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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., Competing Interests: Declaration of interests C.W., M.C.M., S.B., J.C., K.H.K., and S.G. are co-inventors on a patent related to this work., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.

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 RM ) in nonlymphoid tissues triggers vascular permeability, which facilitates rapid distribution of serum antibodies into local tissues. T RM 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 RM in brain and reproductive mucosa are both competent to induce rapid antibody exudation. T RM 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 RM antigen recognition can enhance local humoral immunity., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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-3 lo 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., (Copyright © 2022 by The American Association of Immunologists, Inc.)

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., (© 2022 John Wiley & Sons Ltd.)

Tissue resident memory T cells (T RM ) are enriched in non-lymphoid tissues and represent a formidable barrier against invading pathogens and tumors. T RM 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 RM 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 RM establishment and function. A deeper understanding of these processes is instrumental in designing new means to target T RM for desirable outcomes in infectious diseases, cancers and autoimmunity., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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 RM ) 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 RM 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 RM 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 RM .

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 1,2 , 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.

Optimal ex vivo expansion protocols of tumor-specific T cells followed by adoptive cell therapy must yield T cells able to home to tumors and effectively kill them. Our previous study demonstrated ex vivo activation in the presence of IL-12-induced optimal CD8 + T cell expansion and melanoma regression; however, adverse side effects, including autoimmunity, can occur. This may be due to transfer of high-avidity self-specific T cells. In this study, we compared mouse low- and high-avidity T cells targeting the tumor Ag tyrosinase-related protein 2 (TRP2). Not surprisingly, high-avidity T cells provide superior tumor control, yet low-avidity T cells can promote tumor regression. The addition of IL-12 during in vitro expansion boosts low-avidity T cell responsiveness, tumor regression, and prevents T cell exhaustion. In this study, we demonstrate that IL-12-primed T cells are resistant to PD-1/PD-L1-mediated suppression and retain effector function. Importantly, IL-12 preconditioning prevented exhaustion as LAG-3, PD-1, and TOX were decreased while simultaneously increasing KLRG1. Using intravital imaging, we also determined that high-avidity T cells have sustained contacts with intratumoral dendritic cells and tumor targets compared with low-avidity T cells. However, with Ag overexpression, this defect is overcome, and low-avidity T cells control tumor growth. Taken together, these data illustrate that low-avidity T cells can be therapeutically beneficial if cocultured with IL-12 cytokine during in vitro expansion and highly effective in vivo if Ag is not limiting. Clinically, low-avidity T cells provide a safer alternative to high-avidity, TCR-engineered T cells, as IL-12-primed, low-avidity T cells cause less autoimmune vitiligo., (Copyright © 2020 by The American Association of Immunologists, Inc.)

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., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2020 Stolley et al.)

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., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Central memory T (T CM ) 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 RM ) cells are parked within single organs, share properties with terminal effectors and contribute to rapid host protection. We observed that reactivated T RM cells rejoined the circulating pool. Epigenetic analyses revealed that T RM cells align closely with conventional memory T cell populations, bearing little resemblance to recently activated effectors. Fully differentiated T RM cells isolated from small intestine epithelium exhibited the potential to differentiate into T CM cells, effector memory T cells and T RM cells on recall. Ex-T RM cells, former intestinal T RM 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 RM cells. Thus, T RM cells can rejoin the circulation but are advantaged to re-form local T RM when called on.

Recent studies have characterized populations of memory CD8 + T cells that do not recirculate through the blood but are, instead, retained in nonlymphoid tissues. Such CD8 + tissue resident memory T cells (T RM ) are critical for pathogen control at barrier sites. Identifying T RM and defining the basis for their tissue residency is therefore of considerable importance for understanding protective immunity and improved vaccine design. Expression of the molecule CD69 is widely used as a definitive marker for T RM , yet it is unclear whether CD69 is universally required for producing or retaining T RM Using multiple mouse models of acute immunization, we found that the functional requirement for CD69 was highly variable, depending on the tissue examined, playing no detectable role in generation of T RM at some sites (such as the small intestine), whereas CD69 was critical for establishing resident cells in the kidney. Likewise, forced expression of CD69 (but not expression of a CD69 mutant unable to bind the egress factor S1PR1) promoted CD8 + T RM generation in the kidney but not in other tissues. Our findings indicate that the functional relevance of CD69 in generation and maintenance of CD8 + T RM varies considerably, chiefly dependent on the specific nonlymphoid tissue studied. Together with previous reports that suggest uncoupling of CD69 expression and tissue residency, these findings prompt caution in reliance on CD69 expression as a consistent marker of CD8 + T RM ., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Regulated activation of the cytokine TGF-β by integrins α v β 6 and α v β 8 expressed on keratinocytes is required for residence of epidermal-resident memory T cells, but whether skin-derived signals also affect recirculating memory cells in the skin remains unclear. Here, we show that after resolution of skin vaccinia virus (VV) infection, antigen-specific circulating memory CD8 + T cells migrated into skin. In mice lacking α v β 6 and α v β 8 integrins (Itgb6 -/- Itgb8 fl/fl -K14-cre), the absence of epidermal-activated TGF-β resulted in a gradual loss of E- or P-selectin-binding central and peripheral memory populations, which were rescued when skin entry was inhibited. Skin recirculating memory cells were required for optimal host defense against skin VV infection. These data demonstrate that skin migration can persist after resolution of local skin infection and that the cytokine environment within this nonlymphoid tissue shapes the differentiation state and persistence of the central and peripheral memory-T-cell pool., (Copyright © 2019 Elsevier Inc. All rights reserved.)

