Circulating KLRG1+ long-lived effector memory T cells retain the flexibility to become tissue resident.

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.

Novel chemokine biomarkers in melanoma†.

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.

Phenotype and function of IL-10 producing NK cells in individuals with malaria experience.

Plasmodium 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 natural killer (NK) cells correlate with reduced parasite load and protection from symptoms. We also previously found that murine NK cell production of IL-10 can protect mice from experimental cerebral malaria. Human NK cells can also secrete IL-10, but it was unknown what NK cell subsets produce IL-10 and if this is affected by malaria experience. We hypothesize that NK cell immunoregulation may lower inflammation and reduce fever induction. Here, we show that NK cells from subjects with malaria experience make significantly more IL-10 than subjects with no malaria experience. We then determined the proportions of NK cells that are cytotoxic and produce interferon gamma and/or IL-10 and identified a signature of adaptive and checkpoint molecules on IL-10-producing NK cells. Lastly, we find that co-culture with primary monocytes, Plasmodium -infected RBCs, and antibody induces IL-10 production by NK cells. These data suggest that NK cells may contribute to protection from malaria symptoms via IL-10 production.

PD1 blockade improves survival and CD8+ cytotoxic capacity, without increasing inflammation, during normal microbial experience in old mice.

By 2030, individuals 65 years of age or older will make up approximately 20% of the world's population1. Older individuals are at the highest risk for mortality from infections, largely due to the pro-inflammatory, dysfunctional immune response, which is collectively known as immunosenescence2. During aging, CD8+ T cells acquire an exhausted phenotype, including increased expression of inhibitory receptors, such as programmed cell death 1 (PD1), a decline in effector function and elevated expression of inflammatory factors3-7. PD1 reduces T cell receptor activity via SHP2-dependent dephosphorylation of multiple pathways; accordingly, inhibiting PD1 activity through monoclonal antibodies increases CD8+ T cell effector response in young mice8-11. Attempts to improve CD8+ T cell responses by blocking inhibitory receptors are attractive; however, they can lead to adverse immune events due to overamplification of T cell receptor signaling and T cell activation12,13. Here we investigated the effect of monoclonal anti-PD1 immunotherapy during normal microbial experience, otherwise known as exposure to dirty mice, to determine whether it either improves exhausted CD8+ T cell responses in old mice or leads to a heightened inflammatory response and increased mortality.

IL-15 Complex-Induced IL-10 Enhances Plasmodium-specific CD4+ T Follicular Helper Differentiation and Antibody Production.

Malaria, which results from infection with Plasmodium parasites, remains a major public health problem. Although 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 Abs play in protection from malaria disease, dysregulated inflammation is thought to contribute to the suboptimal 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, in this study we demonstrate that IL-15C treatment enhances T follicular helper (Tfh) differentiation and modulates cytokine production by CD4+ T cells. 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 Ab 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 Ab 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.

Leveraging mice with diverse microbial exposures for advances in osteoimmunology.

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.

Natural Microbial Exposure from the Earliest Natural Time Point Enhances Immune Development by Expanding Immune Cell Progenitors and Mature Immune Cells.

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.

Ablation of SYK Kinase from Expanded Primary Human NK Cells via CRISPR/Cas9 Enhances Cytotoxicity and Cytokine Production.

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.

Reduced T Cell Priming in Microbially Experienced "Dirty" Mice Results from Limited IL-27 Production by XCR1+ Dendritic Cells.

