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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.

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.

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.

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.

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 (TRM) 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 TRM 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 TRM 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 TRM., Irreversible electroporation (IRE), a soft-tissue ablation technique used for tumour ablation, has been suggested to promote systemic immune responses. Here the authors show that IRE, followed by anti-CTLA-4 blockade, elicits the expansion of tumor antigen-specific CD8+ T cells and is associated with tissue residency and improved anti-tumor immune response in a preclinical model of prostate cancer.

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 (TRM) are critical for pathogen control at barrier sites. Identifying TRM 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 TRM, yet it is unclear whether CD69 is universally required for producing or retaining TRM. 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 TRM 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+ TRM generation in the kidney but not in other tissues. Our findings indicate that the functional relevance of CD69 in generation and maintenance of CD8+ TRM 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+ TRM.

CD4 T cell localization impacts function and differentiation. Beura et al. show that memory CD4+ T cells are largely resident in both lymphoid and non-lymphoid tissues, organize local recall responses, and share overlapping transcriptional and location-specific features with CD8+ TRM., This study examines the extent to which memory CD4+ T cells share immunosurveillance strategies with CD8+ resident memory T cells (TRM). 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+ TRM 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+ TRM 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., Graphical Abstract

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 assessed 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 TCRgd and CD8aa T cells showed marked increase in mice receiving the 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.

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 potential1,2, 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. Investigations in mice using parabiosis and cohousing experiments reveal that nonlymphoid organs serve as reservoirs of tissue-autonomous cellular immunity, leading to the decentralization of organism-level immune homeostasis.

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.

Tissue resident memory T cells (TRM) 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, TRM reside permanently within tissues. As the main surveyors of non-lymphoid tissues, TRM are positioned to rapidly respond upon reinfection at barrier sites. During a viral reinfection, TRM 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 TRM 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 TRM in multiple viral challenge models and contextualize the implications of these findings for vaccine development.

Stolley et al. demonstrate that the decay of lung CD8+ T cell responses after primary influenza virus infection is contemporized by the egress of TRM-phenotype T cells from the lung to the draining mediastinal LN by retrograde migration, where they form more stable residents., 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., Graphical Abstract

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

Regulated activation of the cytokine TGFβ 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 TGFβ resulted in a gradual loss of E- or P-selectin-binding central and peripheral memory populations, that was 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.

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.

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.

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.

International audience; 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-beta1 (TGF-beta) for epidermal residence. Here we found that integrins alphavbeta6 and alphavbeta8 were expressed in non-overlapping patterns by keratinocytes (KCs) and maintained the epidermal residence of LCs and TRM cells by activating latent TGF-beta. Similarly, the residence of dendritic cells and TRM cells in the small intestine epithelium also required alphavbeta6. Treatment of the skin with ultraviolet irradiation decreased integrin expression on KCs and reduced the availability of active TGF-beta, which resulted in LC migration. Our data demonstrated that regulated activation of TGF-beta by stromal cells was able to directly control epithelial residence of cells of the immune system through a novel mechanism of intercellular communication

Lymphocytic choriomeningitis virus (LCMV) is a natural mouse pathogen. LCMV Armstrong, an acutely resolved strain, and LCMV Clone13, 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. Here, we describe LCMV Minnesota (LCMV-MN), which was 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 widely disseminated viral replication and viremia that was controlled within 15–30 days; which is of intermediate duration between LCMV Armstrong and Clone13. The magnitude of the LCMV-MN specific CD8+ T cell was maintained at much higher levels than that observed after LCMV Armstrong or Clone13 infections. LCMV-MN responding CD8+ T cells were further associated with significantly biased differentiation towards the recently described PD1+ CXCR5+ Tim-3lo stem-like CD8+ T cell population that was previously shown to be largely responsible for responsiveness to PD-1 inhibitory checkpoint blockade. In contrast to LCMV Clone13-induced responses, this subset persisted after resolution of LCMV-MN viremia, yet transcriptionally, phenotypically and functionally resembled PD1+ TCF1+ stem-like CD8+ T cells maintained by LCMV Clone13 infection. Together with existing models, LCMV-MN may contribute to a better understanding of the breadth of immune response in different chronic infections or tumor settings as well as the regulation of responsiveness to PD-1 blockade.

