Programs for the persistence, vigilance and control of human CD8+ lung-resident memory T cells.

Tissue-resident memory T cells (TRM cells) in the airways mediate protection against respiratory infection. We characterized TRM cells expressing integrin αE (CD103) that reside within the epithelial barrier of human lungs. These cells had specialized profiles of chemokine receptors and adhesion molecules, consistent with their unique localization. Lung TRM cells were poised for rapid responsiveness by constitutive expression of deployment-ready mRNA encoding effector molecules, but they also expressed many inhibitory regulators, suggestive of programmed restraint. A distinct set of transcription factors was active in CD103+ TRM cells, including Notch. Genetic and pharmacological experiments with mice revealed that Notch activity was required for the maintenance of CD103+ TRM cells. We have thus identified specialized programs underlying the residence, persistence, vigilance and tight control of human lung TRM cells.

A central role for Notch in effector CD8(+) T cell differentiation.

Activated CD8(+) T cells choose between terminal effector cell (TEC) or memory precursor cell (MPC) fates. We found that the signaling receptor Notch controls this 'choice'. Notch promoted the differentiation of immediately protective TECs and was correspondingly required for the clearance of acute infection with influenza virus. Notch activated a major portion of the TEC-specific gene-expression program and suppressed the MPC-specific program. Expression of Notch was induced on naive CD8(+) T cells by inflammatory mediators and interleukin 2 (IL-2) via pathways dependent on the metabolic checkpoint kinase mTOR and the transcription factor T-bet. These pathways were subsequently amplified downstream of Notch, creating a positive feedback loop. Notch thus functions as a central hub where information from different sources converges to match effector T cell differentiation to the demands of an infection.

Classical DC2 subsets and monocyte-derived DC: Delineating the developmental and functional relationship.

To specifically tailor immune responses to a given pathogenic threat, dendritic cells (DC) are highly heterogeneous and comprise many specialized subtypes, including conventional DC (cDC) and monocyte-derived DC (MoDC), each with distinct developmental and functional characteristics. However, the functional relationship between cDC and MoDC is not fully understood, as the overlapping phenotypes of certain type 2 cDC (cDC2) subsets and MoDC do not allow satisfactory distinction of these cells in the tissue, particularly during inflammation. However, precise cDC2 and MoDC classification is required for studies addressing how these diverse cell types control immune responses and is therefore currently one of the major interests in the field of cDC research. This review will revise murine cDC2 and MoDC biology in the steady state and under inflammatory conditions and discusses the commonalities and differences between ESAMlo cDC2, inflammatory cDC2, and MoDC and their relative contribution to the initiation, propagation, and regulation of immune responses.

Guidelines for DC preparation and flow cytometry analysis of mouse nonlymphoid tissues.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various nonlymphoid tissues. DC are sentinels of the immune system present in almost every mammalian organ. Since they represent a rare cell population, DC need to be extracted from organs with protocols that are specifically developed for each tissue. This article provides detailed protocols for the preparation of single-cell suspensions from various mouse nonlymphoid tissues, including skin, intestine, lung, kidney, mammary glands, oral mucosa and transplantable tumors. Furthermore, our guidelines include comprehensive protocols for multiplex flow cytometry analysis of DC subsets and feature top tricks for their proper discrimination from other myeloid cells. With this collection, we provide guidelines for in-depth analysis of DC subsets that will advance our understanding of their respective roles in healthy and diseased tissues. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all coauthors, making it an essential resource for basic and clinical DC immunologists.

Posttranslational modifications by ADAM10 shape myeloid antigen-presenting cell homeostasis in the splenic marginal zone.