This study examines the extent to which memory CD4 + T cells share immunosurveillance strategies with CD8 + resident memory T cells (T RM ). After acute viral infection, memory CD4 + T cells predominantly used residence to survey nonlymphoid tissues, albeit not as stringently as observed for CD8 + T cells. In contrast, memory CD4 + T cells were more likely to be resident within lymphoid organs than CD8 + T cells. Migration properties of memory-phenotype CD4 + T cells in non-SPF parabionts were similar, generalizing these results to diverse infections and conditions. CD4 + and CD8 + T RM shared overlapping transcriptional signatures and location-specific features, such as granzyme B expression in the small intestine, revealing tissue-specific and migration property-specific, in addition to lineage-specific, differentiation programs. Functionally, mucosal CD4 + T RM reactivation locally triggered both chemokine expression and broad immune cell activation. Thus, residence provides a dominant mechanism for regionalizing CD4 + T cell immunity, and location enforces shared transcriptional, phenotypic, and functional properties with CD8 + T cells., (© 2019 Beura et al.)

The migratory capacity of adaptive CD8αβ T cells dictates their ability to locate target cells and exert cytotoxicity, which is the basis of immune surveillance for the containment of microbes and disease. The small intestine (SI) is the largest mucosal surface and is a primary site of pathogen entrance. Using two-photon laser scanning microscopy, we found that motility of antigen (Ag)-specific CD8αβ T cells in the SI is dynamic and varies with the environmental milieu. Pathogen-specific CD8αβ T cell movement differed throughout infection, becoming locally confined at memory. Motility was not dependent on CD103 but was influenced by micro-anatomical locations within the SI and by inflammation. CD8 T cells responding to self-protein were initially affected by the presence of self-Ag, but this was altered after complete tolerance induction. These studies identify multiple factors that affect CD8αβ T cell movement in the intestinal mucosa and show the adaptability of CD8αβ T cell motility., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Many US immigrant populations develop metabolic diseases post immigration, but the causes are not well understood. Although the microbiome plays a role in metabolic disease, there have been no studies measuring the effects of US immigration on the gut microbiome. We collected stool, dietary recalls, and anthropometrics from 514 Hmong and Karen individuals living in Thailand and the United States, including first- and second-generation immigrants and 19 Karen individuals sampled before and after immigration, as well as from 36 US-born European American individuals. Using 16S and deep shotgun metagenomic DNA sequencing, we found that migration from a non-Western country to the United States is associated with immediate loss of gut microbiome diversity and function in which US-associated strains and functions displace native strains and functions. These effects increase with duration of US residence and are compounded by obesity and across generations., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Although CD8 T-cell vaccines do not have the record of success of humoral-mediated vaccines, they do not receive the same degree of effort. Many diseases, including malaria, tuberculosis, and acquired immune deficiency syndrome (AIDS) have not yielded to vaccines, and intrinsic barriers may impede approaches limited solely to generating antibodies. Moreover, population growth and modernization are driving an increased pace of new emerging global health threats (human immunodeficiency virus [HIV] is a recent example), which will create unpredictable challenges for vaccinologists. Vaccine-elicited CD8 T cells may contribute to protective modalities, although their development will require a more thorough understanding of CD8 T-cell biology, practices for manufacturing and delivering CD8 T-cell-eliciting vectors that have acceptable safety profiles, and, ultimately, the political will and faith of those that make vaccine research funding decisions., (Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.)

