The immgenT collaborative project generated a comprehensive molecular atlas of T cells spanning virtually all mouse organs and disease states, profiling ~800,000 cells from 750 samples with RNA, 128-plex surface protein, and αβTCR sequence. Applying a deep generative model to joint RNA and protein data defined a finite landscape of T-cell states organized into eight lineages and 110 robust clusters, integrating identical cells from different contexts, and resolving prior nomenclatures. Analysis of effector molecules, transcription factors and modules showed that both immunological functions and regulatory programs are shared across cell states. This framework provides a stable, reusable reference, demonstrated by computationally integrating 16 external datasets from diverse biological contexts. A set of public web tools supports browsing of these data, allows mapping of any dataset onto the immgenT framework. These results propose a molecular classification of T cells organized around a set of shared states reused across immunological contexts. ### Competing Interest Statement The authors have declared no competing interest.

Mice with normalized microbial exposure (NME) harbor an immune system that more accurately reflects that of humans compared to mice maintained as specific pathogen-free (SPF). An explanation for the observed alterations in the composition of the T cell compartment in NME mice has not been reported. We compared the T cell landscape in NME versus SPF mice at baseline and after acute LCMV infection. Using the immgenT dataset, we found no unique T cell populations in NME, but the landscape shifted towards activated T cells with increased propensity for effector functions and improved pathogen clearance. CD8+ KLRG1+ cells (immgenT CD8_cl12) are significantly expanded in NME mice. Their predominance was a result of both increased formation and the conversion of other memory populations to a KLRG1+ phenotype. Thus, NME mice provide insight into a diverse T cell compartment rich with cells previously found to be limited in SPF mice.

Mice with natural microbial exposures (NMEs) have been proposed to be superior to specific pathogen-free (SPF) laboratory mice for modeling adult human immune and inflammatory profiles. Research using NME mice has focused on the effects of sustained exposure on peripheral blood and lymphoid and nonlymphoid organs. The effect of short-term NME is not known, in particular the impact to bone marrow immune composition. Male and female C57Bl/6J mice were exposed to SPF conditions for 6 wk (age 4 to 10 wk) with or without exposure to dirty bedding from pet store mice for the first week. At sacrifice, substantial shifts in the fecal bacteriome, at the phylum, order, and genus levels were evident in male and female NME mice, confirming induction of a new exposome with NME. However, only female NME mice had pathogens in their feces detectable by polymerase chain reaction (2 of 35). Numerous sex-dependent differences in T cell and myeloid composition in bone marrow and spleens were observed. We report several novel immune composition findings by sex, including higher proportions of Ki67+ regulatory and CD8+ T cells in female bone marrow. We detected 2 differences resulting from NME in the spleens of females, including increased PD-1 in CD44+CD8+ T cells and higher mean Foxp3 expression in regulatory T cells. We conclude that short-term exposure to pet store bedding is insufficient to induce robust changes in immune composition in spleens or bone marrow. Future research with the NME model should focus on sustained exposure and investigate exposure timing.

Memory T (TMEM) cells in the circulation (Tcircm cells) comprise a spectrum of populations that vary in their differentiation, durability, and function. While recirculation was once considered a feature of all TMEM cells, considerable advances in understanding the role of non-circulating resident memory (Trm) T cells have raised the question of how Tcircm populations integrate and complement Trm cells in mediating responses to pathogens and tumors. This review discusses current understanding about the functional properties of Tcircm subsets, their identifying characteristics and distinct pathways of differentiation and homeostasis, and the role of Tcircm cells in providing versatile, safe, and effective immunity.