Successful vaccination strategies offer the potential for lifelong immunity against infectious diseases and cancer. There has been increased attention regarding the limited translation of some preclinical findings generated using specific pathogen-free (SPF) laboratory mice to humans. One potential reason for the difference between preclinical and clinical findings lies in maturation status of the immune system at the time of challenge. In this study, we used a "dirty" mouse model, where SPF laboratory mice were cohoused (CoH) with pet store mice to permit microbe transfer and immune system maturation, to investigate the priming of a naive T cell response after vaccination with a peptide subunit mixed with polyinosinic-polycytidylic acid and agonistic anti-CD40 mAb. Although this vaccination platform induced robust antitumor immunity in SPF mice, it failed to do so in microbially experienced CoH mice. Subsequent investigation revealed that despite similar numbers of Ag-specific naive CD4 and CD8 T cell precursors, the expansion, differentiation, and recall responses of these CD4 and CD8 T cell populations in CoH mice were significantly reduced compared with SPF mice after vaccination. Evaluation of the dendritic cell compartment revealed reduced IL-27p28 expression by XCR1+ dendritic cells from CoH mice after vaccination, correlating with reduced T cell expansion. Importantly, administration of recombinant IL-27:EBI3 complex to CoH mice shortly after vaccination significantly boosted Ag-specific CD8 and CD4 T cell expansion, further implicating the defect to be T cell extrinsic. Collectively, our data show the potential limitation of exclusive use of SPF mice when testing vaccine efficacy.

Responsiveness to interleukin-15 therapy is shared between tissue-resident and circulating memory CD8+ T cell subsets.

Interleukin-15 (IL-15) is often considered a central regulator of memory CD8+ T cells, based primarily on studies of recirculating subsets. However, recent work identified IL-15-independent CD8+ T cell memory populations, including tissue-resident memory CD8+ T cells (TRM) in some nonlymphoid tissues (NLTs). Whether this reflects the existence of IL-15-insensitive memory CD8+ T cells is unclear. We report that IL-15 complexes (IL-15c) stimulate rapid proliferation and expansion of both tissue-resident and circulating memory CD8+ T cell subsets across lymphoid and nonlymphoid tissues with varying magnitude by tissue and memory subset, in some sites correlating with differing levels of the IL-2Rß. This was conserved for memory CD8+ T cells recognizing distinct antigens and elicited by different pathogens. Following IL-15c-induced expansion, divided cells contracted to baseline numbers and only slowly returned to basal proliferation, suggesting a mechanism to transiently amplify memory populations. Through parabiosis, we showed that IL-15c drive local proliferation of TRM, with a degree of recruitment of circulating cells to some NLTs. Hence, irrespective of homeostatic IL-15 dependence, IL-15 sensitivity is a defining feature of memory CD8+ T cell populations, with therapeutic potential for expansion of TRM and other memory subsets in an antigen-agnostic and temporally controlled fashion.

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

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.

Mice with diverse microbial exposure histories as a model for preclinical vaccine testing.

Laboratory mice comprise an expeditious model for preclinical vaccine testing; however, vaccine immunogenicity in these models often inadequately translates to humans. Reconstituting physiologic microbial experience to specific pathogen-free (SPF) mice induces durable immunological changes that better recapitulate human immunity. We examined whether mice with diverse microbial experience better model human responses post vaccination. We co-housed laboratory mice with pet-store mice, which have varied microbial exposures, and then assessed immune responses to influenza vaccines. Human transcriptional responses to influenza vaccination are better recapitulated in co-housed mice. Although SPF and co-housed mice were comparably susceptible to acute influenza infection, vaccine-induced humoral responses were dampened in co-housed mice, resulting in poor control upon challenge. Additionally, protective heterosubtypic T cell immunity was compromised in co-housed mice. Because SPF mice exaggerated humoral and T cell protection upon influenza vaccination, reconstituting microbial experience in laboratory mice through co-housing may better inform preclinical vaccine testing.

Senolytics reduce coronavirus-related mortality in old mice.

The COVID-19 pandemic has revealed the pronounced vulnerability of the elderly and chronically ill to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced morbidity and mortality. Cellular senescence contributes to inflammation, multiple chronic diseases, and age-related dysfunction, but effects on responses to viral infection are unclear. Here, we demonstrate that senescent cells (SnCs) become hyper-inflammatory in response to pathogen-associated molecular patterns (PAMPs), including SARS-CoV-2 spike protein-1, increasing expression of viral entry proteins and reducing antiviral gene expression in non-SnCs through a paracrine mechanism. Old mice acutely infected with pathogens that included a SARS-CoV-2-related mouse ß-coronavirus experienced increased senescence and inflammation, with nearly 100% mortality. Targeting SnCs by using senolytic drugs before or after pathogen exposure significantly reduced mortality, cellular senescence, and inflammatory markers and increased antiviral antibodies. Thus, reducing the SnC burden in diseased or aged individuals should enhance resilience and reduce mortality after viral infection, including that of SARS-CoV-2.