Lymphocytes enter tissues from blood vessels through a well-characterized three-step process of extravasation. To our knowledge, nonvascular routes of lymphocyte entry have not been described. In this article, we report that Ag-experienced CD8 T cells in mice recirculate from blood through the peritoneal cavity. In the event of infection, Ag-experienced CD8 T cell subsets adhered to visceral organs, indicating potential transcapsular immunosurveillance. Focusing on the male genital tract (MGT), we observed Ag-experienced CD8 T cell migration from the peritoneal cavity directly to the infected MGT across the capsule, which was dependent on the extracellular matrix receptor CD44. We also observed that, following clearance of infection, the MGT retained functional resident memory CD8 T cells. These data suggest that recirculation through body cavities may provide T cells with opportunities for broad immunosurveillance and potential nonvascular mechanisms of entry., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Extracellular ATP (eATP) is an ancient 'danger signal' used by eukaryotes to detect cellular damage 1 . In mice and humans, the release of eATP during inflammation or injury stimulates both innate immune activation and chronic pain through the purinergic receptor P2RX7 2-4 . It is unclear, however, whether this pathway influences the generation of immunological memory, a hallmark of the adaptive immune system that constitutes the basis of vaccines and protective immunity against re-infection 5,6 . Here we show that P2RX7 is required for the establishment, maintenance and functionality of long-lived central and tissue-resident memory CD8 + T cell populations in mice. By contrast, P2RX7 is not required for the generation of short-lived effector CD8 + T cells. Mechanistically, P2RX7 promotes mitochondrial homeostasis and metabolic function in differentiating memory CD8 + T cells, at least in part by inducing AMP-activated protein kinase. Pharmacological inhibitors of P2RX7 provoked dysregulated metabolism and differentiation of activated mouse and human CD8 + T cells in vitro, and transient P2RX7 blockade in vivo ameliorated neuropathic pain but also compromised production of CD8 + memory T cells. These findings show that activation of P2RX7 by eATP provides a common currency that both alerts the nervous and immune system to tissue damage, and promotes the metabolic fitness and survival of the most durable and functionally relevant memory CD8 + T cell populations.

Immunosurveillance of secondary lymphoid organs (SLO) is performed by central memory T cells that recirculate through blood. Resident memory T (Trm) cells remain parked in nonlymphoid tissues and often stably express CD69. We recently identified Trm cells within SLO, but the origin and phenotype of these cells remains unclear. Using parabiosis of "dirty" mice, we found that CD69 expression is insufficient to infer stable residence of SLO Trm cells. Restimulation of nonlymphoid memory CD8 + T cells within the skin or mucosa resulted in a substantial increase in bona fide Trm cells specifically within draining lymph nodes. SLO Trm cells derived from emigrants from nonlymphoid tissues and shared some transcriptional and phenotypic signatures associated with nonlymphoid Trm cells. These data indicate that nonlymphoid cells can give rise to SLO Trm cells and suggest vaccination strategies by which memory CD8 + T cell immunosurveillance can be regionalized to specific lymph nodes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

CD8 + T cell immunosurveillance dynamics influence the outcome of intracellular infections and cancer. Here we used two-photon intravital microscopy to visualize the responses of CD8 + resident memory T cells (T RM cells) within the reproductive tracts of live female mice. We found that mucosal T RM cells were highly motile, but paused and underwent in situ division after local antigen challenge. T RM cell reactivation triggered the recruitment of recirculating memory T cells that underwent antigen-independent T RM cell differentiation in situ. However, the proliferation of pre-existing T RM cells dominated the local mucosal recall response and contributed most substantially to the boosted secondary T RM cell population. We observed similar results in skin. Thus, T RM cells can autonomously regulate the expansion of local immunosurveillance independently of central memory or proliferation in lymphoid tissue.

Background: Human microbiota-associated (HMA) animal models relying on germ-free recipient mice are being used to study the relationship between intestinal microbiota and human disease. However, transfer of microbiota into germ-free animals also triggers global developmental changes in the recipient intestine, which can mask disease-specific attributes of the donor material. Therefore, a simple model of replacing microbiota into a developmentally mature intestinal environment remains highly desirable., Results: Here we report on the development of a sequential, three-course antibiotic conditioning regimen that allows sustained engraftment of intestinal microorganisms following a single oral gavage with human donor microbiota. SourceTracker, a Bayesian, OTU-based algorithm, indicated that 59.3 ± 3.0% of the fecal bacterial communities in treated mice were attributable to the donor source. This overall degree of microbiota engraftment was similar in mice conditioned with antibiotics and germ-free mice. Limited surveys of systemic and mucosal immune sites did not show evidence of immune activation following introduction of human microbiota., Conclusions: The antibiotic treatment protocol described here followed by a single gavage of human microbiota may provide a useful, complimentary HMA model to that established in germ-free facilities. The model has the potential for further in-depth translational investigations of microbiota in a variety of human disease states.