The spleen contains phenotypically and functionally distinct conventional dendritic cell (cDC) subpopulations, termed cDC1 and cDC2, which each can be divided into several smaller and less well-characterized subsets. Despite advances in understanding the complexity of cDC ontogeny by transcriptional programming, the significance of posttranslational modifications in controlling tissue-specific cDC subset immunobiology remains elusive. Here, we identified the cell-surface-expressed A-disintegrin-and-metalloproteinase 10 (ADAM10) as an essential regulator of cDC1 and cDC2 homeostasis in the splenic marginal zone (MZ). Mice with a CD11c-specific deletion of ADAM10 (ADAM10ΔCD11c) exhibited a complete loss of splenic ESAMhi cDC2A because ADAM10 regulated the commitment, differentiation, and survival of these cells. The major pathways controlled by ADAM10 in ESAMhi cDC2A are Notch, signaling pathways involved in cell proliferation and survival (e.g., mTOR, PI3K/AKT, and EIF2 signaling), and EBI2-mediated localization within the MZ. In addition, we discovered that ADAM10 is a molecular switch regulating cDC2 subset heterogeneity in the spleen, as the disappearance of ESAMhi cDC2A in ADAM10ΔCD11c mice was compensated for by the emergence of a Clec12a+ cDC2B subset closely resembling cDC2 generally found in peripheral lymph nodes. Moreover, in ADAM10ΔCD11c mice, terminal differentiation of cDC1 was abrogated, resulting in severely reduced splenic Langerin+ cDC1 numbers. Next to the disturbed splenic cDC compartment, ADAM10 deficiency on CD11c+ cells led to an increase in marginal metallophilic macrophage (MMM) numbers. In conclusion, our data identify ADAM10 as a molecular hub on both cDC and MMM regulating their transcriptional programming, turnover, homeostasis, and ability to shape the anatomical niche of the MZ.

Guidelines for DC preparation and flow cytometry analysis of mouse lymphohematopoietic tissues.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human DC from lymphoid organs, and various non-lymphoid tissues. Within this chapter, detailed protocols are presented that allow for the generation of single-cell suspensions from mouse lymphohematopoietic tissues including spleen, peripheral lymph nodes, and thymus, with a focus on the subsequent analysis of DC by flow cytometry. However, prepared single-cell suspensions can be subjected to other applications including sorting and cellular enrichment procedures, RNA sequencing, Western blotting, and many more. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.

Guidelines for mouse and human DC functional assays.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. Recent studies have provided evidence for an increasing number of phenotypically distinct conventional DC (cDC) subsets that on one hand exhibit a certain functional plasticity, but on the other hand are characterized by their tissue- and context-dependent functional specialization. Here, we describe a selection of assays for the functional characterization of mouse and human cDC. The first two protocols illustrate analysis of cDC endocytosis and metabolism, followed by guidelines for transcriptomic and proteomic characterization of cDC populations. Then, a larger group of assays describes the characterization of cDC migration in vitro, ex vivo, and in vivo. The final guidelines measure cDC inflammasome and antigen (cross)-presentation activity. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.

Revisiting Current Concepts on the Tolerogenicity of Steady-State Dendritic Cell Subsets and Their Maturation Stages.

The original concept stated that immature dendritic cells (DC) act tolerogenically whereas mature DC behave strictly immunogenically. Meanwhile, it is also accepted that phenotypically mature stages of all conventional DC subsets can promote tolerance as steady-state migratory DC by transporting self-antigens to lymph nodes to exert unique functions on regulatory T cells. We propose that in vivo 1) there is little evidence for a tolerogenic function of immature DC during steady state such as CD4 T cell anergy induction, 2) all tolerance as steady-state migratory DC undergo common as well as subset-specific molecular changes, and 3) these changes differ by quantitative and qualitative markers from immunogenic DC, which allows one to clearly distinguish tolerogenic from immunogenic migratory DC.

Notch in T Cell Differentiation: All Things Considered.

Differentiation of naïve T cells into effector cells is required for optimal protection against different classes of microbial pathogen and for the development of immune memory. Recent findings have revealed important roles for the Notch signaling pathway in T cell differentiation into all known effector subsets, raising the question of how this pathway controls such diverse differentiation programs. Studies in preclinical models support the therapeutic potential of manipulating the Notch pathway to alleviate immune pathology, highlighting the importance of understanding the mechanisms through which Notch regulates T cell differentiation and function. We review these findings here, and outline both unifying principles involved in Notch-mediated T cell fate decisions and cell type- and context-specific differences that may present the most suitable points for therapeutic intervention.