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

Immune cells develop in specialized lymphoid organs, but the mature immune system is integrated throughout the body and regulates tissue homeostasis and defense. This study examines how immunity persists within adult murine tissues while addressing issues of tissue autonomy, resistance to perturbations, and numerical contribution to the cellularity of visceral organs. Using quantitative immunofluorescent microscopy, we report that the immune system is expansible in nonlymphoid tissues following microbial experience, and hematopoietic cells comprise up to 30% of cells in visceral organs of laboratory mice housed in non-SPF conditions. Most hematopoietic cells in nonlymphoid tissues were autonomously maintained for >30 days, without contribution from circulation or primary lymphoid organs. Focusing on antiviral T cell immunity, we demonstrate tissue-autonomous maintenance of resident memory T cells for >200 days. Resident T cell memory was largely durable following significant environmental perturbations and de novo immune responses. Once established, resident T cells did not require the T cell receptor for survival, longevity, or retention of a poised effector-like phenotype. Ultimately, T cell memory did decay in some organs, not others, and gradually increased in circulation. Surgical separation of parabiotic mice unexpectedly revealed a tissue-resident provenance for memory T cells in peripheral blood. We propose a model whereby circulating memory T cells do not replenish resident populations, but instead a tissue-resident reservoir supports long-term maintenance of the bloodborne pool. Collectively, these data demonstrate the durability and autonomous nature of the tissue-integrated immune system.

Many United States 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 U.S. immigration on the gut microbiome. We collected stool, dietary recalls, and anthropometrics from 514 Hmong and Karen individuals living in Thailand and the U.S., including first- and second-generation immigrants and 19 Karen individuals sampled before and after immigration, as well as from 36 U.S.-born European American individuals. Using 16S and deep shotgun metagenomic DNA sequencing, we found that migration from a non-Western country to the U.S. is associated with immediate loss of gut microbiome diversity and function, in which U.S.-associated strains and functions displace native strains and functions. These effects increase with duration of U.S. residence, and are compounded by obesity and across generations.

Current paradigms of CD8 + T cell–mediated protection in HIV infection center almost exclusively on studies of peripheral blood, which is thought to provide a window into immune activity at the predominant sites of viral replication in lymphoid tissues (LTs). Through extensive comparison of blood, thoracic duct lymph (TDL), and LTs in different species, we show that many LT memory CD8 + T cells bear phenotypic, transcriptional, and epigenetic signatures of resident memory T cells (T RMs ). Unlike their circulating counterparts in blood or TDL, most of the total and follicular HIV-specific CD8 + T cells in LTs also resemble T RMs . Moreover, high frequencies of HIV-specific CD8 + T RMs with skewed clonotypic profiles relative to matched blood samples are present in LTs of individuals who spontaneously control HIV replication in the absence of antiretroviral therapy (elite controllers). Single-cell RNA sequencing analysis confirmed that HIV-specific T RMs are enriched for effector-related immune genes and signatures compared with HIV-specific non-T RMs in elite controllers. Together, these data indicate that previous studies in blood have largely failed to capture the major component of HIV-specific CD8 + T cell responses resident within LTs.

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.

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 (TRM cells) within the reproductive tracts of live female mice. We found that mucosal TRM cells were highly motile, but paused and underwent in situ division after local antigen challenge. TRM cell reactivation triggered the recruitment of recirculating memory T cells that underwent antigen-independent TRM cell differentiation in situ. However, the proliferation of pre-existing TRM cells dominated the local mucosal recall response and contributed most substantially to the boosted secondary TRM cell population. We observed similar results in skin. Thus, TRM cells can autonomously regulate the expansion of local immunosurveillance independently of central memory or proliferation in lymphoid tissue. Masopust and colleagues show that mucosal tissue-resident memory T cells proliferate in situ in response to local antigen and dominate the local recall response.