Recent studies have established resident memory T cells (T RM ) as the dominant memory lymphocyte population surveying most nonlymphoid tissues. Unlike other memory T cell lineages, T RM do not recirculate through blood and are permanently confined to their tissue of residence. T RM orchestrate local immune responses and have been shown to accelerate local pathogen control in many experimental infection models. Here we briefly summarize recent advances in T RM differentiation, maintenance, and their protective function. While little is known, we have speculated on the potential implications of T RM for transplantation biology. Areas of emphasis include the role of passenger T RM in controlling latent viral recrudescence in donor organs, donor T RM as a source of graft-versus-host disease, the ability of T RM to potently induce inflammation through sensing and alarm functions, and differentiation of host T cells into T RM in response to local cues inside the allograft. Further investigation of T RM in the context of transplantation might identify therapeutic targets to prolong graft survival., (© 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Tissue resident memory T cells (T RM ) constitute a recently identified T cell lineage that is responsible for frontline defense against viral infections. In contrast to central and effector memory T cells, which constitutively recirculate between tissues and blood, T RM reside permanently within tissues. As the main surveyors of non-lymphoid tissues, T RM are positioned to rapidly respond upon reinfection at barrier sites. During a viral reinfection, T RM trigger the local tissue environment to activate and recruit immune cells and establish an antiviral state. Consistent with this function, there is empirical evidence that T RM accelerate control in the event of reinfection or possible reactivation of latent infections in solid organs and barrier tissues. Here we review recent literature highlighting the protective functions of T RM in multiple viral challenge models and contextualize the implications of these findings for vaccine development., (Copyright © 2016 Elsevier B.V. All rights reserved.)

During acute infections, naive Ag-specific CD8 T cells are activated and differentiate into effector T cells, most of which undergo contraction after pathogen clearance. A small population of CD8 T cells persists as memory to protect against future infections. We investigated the role of adhesion- and degranulation-promoting adapter protein (ADAP) in promoting CD8 T cell responses to a systemic infection. Naive Ag-specific CD8 T cells lacking ADAP exhibited a modest expansion defect early after Listeria monocytogenes or vesicular stomatitis virus infection but comparable cytolytic function at the peak of response. However, reduced numbers of ADAP-deficient CD8 T cells were present in the spleen after the peak of the response. ADAP deficiency resulted in a greater frequency of CD127(+) CD8 memory precursors in secondary lymphoid organs during the contraction phase. Reduced numbers of ADAP-deficient killer cell lectin-like receptor G1(-) CD8 resident memory T (TRM) cell precursors were present in a variety of nonlymphoid tissues at the peak of the immune response, and consequently the total numbers of ADAP-deficient TRM cells were reduced at memory time points. TRM cells that did form in the absence of ADAP were defective in effector molecule expression. ADAP-deficient TRM cells exhibited impaired effector function after Ag rechallenge, correlating with defects in their ability to form T cell-APC conjugates. However, ADAP-deficient TRM cells responded to TGF-β signals and recruited circulating memory CD8 T cells. Thus, ADAP regulates CD8 T cell differentiation events following acute pathogen challenge that are critical for the formation and selected functions of TRM cells in nonlymphoid tissues., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Immune responses differ between laboratory mice and humans. Chronic infection with viruses and parasites are common in humans, but are absent in laboratory mice, and thus represent potential contributors to inter-species differences in immunity. To test this, we sequentially infected laboratory mice with herpesviruses, influenza, and an intestinal helminth and compared their blood immune signatures to mock-infected mice before and after vaccination against yellow fever virus (YFV-17D). Sequential infection altered pre- and post-vaccination gene expression, cytokines, and antibodies in blood. Sequential pathogen exposure induced gene signatures that recapitulated those seen in blood from pet store-raised versus laboratory mice, and adult versus cord blood in humans. Therefore, basal and vaccine-induced murine immune responses are altered by infection with agents common outside of barrier facilities. This raises the possibility that we can improve mouse models of vaccination and immunity by selective microbial exposure of laboratory animals to mimic that of humans., (Copyright © 2016 Elsevier Inc. All rights reserved.)

