we screened the gut mycobiome of pet store mice, and identified  as a dominant fungus in pet store mice across various geographical locations.  strains isolated from mice in geographically distinct pet stores stably colonize the gastrointestinal tract of laboratory mice, independent of gut bacterial composition, maintaining high fungal burdens for extended periods.  rapidly became the dominant fungus in the mouse gut in conventional, antibiotic, and germ-free settings, outcompeting other non-murine fungal strains. Pet store-derived  exhibited unique immunological properties distinct from typical anti-fungal responses. Unlike  colonization,  did not induce circulating neutrophil expansion or Th17 cell populations in the gut mucosa. When administered systemically, -infected mice showed 100% survival with minimal fungal burden in kidneys, contrasting sharply with lethal  infections. Adaptive immune deficiency (Rag1 knockout mice) did not affect  colonization or host response, indicating that B and T cell-mediated immunity does not restrain this fungus.  colonization provided no cross-protection against systemic candidiasis further establishing lack of immune activation. These findings demonstrate that  establishes a benign host-fungal relationship through neutrophil-independent mechanisms, avoiding classical anti-fungal immune activation while maintaining stable gut colonization. Instead, it selectively induces strong type 2 mucosal immune responses, increasing tuft and goblet cell counts and stimulating Th2 and group 2 innate lymphoid cell (ILC2) populations. This immune profile confers notable protection against intestinal nematode infection, demonstrated by reduced  egg counts. Altogether,  serves as an exemplary model for commensal mycobiota, revealing distinct mechanisms for host tolerance and immune modulation.

Article	Year
Developmental plasticity allows outside-in immune responses by resident memory T cells. Nature immunology\| PubMed ID: 32066954	2020
Retrograde migration supplies resident memory T cells to lung-draining LN after influenza infection. The Journal of experimental medicine\| PubMed ID: 32568362	2020
New Insights into the Immune System Using Dirty Mice. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 32571979	2020
Expansible residence decentralizes immune homeostasis. Nature\| PubMed ID: 33731934	2021
Irreversible electroporation augments checkpoint immunotherapy in prostate cancer and promotes tumor antigen-specific tissue-resident memory CD8+ T cells. Nature communications\| PubMed ID: 34162858	2021
Cellular interactions in resident memory T cell establishment and function. Current opinion in immunology\| PubMed ID: 34794039	2022
The immune response to COVID-19: Does sex matter? Immunology\| PubMed ID: 35470422	2022
Novel Lymphocytic Choriomeningitis Virus Strain Sustains Abundant Exhausted Progenitor CD8 T Cells without Systemic Viremia. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 36122933	2022
Tissue-resident memory T cells trigger rapid exudation and local antibody accumulation. Mucosal immunology\| PubMed ID: 36657662	2023
Industrialized human gut microbiota increases CD8+ T cells and mucus thickness in humanized mouse gut. Gut microbes\| PubMed ID: 37853762	2023
Epithelial organoid supports resident memory CD8 T cell differentiation. bioRxiv : the preprint server for biology\| PubMed ID: 38076957	2023
Spatial analysis of murine microbiota and bile acid metabolism during amoxicillin treatment. Cell reports\| PubMed ID: 39116202	2024
Epithelial organoid supports resident memory CD8 T cell differentiation. Cell reports\| PubMed ID: 39153200	2024
Vaginal lactobacilli produce anti-inflammatory β-carboline compounds. Cell host & microbe\| PubMed ID: 39423813	2024
Monocyte-derived TGF-β shapes memory CD8+ T cells. Cellular & molecular immunology\| PubMed ID: 40634637	2025
Protocol for the coculture of murine vaginal epithelial organoids and T cells to induce resident memory CD8 T cell differentiation. STAR protocols\| PubMed ID: 40884788	2025
A host-adapted commensal fungus from pet store mice drives type 2 immunity and cross-kingdom protection. bioRxiv : the preprint server for biology\| PubMed ID: 41542591	2026

Lalit K. Beura

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