Extracellular ATP (eATP) is an ancient 'danger signal' used by eukaryotes to detect cellular damage1. In mice and humans, the release of eATP during inflammation or injury stimulates both innate immune activation and chronic pain through the purinergic receptor P2RX72-4. 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-infection5,6. 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.

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. Electronic supplementary material The online version of this article (doi:10.1186/s40168-017-0306-2) contains supplementary material, which is available to authorized users.

Characterization of the cellular participants in tissue immune responses is crucial to understanding infection, cancer, autoimmunity, allergy, graft rejection and other immunological processes. Previous reports indicate that leukocytes in lung vasculature fail to be completely removed by perfusion. Several studies suggest that intravascular staining may discriminate between tissue-localized and blood-borne cells in the mouse lung. Here we outline a protocol for the validation and use of intravascular staining to define innate and adaptive immune cells in mice. We demonstrate application of this protocol to leukocyte analyses in many tissues and we describe its use in the contexts of lymphocytic choriomeningitis virus and Mycobacterium tuberculosis infections or solid tumors. Intravascular staining and organ isolation usually takes 5-30 min per mouse, with additional time required for any subsequent leukocyte isolation, staining and analysis. In summary, this simple protocol should help enable interpretable analyses of tissue immune responses.

Recent studies have established resident memory T cells (TRM) as the dominant memory lymphocyte population surveying most non-lymphoid tissues. Unlike other memory T cell lineages, TRM do not recirculate through blood and are permanently confined to their tissue of residence. TRM 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 TRM differentiation, maintenance and their protective function. While little is known, we have speculated on the potential implications of TRM for transplantation biology. Areas of emphasis include the role of passenger TRM in controlling latent viral recrudescence in donor organs, donor TRM as a source of graft-versus-host disease (GVHD), the ability of TRM to potently induce inflammation through sensing and alarm functions and differentiation of host T cells into TRM in response to local cues inside the allograft. Further investigation of TRM in the context of transplantation might identify therapeutic targets to prolong graft survival.

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.

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.

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.

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.

Summary 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.

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-β) promoter activation. The strongest inhibitory effect was exhibited by NSP1 followed by, NSP2, NSP11, and NSP4. We focused on NSP1α and NSP1β (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. NSP1β was found to inhibit both IFN regulatory factor 3 (IRF3)- and NF-κB-dependent gene induction by dsRNA and Sendai virus. Mechanistically, the dsRNA-induced phosphorylation and nuclear translocation of IRF3 were strongly inhibited by NSP1β. Moreover, when tested in a porcine myelomonocytic cell line, NSP1β inhibited Sendai virus-mediated activation of porcine IFN-β promoter activity. We propose that this NSP1β-mediated subversion of the host innate immune response plays an important role in PRRSV pathogenesis.

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.

CD8+ T cells play a critical role limiting peripheral virus replication, yet how they locate virus-infected cells within tissues is unknown. Here, we have examined the environmental signals that CD8+ T cells use to localize and eliminate virus-infected skin cells. Epicutaneous vaccinia virus (VV) infection, mimicking human smallpox vaccination, greatly increased expression of the CXCR3 chemokine receptor ligands CXCL9 and -10 in VV-infected skin. Despite normal T cell numbers in the skin, Cxcr3−/− mice exhibited dramatically impaired CD8+ T cell-dependent virus clearance. Intravital microscopy revealed that Cxcr3−/− T cells were markedly deficient in locating, engaging, and killing virus-infected cells. Further, transfer of wild-type CD8+ T cells restored viral clearance in Cxcr3−/−animals. These findings demonstrate a function for CXCR3 in enhancing the ability of tissue-localized CD8+ T cells to locate virus-infected cells and thereby exert anti-viral effector functions.