Notch controls the magnitude of T helper cell responses by promoting cellular longevity.

Generation of effective immune responses requires expansion of rare antigen-specific CD4(+) T cells. The magnitude of the responding population is ultimately determined by proliferation and survival. Both processes are tightly controlled to limit responses to innocuous antigens. Sustained expansion occurs only when innate immune sensors are activated by microbial stimuli or by adjuvants, which has important implications for vaccination. The molecular identity of the signals controlling sustained T-cell responses is not fully clear. Here, we describe a prominent role for the Notch pathway in this process. Coactivation of Notch allows accumulation of far greater numbers of activated CD4(+) T cells than stimulation via T-cell receptor and classic costimulation alone. Notch does not overtly affect cell cycle entry or progression of CD4(+) T cells. Instead, Notch protects activated CD4(+) T cells against apoptosis after an initial phase of clonal expansion. Notch induces a broad antiapoptotic gene expression program that protects against intrinsic, as well as extrinsic, apoptosis pathways. Both Notch1 and Notch2 receptors and the canonical effector RBPJ (recombination signal binding protein for immunoglobulin kappa J region) are involved in this process. Correspondingly, CD4(+) T-cell responses to immunization with protein antigen are strongly reduced in mice lacking these components of the Notch pathway. Our findings, therefore, show that Notch controls the magnitude of CD4(+) T-cell responses by promoting cellular longevity.

Decisions on the road to memory.

A fundamental property of the adaptive immune system is the ability to generate antigen-specific memory, which protects against repeated infections with the same pathogens and determines the success of vaccination. Immune memory is built up alongside a response providing direct protection during the course of a primary immune response. For CD8 T cells, this involves the generation of two distinct types of effector cells. Short lived effector cells (SLECs) confer immediate protection, but contribute little to the memory repertoire. Memory precursor effector cells (MPECs) have the ability to respond to survival signals and develop into memory cells. These two types of cells can be distinguished on the basis of surface markers and express distinct genetic programs. A single naive CD8 T cell can give rise to both MPEC and SLEC daughter cells. This may involve an initial asymmetric division or depend on later instructive signals acting on equipotent daughter cells. Strong inflammatory signals favor the generation of SLECs and weaker inflammation favors the generation of MPECs. A distinguishing feature of MPECs is their ability to persist when most effector cells die. This survival depends on signals from the IL-7 receptor, which induce expression of anti-apoptotic factors. MPECs are therefore characterized by expression of the IL-7 receptor as well as the CCR7 chemokine receptor, which allows homing to areas in lymphoid organs where IL-7 is produced. Critical for persistence of MPECs is further their responsiveness to myeloid cell derived IL-15, which instructs these cells to switch their metabolic programs from glycolysis associated with rapid proliferation to fatty acid oxidation required during a more resting state. As the mechanisms determining generation of immunological memory are unraveled, opportunities will emerge for the improvement of vaccination strategies.

Effective collaboration between marginal metallophilic macrophages and CD8+ dendritic cells in the generation of cytotoxic T cells.

The spleen is the lymphoid organ that induces immune responses toward blood-borne pathogens. Specialized macrophages in the splenic marginal zone are strategically positioned to phagocytose pathogens and cell debris, but are not known to play a role in the activation of T-cell responses. Here we demonstrate that splenic marginal metallophilic macrophages (MMM) are essential for cross-presentation of blood-borne antigens by splenic dendritic cells (DCs). Our data demonstrate that antigens targeted to MMM as well as blood-borne adenoviruses are efficiently captured by MMM and exclusively transferred to splenic CD8(+) DCs for cross-presentation and for the activation of cytotoxic T lymphocytes. Depletion of macrophages in the marginal zone prevents cytotoxic T-lymphocyte activation by CD8(+) DCs after antibody targeting or adenovirus infection. Moreover, we show that tumor antigen targeting to MMM is very effective as antitumor immunotherapy. Our studies point to an important role for splenic MMM in the initial steps of CD8(+) T-cell immunity by capturing and concentrating blood-borne antigens and the transfer to cross-presenting DCs which can be used to design vaccination strategies to induce antitumor cytotoxic T-cell immunity.