IL-15 regulates central and effector memory CD8 T cell (TCM and TEM, respectively) homeostatic proliferation, maintenance, and longevity. Consequently, IL-15 availability hypothetically defines the carrying capacity for total memory CD8 T cells within the host. In conflict with this hypothesis, previous observations demonstrated that boosting generates preternaturally abundant TEM that increases the total quantity of memory CD8 T cells in mice. In this article, we provide a potential mechanistic explanation by reporting that boosted circulating TEM do not require IL-15 for maintenance. We also investigated tissue-resident memory CD8 T cells (TRM), which protect nonlymphoid tissues from reinfection. We observed up to a 50-fold increase in the total magnitude of TRM in mouse mucosal tissues after boosting, suggesting that the memory T cell capacity in tissues is flexible and that TRM may not be under the same homeostatic regulation as primary central memory CD8 T cells and TEM Further analysis identified distinct TRM populations that depended on IL-15 for homeostatic proliferation and survival, depended on IL-15 for homeostatic proliferation but not for survival, or did not depend on IL-15 for either process. These observations on the numerical regulation of T cell memory indicate that there may be significant heterogeneity among distinct TRM populations and also argue against the common perception that developing vaccines that confer protection by establishing abundant TEM and TRM will necessarily erode immunity to previously encountered pathogens as the result of competition for IL-15., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Our current understanding of immunology was largely defined in laboratory mice, partly because they are inbred and genetically homogeneous, can be genetically manipulated, allow kinetic tissue analyses to be carried out from the onset of disease, and permit the use of tractable disease models. Comparably reductionist experiments are neither technically nor ethically possible in humans. However, there is growing concern that laboratory mice do not reflect relevant aspects of the human immune system, which may account for failures to translate disease treatments from bench to bedside. Laboratory mice live in abnormally hygienic specific pathogen free (SPF) barrier facilities. Here we show that standard laboratory mouse husbandry has profound effects on the immune system and that environmental changes produce mice with immune systems closer to those of adult humans. Laboratory mice--like newborn, but not adult, humans--lack effector-differentiated and mucosally distributed memory T cells. These cell populations were present in free-living barn populations of feral mice and pet store mice with diverse microbial experience, and were induced in laboratory mice after co-housing with pet store mice, suggesting that the environment is involved in the induction of these cells. Altering the living conditions of mice profoundly affected the cellular composition of the innate and adaptive immune systems, resulted in global changes in blood cell gene expression to patterns that more closely reflected the immune signatures of adult humans rather than neonates, altered resistance to infection, and influenced T-cell differentiation in response to a de novo viral infection. These data highlight the effects of environment on the basal immune state and response to infection and suggest that restoring physiological microbial exposure in laboratory mice could provide a relevant tool for modelling immunological events in free-living organisms, including humans.

Cells of the immune system that reside in barrier epithelia provide a first line of defense against pathogens. Langerhans cells (LCs) and CD8(+) tissue-resident memory T cells (TRM cells) require active transforming growth factor-β1 (TGF-β) for epidermal residence. Here we found that integrins αvβ6 and αvβ8 were expressed in non-overlapping patterns by keratinocytes (KCs) and maintained the epidermal residence of LCs and TRM cells by activating latent TGF-β. Similarly, the residence of dendritic cells and TRM cells in the small intestine epithelium also required αvβ6. Treatment of the skin with ultraviolet irradiation decreased integrin expression on KCs and reduced the availability of active TGF-β, which resulted in LC migration. Our data demonstrated that regulated activation of TGF-β by stromal cells was able to directly control epithelial residence of cells of the immune system through a novel mechanism of intercellular communication., Competing Interests: The authors declare no competing financial interests.