The small intestine (SI) represents the largest mucosal barrier between the environment and the host. As such, the SI limits both normal and pathogenic microbe invasion and must balance immune tolerance and activation. CD8ab T cells constitute a dominant lymphocyte population in the SI and contribute to both of these processes but detailed investigation into their in situ behavior in response to various stimuli is lacking. We visualized Ag-specific CD8 T cell migration in the SI using two-photon microscopy to understand CD8 T cell immunosurveillance. We found that pathogen-specific CD8 T cells are initially slowed early after infection but regain motility directly after Ag clearance. At a memory time point, CD8 T cells exhibited slower migration concurrent with acquisition of a resident memory phenotype. Current studies are focused on determining what is required for CD8 T cell motility throughout the course of infection. Additionally, as tolerance reversal and expansion of self-specific CD8 T cells has recently become a focus of cancer therapy, we characterized in situ behavior of self-specific CD8 T cells. Using a mouse expressing a model Ag in the SI as a self-protein, we observed self-specific CD8 T cells before and after tolerance reversal. Tolerant CD8 T cells migrate significantly faster than pathogen-specific cells at acute time points after infection. Contrastingly, ex-tolerant CD8 T cells were comparable to pathogen-specific CD8 T cells indicating that indeed their in situ scanning behavior had been altered upon tolerance reversal. These observations have provided valuable insight into CD8 T cell motility within the SI and can inform targeted treatment options and vaccines to ensure effective immunosurveillance of this tissue.

CD8 T cells survey tissues for the presence of abnormal cells. The parameters of CD8 T cell search patterns and immunosurveillance determines outcome of infections and cancer (elimination, controlled persistence, or death). We established a two-photon intravital microscopy model to visualize resident memory CD8 T cell (TRM) behavior and recall responses within the female reproductive tract (FRT) mucosa of living mice. Unlike what has been described in skin epidermis, FRT TRM were highly motile (10 μm/min). Local antigen recognition induced pausing and initiation of in situ TRM division, and this appeared CD4 T cell and dendritic cell independent. Expansion by dividing TRM dominated the local recall response compared to recruitment of recirculating cells or progeny of reactivated central memory T cells. TRM reactivation also triggered recruitment of Ag-specific and bystander recirculating memory cells, and these underwent a de novo TRM differentiation program in situ. These results demonstrate that 1) T cell migration patterns and differentiation may be permanently altered by unrelated infectious experience, 2) that cognate antigen within the FRT is not required to induce local TRM differentiation, 3) TRM undergo in situ division upon recall that 4) may dominate the local secondary effector and memory response.

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.

SummaryMemory 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.

Resident memory T cells sound the alarm Immunological memory protects against reinfection. Resident memory T cells (T RM ) are long-lived and remain in the tissues where they first encountered a pathogen (see the Perspective by Carbone and Gebhardt). Schenkel et al. and Ariotti et al. found that CD8 + T RM cells act like first responders in the female reproductive tissue or the skin of mice upon antigen reencounter. By secreting inflammatory proteins, T RM cells rapidly activated local immune cells to respond, so much so that they protected against infection with an unrelated pathogen. Iijima and Iwasaki found that CD4 + T RM cells protected mice against reinfection with intravaginal herpes simplex virus 2. Science , this issue p. 98 , p. 101 , p. 93 ; see also p. 40

Differentiation and maintenance of recirculating effector memory CD8 T cells (TEM) depends on prolonged cognate Ag stimulation. Whether similar pathways of differentiation exist for recently identified tissue-resident effector memory T cells (TRM), 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 TRM, 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 TRM 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 TRM maintenance within intestinal epithelium in vivo. Thus, induction and maintenance of long-lived effector-like intestinal TRM differed from classic models of TEM ontogeny and were programmed through a novel location-dependent pathway that was required for the persistence of local immunological memory.