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The elderly are particularly susceptible to infectious and neoplastic diseases of the lung and it is unclear how lifelong exposure to environmental pollutants affects respiratory immune function. In an analysis of human lymph nodes (LNs) from 84 organ donors aged 11-93years, we found a specific age-related decline in lung-associated, but not gut-associated, LN immune function linked to the accumulation of inhaled atmospheric particulate matter. Increasing densities of particulates were found in lung-associated LNs with age, but not in the corresponding gut-associated LNs. Particulates were specifically contained within CD68(+)CD169(−) macrophages, which exhibited reduced activation, phagocytic capacity, and altered cytokine production compared to non-particulate-containing macrophages. The structures of B cell follicles and lymphatic drainage were disrupted in lung-associated LN with particulates. Our results reveal that the cumulative effects of environmental exposure with age may compromise immune surveillance of the lung via direct effects on immune cell function and lymphoid architecture.

Insect nicotinic acetylcholine receptors (nAChRs) require cofactors for functional heterologous expression. A previous study revealed that TMX3 was crucial for the functional expression of Drosophila melanogaster Dα1/Dβ1 nAChRs in Xenopus laevis oocytes, while UNC-50 and RIC-3 enhanced the acetylcholine (ACh)-induced responses of the nAChRs. However, it is unclear whether the coexpression of UNC-50 and RIC-3 with TMX3 and the subunit stoichiometry affect pharmacology of Dα1/Dβ1 nAChRs when expressed in X. laevis oocytes. We have investigated the effects of coexpressing UNC-50 and RIC-3 with TMX3 as well as changing the subunit stoichiometry on the agonist activity of ACh and imidacloprid on the Dα1/Dβ1 nAChRs. UNC-50 and RIC-3 hardly affected the agonist affinity of ACh and imidacloprid for the Dα1/Dβ1 nAChRs formed by injecting into X. laevis oocytes with an equal amount mixture of the subunit cRNAs, but enhanced current amplitude of the ACh-induced response. Imidacloprid showed higher affinity for the Dβ1 subunit-excess Dα1/Dβ1 (Dα1/Dβ1 = 1/5) nAChRs than the Dα1 subunit-excess Dα1/Dβ1 (Dα1/Dβ1 = 5/1) nAChRs, suggesting that imidacloprid prefers the Dα1-Dβ1 orthosteric site over the Dα1-Dα1 orthosteric site.

International audience; Clinical manifestations of COVID-19 caused by the new coronavirus SARS-CoV-2 are associated with age1,2. Adults develop respiratory symptoms, which can progress to acute respiratory distress syndrome (ARDS) in the most severe form, while children are largely spared from respiratory illness but can develop a life-threatening multisystem inflammatory syndrome (MIS-C)3-5. Here, we show distinct antibody responses in children and adults after SARS-CoV-2 infection. Adult COVID-19 cohorts had anti-spike (S) IgG, IgM and IgA antibodies, as well as anti-nucleocapsid (N) IgG antibody, while children with and without MIS-C had reduced breadth of anti-SARS-CoV-2-specific antibodies, predominantly generating IgG antibodies specific for the S protein but not the N protein. Moreover, children with and without MIS-C had reduced neutralizing activity as compared to both adult COVID-19 cohorts, indicating a reduced protective serological response. These results suggest a distinct infection course and immune response in children independent of whether they develop MIS-C, with implications for developing age-targeted strategies for testing and protecting the population.