Developing vaccine strategies to generate high numbers of Ag-specific CD8 T cells may be necessary for protection against recalcitrant pathogens. Heterologous prime-boost-boost immunization has been shown to result in large quantities of functional memory CD8 T cells with protective capacities and long-term stability. Completing the serial immunization steps for heterologous prime-boost-boost can be lengthy, leaving the host vulnerable for an extensive period of time during the vaccination process. We show in this study that shortening the intervals between boosting events to 2 wk results in high numbers of functional and protective Ag-specific CD8 T cells. This protection is comparable to that achieved with long-term boosting intervals. Short-boosted Ag-specific CD8 T cells display a canonical memory T cell signature associated with long-lived memory and have identical proliferative potential to long-boosted T cells Both populations robustly respond to antigenic re-exposure. Despite this, short-boosted Ag-specific CD8 T cells continue to contract gradually over time, which correlates to metabolic differences between short- and long-boosted CD8 T cells at early memory time points. Our studies indicate that shortening the interval between boosts can yield abundant, functional Ag-specific CD8 T cells that are poised for immediate protection; however, this is at the expense of forming stable long-term memory., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Memory CD8 T cells protect against intracellular pathogens by scanning host cell surfaces; thus, infection detection rates depend on memory cell number and distribution. Population analyses rely on cell isolation from whole organs, and interpretation is predicated on presumptions of near complete cell recovery. Paradigmatically, memory is parsed into central, effector, and resident subsets, ostensibly defined by immunosurveillance patterns but in practice identified by phenotypic markers. Because isolation methods ultimately inform models of memory T cell differentiation, protection, and vaccine translation, we tested their validity via parabiosis and quantitative immunofluorescence microscopy of a mouse memory CD8 T cell population. We report three major findings: lymphocyte isolation fails to recover most cells and biases against certain subsets, residents greatly outnumber recirculating cells within non-lymphoid tissues, and memory subset homing to inflammation does not conform to previously hypothesized migration patterns. These results indicate that most host cells are surveyed for reinfection by segregated residents rather than by recirculating cells that migrate throughout the blood and body., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Effector CD8(+) T cells scan tissues to locate and kill infected host cells. In this issue of Immunity, Hickman et al. (2015) show that the exploration is not random: infected monocytes attract their assassins by secreting chemokines, which accelerates clearance of epicutaneous vaccinia virus infection., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Vaccines are desired that maintain abundant memory T cells at nonlymphoid sites of microbial exposure, where they may be anatomically positioned for immediate pathogen interception. Here, we test the impact of antigen persistence on mouse CD8 and CD4 T cell distribution and differentiation by comparing responses to infections with different strains of LCMV that cause either acute or chronic infections. We used in vivo labeling techniques that discriminate between T cells present within tissues and abundant populations that fail to be removed from vascular compartments, despite perfusion. LCMV persistence caused up to ∼30-fold more virus-specific CD8 T cells to distribute to the lung compared with acute infection. Persistent infection also maintained mucosal-homing α4β7 integrin expression, higher granzyme B expression, alterations in the expression of the TRM markers CD69 and CD103, and greater accumulation of virus-specific CD8 T cells in the large intestine, liver, kidney, and female reproductive tract. Persistent infection also increased LCMV-specific CD4 T cell quantity in mucosal tissues and induced maintenance of CXCR4, an HIV coreceptor. This study clarifies the relationship between viral persistence and CD4 and CD8 T cell distribution and mucosal phenotype, indicating that chronic LCMV infection magnifies T cell migration to nonlymphoid tissues., (© Society for Leukocyte Biology.)

The pathogen recognition theory dictates that, upon viral infection, the innate immune system first detects microbial products and then responds by providing instructions to adaptive CD8 T cells. Here, we show in mice that tissue resident memory CD8 T cells (T(RM) cells), non-recirculating cells located at common sites of infection, can achieve near-sterilizing immunity against viral infections by reversing this flow of information. Upon antigen resensitization within the mouse female reproductive mucosae, CD8(+) T(RM) cells secrete cytokines that trigger rapid adaptive and innate immune responses, including local humoral responses, maturation of local dendritic cells, and activation of natural killer cells. This provided near-sterilizing immunity against an antigenically unrelated viral infection. Thus, CD8(+) T(RM) cells rapidly trigger an antiviral state by amplifying receptor-derived signals from previously encountered pathogens., (Copyright © 2014, American Association for the Advancement of Science.)

Resident memory T cells (TRM) comprise a subset of nonrecirculating memory T cells that remain positioned at common portals of reinfection. These include barrier tissues such as the mucosae and skin. TRM orchestrate the initial response to pathogens re-encountered at these locales, thereby accelerating protective immune responses., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Previous studies from our laboratory revealed that cellular poly(C) binding protein 2 (PCBP2) downregulates vesicular stomatitis virus (VSV) gene expression. We show here that VSV infection induces the formation of granular structures in the cytoplasm containing cellular RNA-binding proteins, including PCBP2, T-cell-restricted intracellular antigen 1 (TIA1), and TIA1-related protein (TIAR). Depletion of TIA1 via small interfering RNAs (siRNAs), but not depletion of TIAR, results in enhanced VSV growth and gene expression. The VSV-induced granules appear to be similar to the stress granules (SGs) generated in cells triggered by heat shock or oxidative stress but do not contain some of the bona fide SG markers, such as eukaryotic initiation factor 3 (eIF3) or eIF4A, or the processing body (PB) markers, such as mRNA-decapping enzyme 1A (DCP1a), and thus may not represent canonical SGs or PBs. Our results revealed that the VSV-induced granules, called SG-like structures here, contain the viral replicative proteins and RNAs. The formation and maintenance of the SG-like structures required viral replication and ongoing protein synthesis, but an intact cytoskeletal network was not necessary. These results suggest that cells respond to VSV infection by aggregating the antiviral proteins, such as PCBP2 and TIA1, to form SG-like structures. The functional significance of these SG-like structures in VSV-infected cells is currently under investigation.