E-Cadherin is Dispensable to Maintain Langerhans Cells in the Epidermis.

The cell adhesion molecule E-cadherin is a major component of adherens junctions and marks Langerhans cells (LC), the only dendritic cell (DC) population of the epidermis. LC form a dense network and attach themselves to the surrounding keratinocytes via homophilic E-cadherin binding. LC activation, mobilization, and migration require a reduction in LC E-cadherin expression. To determine whether E-cadherin plays a role in regulating LC homeostasis and function, we generated CD11c-specific E-cadherin knockout mice (CD11c-Ecaddel). In the absence of E-cadherin-mediated cell adhesion, LC numbers remained stable and similar as in control mice, even in aged animals. Intriguingly, E-cadherin-deficient LC displayed a dramatically changed morphology characterized by a more rounded cell body and fewer dendrites than wild-type cells. Nevertheless, maturation and migration of LC lacking E-cadherin was not altered, neither under steady-state nor inflammatory conditions. Accordingly, CD11c-Ecaddel and control mice developed comparable contact hypersensitivity reactions and imiquimod-triggered psoriatic skin inflammation, indicating that E-cadherin on LC does not influence their ability to orchestrate T cell-mediated immunity. In conclusion, our data demonstrate that E-cadherin is dispensable to maintain LC in the epidermis and does not regulate LC maturation, migration, and function.

Langerin+CD8+ Dendritic Cells in the Splenic Marginal Zone: Not So Marginal After All.

Dendritic cells (DC) fulfill an essential sentinel function within the immune system, acting at the interface of innate and adaptive immunity. The DC family, both in mouse and man, shows high functional heterogeneity in order to orchestrate immune responses toward the immense variety of pathogens and other immunological threats. In this review, we focus on the Langerin+CD8+ DC subpopulation in the spleen. Langerin+CD8+ DC exhibit a high ability to take up apoptotic/dying cells, and therefore they are essential to prime and shape CD8+ T cell responses. Next to the induction of immunity toward blood-borne pathogens, i.e., viruses, these DC are important for the regulation of tolerance toward cell-associated self-antigens. The ontogeny and differentiation pathways of CD8+CD103+ DC should be further explored to better understand the immunological role of these cells as a prerequisite of their therapeutic application.

The Fate Choice Between Effector and Memory T Cell Lineages: Asymmetry, Signal Integration, and Feedback to Create Bistability.

CD8+ T cells clear primary infections with intracellular pathogens and provide long-term immunity against reinfection. Two different types of CD8+ T cells are responsible for these functions: short-lived effector T cells and memory T cells. The cellular relationship between these two types of CD8+ T cells has been subject to much investigation. Both cell types can derive from a single naïve CD8+ T cell precursor. Their generation requires a fate choice early during a T cell response. As a result, two populations of T cells emerge. One of these consists of terminally differentiated short-lived effector T cells. The other contains cells able to develop into long-lived memory T cells. A foundation for development of these two populations may be laid during the first division of an activated naïve T cell precursor, as a consequence of asymmetric segregation of fate-determining factors into the daughter cells. Nonetheless, the binary choice between the two lineages is strongly influenced by signals, which ensure that the differentiation process is matched with the needs posed by the infection. Here, we will discuss the genetic and metabolic programs governing differentiation of these two lineages as well as the processes leading to their induction and consolidation to create bistability. These processes involve extensive lateral inhibition between the programs as well as positive feedback between the genetic programs and the signaling pathways responsible for their induction. These features will be highlighted by discussing the role of the Notch signaling pathway in guiding the decision between the two lineages.

Evaluation of the DRI oxycodone immunoassay for the detection of oxycodone in urine.