Memory CD8+ T (Tmem) cells are activated into innate-like killers by cytokines, including interleukin-12 (IL-12), IL-15 and IL-18; but mechanisms regulating this phenomenon (termed bystander activation) are unclear. Here we show that basal IL-4 signals antagonize IL-18 sensing and subsequent interferon-γ production during Tmem cell bystander activation. IL-4 treatment can act directly on Tmem cells in a STAT6-dependent manner to limit interferon-γ-mediated control of a bystander bacterial infection. IL-4 does not simply block bystander activation but tunes effector molecule expression. Strain-specific defects in bystander activation of homeostatic Tmem cells partially relates to IL-4 exposure, but these differences are erased in Tmem cells produced by T cell antigen receptor activation, leading to uniform IL-18 receptor expression and capacity for bystander activation/cytotoxicity. Our data demonstrate that bystander activation by inflammatory cytokines is subject to regulation by both IL-4 and prior antigen experience. These findings underscore the importance of the cytokine milieu in dictating bystander-mediated pathogen control.

Parasitic infections are a major worldwide health burden, yet most studies of CD8 T cell differentiation focus on acute viral and bacterial infections. To understand effector and memory CD8 T cell responses during erythrocytic malaria infection in mice, we utilized transgenic OT-I T cells and compared CD8 T cell responses between infection with OVA-expressing strains of Listeria monocytogenes (Lm) and Plasmodium berghei ANKA (PbA). We find that CD8 T cells expand vigorously during both infections. However, in contrast to Lm infection, PbA infection induces T cells that are heavily biased toward an IL-7Ra-deficient and KLRG1+ short-lived effector cell (SLEC) phenotype at the expense of memory precursor effector cell (MPECs) formation. PbA-induced inflammation, including IFNγ, is partially responsible for this outcome. Following treatment with antimalarial drugs and T cell contraction, PbA-primed memory T cells are rarely found in the blood and peripheral tissues but do maintain a low presence in the spleen and bone marrow. Despite these poor numbers, PbA memory T cells robustly expand upon vaccination or viral infection, control pathogen burden, and form secondary memory pools. Thus, despite PbA enforced SLEC formation and limited memory, effective secondary responses can still proceed.

CD8αα intestinal intraepithelial lymphocytes (IEL) are unconventional T cells involved in maintaining gut homeostasis. This elusive lineage remains underexplored, partly due to controversies regarding their existence in humans. To shed more light on CD8αα IEL, we conducted a studies using both mouse models and human gut tissue. Fate mapping revealed compartmentalized ontogeny and persistence patterns, where CD8aa IEL in the small intestine, but not in the colon, exhibited a disproportionately high representation of cells generated prior to sexual maturity. Early life derived CD8αα IEL were sustained into old age (over 1.5 years) in the small- but not the large intestine. Parabiosis studies indicated enhanced residency of CD8αα IEL in adult and aged animals as compared to conventional CD8αβ IEL. Guided by findings from our microbially enriched (“dirty”) mouse colonies – a model that better mimics adult human immune responses than SPF mice – we identified a population of CD8αα IEL in the human intestine. Encouraged by these discoveries, we sought to develop tools to study CD8αα IEL more comprehensively and established culture conditions to maintain CD8αα IEL ex vivo. In addition, single-cell analysis of > 250 surface markers employing machine learning uncovered a surface marker that, when targeted, depleted CD8αα but not CD4 or CD8αβ IEL. Our findings illuminate the significance of CD8αα IEL, suggesting its potential as a target for immunotherapeutic or vaccination strategies. National Health and Medical Research Council, Australia (NHMRC, APP2008853) Mucosal and Regional Immunology (MUC)