The non-structural protein 1 (nsp1) of porcine reproductive and respiratory syndrome virus is partly responsible for inhibition of type I interferon (IFN) response by the infected host. By performing alanine-scanning mutagenesis, we have identified amino acid residues in nsp1α and nsp1β (the proteolytic products of nsp1) that when substituted with alanine(s) exhibited significant relief of IFN-suppression. A mutant virus (16-5A, in which residues 16-20 of nsp1β were substituted with alanines) encoding mutant nsp1β recovered from infectious cDNA clone was shown to be attenuated for growth in vitro and induced significantly higher amount of type I IFN transcripts in infected macrophages. In infected pigs, the 16-5A virus exhibited reduced growth at early times after infection but quickly regained wild type growth properties as a result of substitutions within the mutated sequences. The results indicate a strong selection pressure towards maintaining the IFN-inhibitory property of the virus for successful propagation in pigs., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Porcine reproductive and respiratory syndrome virus (PRRSV) suppresses tumor necrosis factor-alpha (TNF-α) production at both transcriptional and post-transcriptional levels by its non-structural proteins 1α and 1β (Nsp1α and Nsp1β). To identify the amino acid residues responsible for this activity, we generated several alanine substitution mutants of Nsp1α and Nsp1β. Examination of the mutant proteins revealed that Nsp1α residues Gly90, Asn91, Arg97, Arg100 and Arg124 were necessary for TNF-α promoter suppression, whereas several amino acids spanning the entire Nsp1β were found to be required for this activity. Two mutant viruses, with mutations at Nsp1α Gly90 or Nsp1β residues 70-74, generated from infectious cDNA clones, exhibited attenuated viral replication in vitro and TNF-α was found to be up regulated in infected macrophages. In infected pigs, the Nsp1β mutant virus was attenuated in growth. These studies provide insights into how PRRSV evades the effector mechanisms of innate immunity during infection., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Differentiation and maintenance of recirculating effector memory CD8 T cells (T(EM)) depends on prolonged cognate Ag stimulation. Whether similar pathways of differentiation exist for recently identified tissue-resident effector memory T cells (T(RM)), which contribute to rapid local protection upon pathogen re-exposure, is unknown. Memory CD8αβ(+) T cells within small intestine epithelium are well-characterized examples of T(RM), and they maintain a long-lived effector-like phenotype that is highly suggestive of persistent Ag stimulation. This study sought to define the sources and requirements for prolonged Ag stimulation in programming this differentiation state, including local stimulation via cognate or cross-reactive Ags derived from pathogens, microbial flora, or dietary proteins. Contrary to expectations, we found that prolonged cognate Ag stimulation was dispensable for intestinal T(RM) ontogeny. In fact, chronic antigenic stimulation skewed differentiation away from the canonical intestinal T cell phenotype. Resident memory signatures, CD69 and CD103, were expressed in many nonlymphoid tissues including intestine, stomach, kidney, reproductive tract, pancreas, brain, heart, and salivary gland and could be driven by cytokines. Moreover, TGF-β-driven CD103 expression was required for T(RM) maintenance within intestinal epithelium in vivo. Thus, induction and maintenance of long-lived effector-like intestinal T(RM) differed from classic models of T(EM) ontogeny and were programmed through a novel location-dependent pathway that was required for the persistence of local immunological memory.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection of swine results in substantial economic losses to the swine industry worldwide. Identification of cellular factors involved in PRRSV life cycle not only will enable a better understanding of virus biology but also has the potential for the development of antiviral therapeutics. The PRRSV nonstructural protein 1 (nsp1) has been shown to be involved in at least two important functions in the infected hosts: (i) mediation of viral subgenomic (sg) mRNA transcription and (ii) suppression of the host's innate immune response mechanisms. To further our understanding of the role of the viral nsp1 in these processes, using nsp1β, a proteolytically processed functional product of nsp1 as bait, we have identified the cellular poly(C)-binding proteins 1 and 2 (PCBP1 and PCBP2) as two of its interaction partners. The interactions of PCBP1 and PCBP2 with nsp1β were confirmed both by coimmunoprecipitation in infected cells and/or in plasmid-transfected cells and also by in vitro binding assays. During PRRSV infection of MARC-145 cells, the cytoplasmic PCBP1 and PCBP2 partially colocalize to the viral replication-transcription complexes. Furthermore, recombinant purified PCBP1 and PCBP2 were found to bind the viral 5' untranslated region (5'UTR). Small interfering RNA (siRNA)-mediated silencing of PCBP1 and PCBP2 in cells resulted in significantly reduced PRRSV genome replication and transcription without adverse effect on initial polyprotein synthesis. Overall, the results presented here point toward an important role for PCBP1 and PCBP2 in regulating PRRSV RNA synthesis.