Generating Mice with Diverse Microbial Experience.

Laboratory strains of mice are typically housed in specific pathogen-free facilities to minimize exposure to microbes. This method encourages uniformity in responses to experimentally induced parameters and reduces loss of animals, allowing for the survival and study of immunodeficient mice. However, the restrictions also limit physiologic relevance to humans, who are exposed to numerous microbes from birth. Recent evidence from several groups has demonstrated that exposure of laboratory mice to commensal and pathogenic microbes normally found in wild or pet store mice can dramatically impact the cellular makeup and function of the immune system. This article outlines procedures for exposing laboratory strains of mice to the diverse array of microbes typically found in pet store mice. Suggested methods for characterization of the immune system following this exposure are also described. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Cohousing laboratory strains of mice with pet store mice Support Protocol: Antibody staining of circulating immune cells and analysis by flow cytometry Basic Protocol 2: Exposure of laboratory strains of mice to fomite bedding.

KLRG1+ Memory CD8 T Cells Combine Properties of Short-Lived Effectors and Long-Lived Memory.

CD8 effector T cells with a CD127hi KLRG1- phenotype are considered precursors to the long-lived memory pool, whereas KLRG1+CD127low cells are viewed as short-lived effectors. Nevertheless, we and others have shown that a KLRG1+CD127low population persists into the memory phase and that these T cells (termed long-lived effector cells [LLEC]) display robust protective function during acute rechallenge with bacteria or viruses. Whether these T cells represent a true memory population or are instead a remnant effector cell population that failed to undergo initial contraction has remained unclear. In this study, we show that LLEC from mice express a distinct phenotypic and transcriptional signature that shares characteristics of both early effectors and long-lived memory cells. We also find that in contrast to KLRG1+ effector cells, LLEC undergo homeostatic proliferation and are not critically dependent on IL-15 for their maintenance. Furthermore, we find that LLEC are predominantly derived from KLRG1+ effector cells when isolated at day 12 of the response. Our work challenges the concept that the KLRG1+CD127low population is dominated by short-lived cells and shows that KLRG1 downregulation is not a prerequisite to become a long-lived protective memory T cell.

New Insights into the Immune System Using Dirty Mice.

The mouse (Mus musculus) 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.

NK Cell IL-10 Production Requires IL-15 and IL-10 Driven STAT3 Activation.

Natural killer (NK) cells can produce IFNγ or IL-10 to regulate inflammation and immune responses but the factors driving NK cell IL-10 secretion are poorly-defined. Here, we identified NK cell-intrinsic STAT3 activation as vital for IL-10 production during both systemic Listeria monocytogenes (Lm) infection and following IL-15 cytokine/receptor complex (IL15C) treatment for experimental cerebral malaria (ECM). In both contexts, conditional Stat3 deficiency in NK cells abrogated production of IL-10. Initial NK cell STAT3 phosphorylation was driven by IL-15. During Lm infection, this required capture or presentation of IL-15 by NK cell IL-15Rα. Persistent STAT3 activation was required to drive measurable IL-10 secretion and required NK cell expression of IL-10Rα. Survival-promoting effects of IL-15C treatment in ECM were dependent on NK cell Stat3 while NK cell-intrinsic deficiency for Stat3, Il15ra, or Il10ra abrogated NK cell IL-10 production and increased resistance against Lm. NK cell Stat3 deficiency did not impact production of IFNγ, indicating the STAT3 activation initiated by IL-15 and amplified by IL-10 selectively drives the production of anti-inflammatory IL-10 by responding NK cells.