CD8+ T cells exhibit distinct changes with aging, including a diminished naïve cell pool, an expansion of memory and exhausted cells, and altered effector molecule production, altogether leading to increased susceptibility to infection. They have reduced cytotoxicity in vivo, but increased granule content and faster cytotoxic kinetics to target cells in vitro. Whether CD8+ T cells from old mice degranulate when activated in vivo, within the aged environment, is unknown. This study investigates in vitro and in vivo degranulation of CD8+ T cells from young and old mice during supraphysiological aCD3 stimulation and two types of infection. Actively degranulating CD8+ CD44+ T cells were identified by positive labeling after a two-hour exposure to granule-specific fluorescent antibodies (CD107a and CD107b). Surprisingly, CD8+ T cells from old mice challenged with supraphysiological TCR-specific stimulation exhibited higher levels of degranulation as compared to their young counterparts. This effect is more prominent in vitro and can be partially explained by the age-specific increase in CD8+ CD44+ CD62L− cells. However, during microbial exposure or LCMV Armstrong infection, we show that CD8+ CD44+ and antigen-specific T cells from old mice have reduced degranulation, consistent with the diminished cytotoxic capacity. These data highlight the preserved intrinsic cytotoxic capacity of memory CD8+ T cells from old mice and suggest that the aged microenvironment and type of stimulation are contributing factors to the lower degranulation and cytotoxic capacity of these cells. This provides insight into the potential of increasing T cell activation to improve vaccine approaches in the elderly.

P.falciparum infection can trigger high levels of inflammation that lead to fever and sometimes severe disease. People living in malaria-endemic areas gradually develop resistance to symptomatic malaria and control both parasite numbers and the inflammatory response. We previously found that adaptive NK cells correlated with reduced parasite load and protection from symptoms. We also found that murine NK cell production of IL-10 protected mice from experimental cerebral malaria. Human NK cells can also secrete IL-10, but it is unknown what NK cell subsets produce IL-10 or if this is affected by malaria experience. We hypothesized that NK cell immunoregulation may lower inflammation and reduce fever induction. Here, we showed that NK cells from participants with malaria experience make significantly more IL-10 than participants with no malaria experience. We then determined the proportions of NK cells that are cytotoxic and produce IFN-γ and/or IL-10 and identified a signature of adaptive and checkpoint molecules on IL-10-producing NK cells. Lastly, we found that coculture with primary monocytes, Plasmodium-infected RBCs, and antibody induced IL-10 production by NK cells. These data suggest that NK cells may contribute to protection from malaria symptoms via IL-10 production.

ABSTRACT Malaria, which results from infection with Plasmodium parasites, remains a major public health problem. While humans do not develop long-lived, sterilizing immunity, protection against symptomatic disease develops after repeated exposure to Plasmodium parasites and correlates with the acquisition of humoral immunity. Despite the established role antibodies play in protection from malaria disease, dysregulated inflammation is thought to contribute to the sub-optimal immune response to Plasmodium infection. Plasmodium berghei ANKA (PbA) infection results in a fatal severe malaria disease in mice. We previously demonstrated that treatment of mice with IL-15 complex (IL-15C; IL-15 bound to an IL-15Rα-Fc fusion protein) induces IL-10 expression in NK cells, which protects mice from PbA-induced death. Using a novel MHC class II tetramer to identify PbA-specific CD4 + T cells, herein we demonstrate that IL-15C treatment enhances Tfh differentiation. Moreover, genetic deletion of NK cell-derived IL-10 or IL-10R expression on T cells prevents IL-15C-induced Tfh differentiation. Additionally, IL-15C treatment results in increased anti-PbA IgG antibody levels and improves survival following reinfection. Overall, these data demonstrate that IL-15C treatment, via its induction of IL-10 from NK cells, modulates the dysregulated inflammation during Plasmodium infection to promote Tfh differentiation and antibody generation, correlating with improved survival from reinfection. These findings will facilitate improved control of malaria infection and protection from disease by informing therapeutic strategies and vaccine design.

KLRG1 + CD8 T cells persist for months after clearance of acute infections and maintain high levels of effector molecules, contributing protective immunity against systemic pathogens. Upon secondary infection, these long-lived effector cells (LLECs) are incapable of forming other circulating KLRG1 − memory subsets such as central and effector memory T cells. Thus, KLRG1 + memory T cells are frequently referred to as a terminally differentiated population that is relatively short lived. Here, we show that after viral infection of mice, effector cells derived from LLECs rapidly enter nonlymphoid tissues and reduce pathogen burden but are largely dependent on receiving antigen cues from vascular endothelial cells. Single-cell RNA sequencing reveals that secondary memory cells in nonlymphoid tissues arising from either KLRG1 + or KLRG1 − memory precursors develop a similar resident memory transcriptional signature. Thus, although LLECs cannot differentiate into other circulating memory populations, they still retain the flexibility to enter tissues and establish residency.