We evaluated the performance of the DRI Oxycodone (DRI-Oxy) enzyme immunoassay for the detection of oxycodone and its primary metabolite, oxymorphone, in urine, by testing 1523 consecutive urine specimens collected from pain management patients. All 1523 specimens were tested with the DRI-Oxy assay at a cut-off of 100 ng/mL and then analyzed by gas chromatography-mass spectrometry (GC-MS) for opiates, including oxycodone and oxymorphone. Approximately 29% (435) of the 1523 specimens yielded positive results by the DRI-Oxy assay. Of these 435 specimens, GC-MS confirmed the presence of oxycodone and/or oxymorphone at >100 ng/mL in 433 specimens, an agreement of 99.5%. In addition to oxycodone and/or oxymorphone, 189 of the 433 positive specimens contained other opiates including codeine, hydrocodone, hydromorphone, and morphine. These other opiates were also present in 54% (590/1084) of the oxycodone negative specimens. The DRI-Oxy assay demonstrated no cross-reactivity, yielding negative results, with specimens containing concentrations of codeine, >75,000 ng/mL; hydrocodone, >75,000 ng/mL; hydromorphone, >12,000 ng/mL; and morphine, >163,000 ng/mL. From the presented study, the sensitivity of the DRI-Oxy was 0.991 and the selectivity 0.998. The DRI-Oxy assay provided a highly reliable method for the detection of oxycodone and/or oxymorphone in urine specimens.

Direct analysis of opiates in urine by liquid chromatography-tandem mass spectrometry.

A method for the direct analysis of 10 opiate compounds in urine was developed using liquid chromatography-mass spectrometry-mass spectrometry (LC-MS-MS) with electrospray ionization interface (ESI). Opiates included were morphine-3-P-glucuronide, morphine-6--glucuronide, morphine, oxymorphone, hydromorphone, norcodeine, codeine, oxycodone, 6-monoacetylmorphine (6MAM), and hydrocodone. Urine samples were prepared by centrifugation to remove large particles and direct injection into the LC-MS-MS. Separation and detection of all compounds was accomplished within 6 min. Linearity was established for all opiates except 6MAM from 50 ng/mL to 10,000 ng/mL; 6MAM from 0.25 ng/mL to 50 ng/mL with all correlation coefficients (r) > 0.99. Interrun precision (%CV) ranged from 1.1% to 16.7%, and intrarun precision ranged from 1.3% to 16.3%. Accuracy (% bias) ranged from -7.3% to 13.6% and -8.5% to 11.8 for inter- and intrarun, respectively. Eighty-nine urine samples previously analyzed by gas chromatography-MS were re-analyzed by the LC-MS-MS method. The qualitative results found an 88% agreement for negative samples between the two methods and 94% for positive samples. The LC-MS-MS method identified 19 samples with additional opiates in the positive samples. Overall, the direct injection LC-MS-MS method performed well and permitted the rapid analysis of urine samples for several opiates simultaneously without extensive sample preparation.

Urine concentrations of fentanyl and norfentanyl during application of Duragesic transdermal patches.

