Kathryn A. Fraser

Dynamic T cell migration program provides resident memory within intestinal epithelium

Migration to intestinal mucosa putatively depends on local activation because gastrointestinal lymphoid tissue induces expression of intestinal homing molecules, whereas skin-draining lymph nodes do not. This paradigm is difficult to reconcile with reports of intestinal T cell responses after alternative routes of immunization. We reconcile this discrepancy by demonstrating that activation within spleen results in intermediate induction of homing potential to the intestinal mucosa. We further demonstrate that memory T cells within small intestine epithelium do not routinely recirculate with memory T cells in other tissues, and we provide evidence that homing is similarly dynamic in humans after subcutaneous live yellow fever vaccine immunization. These data explain why systemic immunization routes induce local cell-mediated immunity within the intestine and indicate that this tissue must be seeded with memory T cell precursors shortly after activation.

Normalizing the environment recapitulates adult human immune traits in laboratory mice.

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.Our current understanding of immunology was largely defined in laboratory mice because of experimental advantages including inbred homogeneity, tools for genetic manipulation, the ability to perform kinetic tissue analyses starting with the onset of disease, and tractable models. Comparably reductionist experiments are neither technically nor ethically possible in humans. Despite revealing many fundamental principals of immunology, there is growing concern that mice fail to capture relevant aspects of the human immune system, which may account for failures to translate disease treatments from bench to bedside1–8. Laboratory mice live in abnormally hygienic “specific pathogen free” (SPF) barrier facilities. Here we show that the standard practice of laboratory mouse husbandry has profound effects on the immune system and that environmental changes result in better recapitulation of features of adult humans. Laboratory mice lack effector-differentiated and mucosally distributed memory T cells, which more closely resembles neonatal than adult humans. These cell populations were present in free-living barn populations of feral mice, pet store mice with diverse microbial experience, and were induced in laboratory mice after co-housing with pet store mice, suggesting a role for environment. Consequences of altering mouse housing profoundly impacted the cellular composition of the innate and adaptive immune system and resulted in global changes in blood cell gene expression patterns that more closely aligned with immune signatures of adult humans rather than neonates, altered the mouse’s resistance to infection, and impacted T cell differentiation to a de novo viral infection. These data highlight the impact 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 modeling immunological events in free-living organisms, including humans.

Antigen-Independent Differentiation and Maintenance of Effector-like Resident Memory T Cells in Tissues

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.

Sensing and alarm function of resident memory CD8 + T cells

CD8+ T cells eliminate intracellular infections through two contact-dependent effector functions: cytolysis and secretion of antiviral cytokines. Here we identify the following additional function for memory CD8+ T cells that persist at front-line sites of microbial exposure: to serve as local sensors of previously encountered antigens that precipitate innate-like alarm signals and draw circulating memory CD8+ T cells into the tissue. When memory CD8+ T cells residing in the female mouse reproductive tract encountered cognate antigen, they expressed interferon-γ (IFN-γ), potentiated robust local expression of inflammatory chemokines and induced rapid recruitment of circulating memory CD8+ T cells. Anamnestic responses in front-line tissues are thus an integrated collaboration between front-line and circulating populations of memory CD8+ T cells, and vaccines should establish both populations to maximize rapid responses.

Resident memory CD8 T cells trigger protective innate and adaptive immune responses

Resident memory T cells sound the alarm Immunological memory protects against reinfection. Resident memory T cells (TRM) 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+ TRM cells act like first responders in the female reproductive tissue or the skin of mice upon antigen reencounter. By secreting inflammatory proteins, TRM 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+ TRM cells protected mice against reinfection with intravaginal herpes simplex virus 2. Science, this issue p. 98, p. 101, p. 93; see also p. 40 Resident memory CD8+ T cells orchestrate a broad immune response in response to reinfection. [Also see Perspective by Carbone and Gebhardt] 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 (TRM 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+ TRM 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+ TRM cells rapidly trigger an antiviral state by amplifying receptor-derived signals from previously encountered pathogens.

Quantifying Memory CD8 T Cells Reveals Regionalization of Immunosurveillance.

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.

Cutting Edge: Resident Memory CD8 T Cells Occupy Frontline Niches in Secondary Lymphoid Organs

Resident memory CD8 T cells (TRM) are a nonrecirculating subset positioned in nonlymphoid tissues to provide early responses to reinfection. Although TRM are associated with nonlymphoid tissues, we asked whether they populated secondary lymphoid organs (SLO). We show that a subset of virus-specific memory CD8 T cells in SLO exhibit phenotypic signatures associated with TRM, including CD69 expression. Parabiosis revealed that SLO CD69+ memory CD8 T cells do not circulate, defining them as TRM. SLO TRM were overrepresented in IL-15–deficient mice, suggesting independent regulation compared with central memory CD8 T cells and effector memory CD8 T cells. These cells were positioned at SLO entry points for peripheral Ags: the splenic marginal zone, red pulp, and lymph node sinuses. Consistent with a potential role in guarding SLO pathogen entry points, SLO TRM did not vacate their position in response to peripheral alarm signals. These data extend the range of tissue resident memory to SLO.

Preexisting High Frequencies of Memory CD8+ T Cells Favor Rapid Memory Differentiation and Preservation of Proliferative Potential upon Boosting

Memory CD8+ T cell quantity and quality determine protective efficacy against reinfection. Heterologous prime boost vaccination minimizes contraction of anamnestic effectors and maximizes memory CD8+ T cell quantity but reportedly erodes proliferative potential and protective efficacy. This study exploited heterologous prime boost vaccination to discover parameters regulating effector CD8+ T cell contraction and memory differentiation. When abundant memory T cells were established, boosting induced only 5-8 cell divisions, unusually rapid memory T cell differentiation as measured by phenotype and mitochondrial bioenergetic function, long-lived survival of 50% of effector T cells, and preservation of proliferative potential. Conversely, boosting in situations of low memory CD8+ T cell frequencies induced many cell divisions, increased contraction of effector cells, and caused senescence, low mitochondrial membrane potential, and poorly protective memory. Thus, anamnestic memory T cell differentiation is flexible, and abundant quantity can be achieved while maximizing protective efficacy and preserving proliferative potential.

IL-15–Independent Maintenance of Tissue-Resident and Boosted Effector Memory CD8 T Cells

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.

Lymphocytic choriomeningitis virus persistence promotes effector-like memory differentiation and enhances mucosal T cell distribution.

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.