Tissue-resident memory CD8+ T (Trm) cells control infections and cancer and are defined by their lack of recirculation. Because migration is difficult to assess, residence is usually inferred by putative residence-defining phenotypic and gene signature proxies. We assessed the validity and universality of residence proxies by integrating mouse parabiosis, multi-organ sampling, intravascular staining, acute and chronic infection models, dirty mice, and single-cell multi-omics. We report that memory T cells integrate a constellation of inputs—location, stimulation history, antigen persistence, and environment—resulting in myriad differentiation states. Thus, current Trm-defining methodologies have implicit limitations, and a universal residence-specific signature may not exist. However, we define genes and phenotypes that more robustly correlate with tissue residence across the broad range of conditions that we tested. This study reveals broad adaptability of T cells to diverse stimulatory and environmental inputs and provides practical recommendations for evaluating Trm cells.

Fundamental discoveries in many aspects of mammalian physiology have been made using laboratory mice as research models. These studies have been facilitated by the genetic tractability and inbreeding of such mice, the large set of immunological reagents that are available, and the establishment of environmentally controlled, high-throughput facilities. Such facilities typically include barriers to keep the mouse colonies free of pathogens and the frequent re-derivation of the mice severely limits their commensal flora. Because humans have co-evolved with microorganisms and are exposed to a variety of pathogens, a growing community of researchers posits that preclinical disease research can be improved by studying mice in the context of the microbiota and pathogens that they would encounter in the natural world. Here, we provide a perspective of how these different approaches can be combined and integrated to improve existing mouse models to enhance our understanding of disease mechanisms and develop new therapies for humans. We also propose that the term 'mice with natural microbiota' is more appropriate for describing these models than existing terms such as 'dirty mice'.

The melanoma tumor microenvironment is a complex milieu of cancer, inflammatory, and stromal cells. In this context, chemokines play a pivotal role in recruiting inflammatory cells and influence the tumor, exerting both pro‐tumorigenic and anti‐tumoral roles. Interactions between these cells is what ultimately hold together and transform the tumor into an efficient machine. A recent study found that chemokines CCL8, CCL15, and CCL20 were upregulated in melanoma cells when co‐cultured with macrophages and were associated with poor survival rates. CCL8 and CCL15 also stimulated melanoma cell growth, invasion, and metastasis, and were highly expressed in tumors prone to metastasize, suggesting these chemokines are attractive and independent biomarkers. Understanding the intricated interactions within the tumor microenvironment could lead to prognostic biomarkers and to the development of new therapeutic strategies for melanoma. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The skeletal and immune systems are intricately intertwined within the bone marrow microenvironment, a field of study termed osteoimmunology. Osteoimmune interactions are key players in bone homeostasis and remodeling. Despite the critical role of the immune system in bone health, virtually all animal research in osteoimmunology, and more broadly bone biology, relies on organisms with naïve immune systems. Drawing on insights from osteoimmunology, evolutionary anthropology, and immunology, this perspective proposes the use of a novel translational model: the dirty mouse. Dirty mice, characterized by diverse exposures to commensal and pathogenic microbes, have mature immune systems comparable to adult humans, while the naïve immune system of specific-pathogen free mice is akin to a neonate. Investigation into the dirty mouse model will likely yield important insights in our understanding of bone diseases and disorders. A high benefit of this model is expected for diseases known to have a connection between overactivation of the immune system and negative bone outcomes, including aging and osteoporosis, rheumatoid arthritis, HIV/AIDS, obesity and diabetes, bone marrow metastases, and bone cancers.