Immunoprecipitation and subsequent mass spectrometry analysis of the cellular proteins from cells expressing the vesicular stomatitis virus (VSV) P protein identified the poly(C) binding protein 2 (PCBP2) as one of the P protein-interacting proteins. To investigate the role of PCBP2 in the viral life cycle, we examined the effects of depletion or overexpression of this protein on VSV growth. Small interfering RNA-mediated silencing of PCBP2 promoted VSV replication. Conversely, overexpression of PCBP2 in transfected cells suppressed VSV growth. Further studies revealed that PCBP2 negatively regulates overall viral mRNA accumulation and subsequent genome replication. Coimmunoprecipitation and immunofluorescence microscopic studies showed that PCBP2 interacts and colocalizes with VSV P protein in virus-infected cells. The P-PCBP2 interaction did not result in reduced levels of protein complex formation with the viral N and L proteins, nor did it induce degradation of the P protein. In addition, PCBP1, another member of the poly(C) binding protein family with homology to PCBP2, was also found to interact with the P protein and inhibit the viral mRNA synthesis at the level of primary transcription without affecting secondary transcription or genome replication. The inhibitory effects of PCBP1 on VSV replication were less pronounced than those of PCBP2. Overall, the results presented here suggest that cellular PCBP2 and PCBP1 antagonize VSV growth by affecting viral gene expression and highlight the importance of these two cellular proteins in restricting virus infections.

The objective of this study was to identify porcine reproductive and respiratory syndrome virus (PRRSV)-encoded proteins that are responsible for the inhibition of TNF-α expression and the mechanism(s) involved in this phenomenon. Using a TNF-α promoter reporter system, the non-structural protein 1 (Nsp1) was found to strongly suppress the TNF-α promoter activity. Such inhibition takes place especially at the promoter's proximal region. Both Nsp1α and Nsp1β, the two proteolytic fragments of Nsp1, were shown to be involved in TNF-α promoter suppression. Furthermore, using reporter plasmids specific for transcription factors (TFs) that bind to TNF-α promoter, Nsp1α and Nsp1β were demonstrated to inhibit the activity of the TFs that bind CRE-κB(3) and Sp1 elements respectively. Subsequent analyses showed that Nsp1α moderately inhibits NF-κB activation and that Nsp1β completely abrogates the Sp1 transactivation. These findings reveal one of the important mechanisms underlying the innate immune evasion by PRRSV during infection., (Copyright © 2010 Elsevier Inc. All rights reserved.)

We show here that replication of defective interfering (DI) particle RNA in HEK293 cells stably expressing vesicular stomatitis virus (VSV) replication proteins potently activates interferon (IFN) and IFN signaling pathways through upregulation of IFN-beta promoter, IFN-stimulated response element (ISRE) promoter, and NF-kappaB promoter activities. Replication of DI particle RNA, not mere expression of the viral replication proteins, was found to be critical for induction of IFN and IFN signaling. The stable cells supporting replication of DI RNA described in this report will be useful in further examining the innate immune signaling pathways and the host cell functions in viral genome replication.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection of swine leads to a serious disease characterized by a delayed and defective adaptive immune response. It is hypothesized that a suboptimal innate immune response is responsible for the disease pathogenesis. In the study presented here we tested this hypothesis and identified several nonstructural proteins (NSPs) with innate immune evasion properties encoded by the PRRS viral genome. Four of the total ten PRRSV NSPs tested were found to have strong to moderate inhibitory effects on beta interferon (IFN-beta) promoter activation. The strongest inhibitory effect was exhibited by NSP1 followed by, NSP2, NSP11, and NSP4. We focused on NSP1alpha and NSP1beta (self-cleavage products of NSP1 during virus infection) and NSP11, three NSPs with strong inhibitory activity. All of three proteins, when expressed stably in cell lines, strongly inhibited double-stranded RNA (dsRNA) signaling pathways. NSP1beta was found to inhibit both IFN regulatory factor 3 (IRF3)- and NF-kappaB-dependent gene induction by dsRNA and Sendai virus. Mechanistically, the dsRNA-induced phosphorylation and nuclear translocation of IRF3 were strongly inhibited by NSP1beta. Moreover, when tested in a porcine myelomonocytic cell line, NSP1beta inhibited Sendai virus-mediated activation of porcine IFN-beta promoter activity. We propose that this NSP1beta-mediated subversion of the host innate immune response plays an important role in PRRSV pathogenesis.