A study of the urinary concentration of fentanyl (F) and its major metabolite norfentanyl (NF) in chronic pain patients treated with the Duragesic continuous release transdermal patches is presented. These patches are available in 10, 20, 30, and 40 cm(2) sizes releasing 25, 50, 75, and 100 microg/h F, respectively. F is rapidly and extensively metabolized, with NF as the major metabolite. Five hundred-forty six random urine specimens were collected from chronic pain patients wearing 25, 50, 75, or 100 ug F transdermal patches. Urine specimens were collected from hours after application to several days later after continuous F release. Each specimen was analyzed for F, NF, creatinine, and pH. Additionally, each was screened by enzyme immunoassay for the following: amphetamines, barbiturates, benzodiazepines, cocaine metabolite, methadone, phencyclidine, d-propoxyphene, opiates, and marijuana metabolites. All positive screening results were confirmed by gas chromatography-mass spectrometry (GC-MS). F and NF were isolated from urine by solid-phase extraction then identified and quantified by GC-MS in SIM mode. The LODs and LOQs for F and NF were 3 ng/mL, respectively. The results of F and NF analysis of urine form those wearing 25-microg patches (N = 142) was mean F, 47 ng/mL with a range of 0 to 983 ng/mL, and 97% of the specimens contained < 200 ng/mL and mean NF, 175 ng/mL with a range of 0-980 ng/mL, while 95% of the specimens contained < 400 ng/mL. The results of F and NF analysis of urine form those wearing 50 microg patches (N = 184) was: mean F, 74 ng/mL with a range of 0 to 589 ng/mL, and 92% of the specimens contained < 200 ng/mL and mean NF, 257 ng/mL with a range of 0-2200 ng/mL, and 98% of the specimens contained < 1000 ng/mL. The results of F and NF analysis of urine form those wearing 75 microg patches (N = 85) was mean F, 107 ng/mL with a range of 0 to 1280 ng/mL, and 98% of the specimens contained < 400 ng/mL and mean NF, 328 ng/mL with a range of 0-5630 ng/mL, and 99% of the specimens contained < 1000 ng/mL. The results of F and NF analysis of urine form those wearing 100 ug patches (N = 135) was mean F, 100 ng/mL with a range of 0 to 1080 ng/mL, while 96% of the specimens contained < 400 ng/mL and mean NF, 373 ng/mL with a range of 0-5730 ng/mL, and 95% of the specimens contained < 1000 ng/mL. The incidence of other drugs detected as a percentage the specimens was opiates, 48%, benzodiazepines, 43%; barbiturates, 3%; methadone, 4%; marijuana metabolite, 3%; and cocaine metabolite, 1%. With the exception of F and/or NF, no other drugs were detected in 25% of the specimens. These data demonstrate the wide variation in concentrations of F and NF in random urine specimens following application of Duragesic patches. However, these values obtained during therapeutic use far exceed concentrations previously reported in fatal poisoning. In general, one may expect to find urine NF concentrations 3-4 times higher than those of F.

Evaluation of the Triage PPY on-site testing device for the detection of dextropropoxyphene in urine.

A new point-of-care colloidal metal immunoassay urine drugs-of-abuse testing device, the BIOSITE TRIAGE Plus Propoxyphene (TPP), was evaluated for the rapid detection of dextropropoxyphene (PPY) and/or its primary metabolite, norpropoxyphene (NP), in urine at a total PPY/NP concentration of 300 ng/mL or greater. This assay has been added to the Triage device that tests for commonly abused drugs. Adding to drug-free urine PPY and NP established the linearity of the TPP assay at concentrations of 40%, 80%, 120%, and 160% of the cut-off concentration. No significant cross-reactivity was found at 1.0 g/L for 32 drugs commonly encountered in emergency department admissions. Significant cross-reactivity was observed only with diphenhydramine and tricyclic antidepressants. TPP results from 160 urine specimens screened for PPY and/or NP were compared to those obtained by testing with DRI enzyme immunoassay, Emit II plus immunoassay, Abuscreen Online immunoassay and gas chromatography-mass spectrometry (GC-MS). There was a 98.8% agreement of positive or negative results between TPP and both the DRI and OnLine assays. The two discordant TPP results were due to concentrations of NP below the TPP minimum cross-reactivity value of 400 ng/mL. These two specimens yielded GC-MS NP concentrations of 262 and 359 ng/mL. These NP concentrations were within +/- 20% of the cross-reactivity cut-off value for NP for TPP, DRI, and Online. There was only an 88% agreement of positive or negative results between TPP and the Emit assay. Twenty urine specimens yielding PPY positive results when tested by TPP were negative by Emit testing. The discordant TPP results were due to poor cross-reactivity of Emit to NP. A 98.8% agreement of positive PPY results was observed between TPP and GC-MS. Discordant urines were found to contain PPY concentrations below the cut-off value of the assay. TPP was found to be an accurate device for the detection of PPY and NP in urine.