Studies of the CD8 T cell memory compartment in mice have primarily been done in young mice maintained in specific pathogen free (SPF) environment. The murine compartment is skewed towards central memory CD8 T cells, whereas adult human memory T cells are dominated by effector memory T cells. The murine memory compartment is akin to that of neonate humans, suggesting there are factors missing in SPF mice that are required to mature the T cell compartment. We wanted to ask how pathogen exposure changes the memory compartment of mice, so we utilized a model of pet shop mouse co-housing (COH), which exposes mice to a wide variety of normal mouse pathogens. COH mice display a substantial increase in long-lived effector CD8 T cells (LLEC). LLEC are a CD8 T cell memory population described by the Hamilton Lab that is phenotypically more effector-like and are highly effective at clearing systemic infections when compared to other memory T cells. However, LLEC display reduced homeostatic and antigen-driven proliferation in SPF mice leading to a lower representation in the memory pool. The factors in COH that support LLEC expansion and persistence are currently unknown, and it is also unclear if COH impacts LLEC function. Thus, the goal of this study is to understand what intrinsic and environmental factors contribute to LLEC formation and maintenance, as well as determine how LLEC function contributes to memory responses in COH mice. I have found that COH LLEC are more polyfunctional compared to SPF LLEC, and my preliminary work has found that LLEC turnover is increased in COH mice. Future studies will determine what specific inflammatory cues lead to LLEC expansion, and if this process can be altered to improve T cell responses to infection and vaccination. Supported by grants from the NIH (R01 AI155468)

CMV infection alters NK cell phenotype and function toward a more memory-like immune state. These cells, termed adaptive NK cells, typically express CD57 and NKG2C but lack expression of the FcRγ-chain (gene: FCER1G, FcRγ), PLZF, and SYK. Functionally, adaptive NK cells display enhanced Ab-dependent cellular cytotoxicity (ADCC) and cytokine production. However, the mechanism behind this enhanced function is unknown. To understand what drives enhanced ADCC and cytokine production in adaptive NK cells, we optimized a CRISPR/Cas9 system to ablate genes from primary human NK cells. We ablated genes that encode molecules in the ADCC pathway, such as FcRγ, CD3ζ, SYK, SHP-1, ZAP70, and the transcription factor PLZF, and tested subsequent ADCC and cytokine production. We found that ablating the FcRγ-chain caused a modest increase in TNF-α production. Ablation of PLZF did not enhance ADCC or cytokine production. Importantly, SYK kinase ablation significantly enhanced cytotoxicity, cytokine production, and target cell conjugation, whereas ZAP70 kinase ablation diminished function. Ablating the phosphatase SHP-1 enhanced cytotoxicity but reduced cytokine production. These results indicate that the enhanced cytotoxicity and cytokine production of CMV-induced adaptive NK cells is more likely due to the loss of SYK than the lack of FcRγ or PLZF. We found the lack of SYK expression could improve target cell conjugation through enhanced CD2 expression or limit SHP-1-mediated inhibition of CD16A signaling, leading to enhanced cytotoxicity and cytokine production.

Microbial experience fundamentally shapes immunity, particularly during the perinatal period when the immune system is underdeveloped, and novel microbial encounters are common. Most animal models are raised in specific pathogen-free (SPF) conditions with relatively uniform microbial communities. How SPF housing conditions alter early-life immune development relative to natural microbial exposure (NME) has not been thoroughly investigated. In this article, we compare immune development in SPF-raised mice with mice born from immunologically experienced mothers in microbially diverse environments. NME induced broad immune cell expansion, including naive cells, suggesting mechanisms besides activation-induced proliferation contribute to the increase in immune cell numbers. We found NME conditions also expanded immune cell progenitor cell populations in the bone marrow, suggesting microbial experience enhances immune development at the earliest stages of immune cell differentiation. Multiple immune functions characteristically impaired in infants were also enhanced by NME, including T cell memory and Th1 polarization, B cell class switching and Ab production, proinflammatory cytokine expression, and bacterial clearance after Listeria monocytogenes challenge. Collectively, our studies reveal numerous impairments in immune development in SPF conditions relative to natural immune development.