We herein advocate for more extensive utilization of ex vivo resection techniques for otherwise unresectable liver tumors by presenting the largest collective American experience.Advanced in situ resection and vascular reconstruction techniques have made R0 resection possible for otherwise unresectable liver tumors. Ex vivo liver resection may further expand the limits of resectability but remains underutilized due to concerns about technical complexity and vascular thrombosis. However, we believe that the skillset required for ex vivo liver resection is more widespread and the complications less severe than widely assumed, making ex vivo resection a more attractive option in selected case.We retrospectively analyzed 35 cases performed by surgical teams experienced with ex vivo liver resections (at least 4 cases) between 1997 and 2021.We categorized malignancies as highly aggressive (n=18), moderately aggressive (n=14), and low grade (n=3). All patients underwent total hepatectomy, vascular reconstruction and resection in hypothermia on the backtable, and partial liver autotransplantation. Overall survival was 67%/39%/28%, at 1/3/5 years, respectively, with a median survival of 710 days (range: 22-4824). Patient survival for highly aggressive, moderately aggressive, and low-grade tumors was 61%/33%/23%, 67%/40%/22%, and 100%/100%/100% at 1/3/5 years, respectively, with median survival 577 days (range: 22-3873), 444 days (range: 22-4824), and 1825 days (range: 868-3549).Ex vivo resection utilizes techniques commonly practiced in partial liver transplantation, and we demonstrate relatively favorable outcomes in our large collective experience. Therefore, we propose that more liberal use of this technique may benefit selected patients in centers experienced with partial liver transplantation.

Adaptive immune responses to SARS-CoV-2 infection have been extensively characterized in blood; however, most functions of protective immunity must be accomplished in tissues. Here, we report from examination of SARS-CoV-2 seropositive organ donors (ages 10 – 74) that CD4(+) T, CD8(+) T, and B cell memory generated in response to infection is present in bone marrow, spleen, lung, and multiple lymph nodes (LNs) for up to 6 months post-infection. Lungs and lung-associated LNs were the most prevalent sites for SARS-CoV-2-specific memory T and B cells, with significant correlations between circulating and tissue-resident memory T and B cells in all sites. We further identified SARS-CoV-2-specific germinal centers in the lung-associated LNs up to 6 months post-infection. SARS-CoV-2-specific follicular helper T cells were also abundant in lung-associated LNs and lungs. Together, the results indicate local tissue coordination of cellular and humoral immune memory against SARS-CoV-2 for site-specific protection against future infectious challenges.

Immune response dynamics in COVID-19 and its severe manifestations have largely been studied in circulation. Here, we examined the relationship between immune processes in the respiratory tract and circulation through longitudinal phenotypic, transcriptomic and cytokine profiling of paired airway and blood samples from patients with severe COVID-19 relative to heathy controls. In COVID-19 airways, T cells exhibited activated, tissue-resident, and protective profiles; higher T cell frequencies correlated with survival and younger age. Myeloid cells in COVID-19 airways featured hyper-inflammatory signatures and higher frequencies of these cells correlated with mortality and older age. In COVID-19 blood, aberrant CD163+ monocytes predominated over conventional monocytes, and were found in corresponding airway samples and in damaged alveoli. High levels of myeloid chemoattractants in airways suggest recruitment of these cells through a CCL2-CCR2 chemokine axis. Our findings provide insights into immune processes driving COVID-19 lung pathology with therapeutic implications for targeting inflammation in the respiratory tract., Through longitudinal profiling of paired airway and blood from patients with severe COVID-19, Szabo et al. reveal airway immune responses that correlate with age and outcome. They further identify coordinate roles for T and myeloid cells in the respiratory tract and circulation in perpetuating lung pathology and disease pathogenesis.

The persistence and maintenance of immunological memory is critical for long-term immunity generated in response to infection and vaccination. Aging of memory T cells in humans has predominantly been studied in the peripheral blood, and considerably less is known about the aging of memory T cells within tissues. Using tissues obtained from human organ donors, we investigated how tissue localization, expression of the CD103 integrin, and age influence the phenotype of tissue resident memory T cells (TRM). Using multi-parameter flow cytometry, we characterized diverse tissue sites from donors ages 9 to 93 years old and demonstrate that age-associated changes in the T cell compartment exhibit tissue- and subset- specific dynamics. CD8 T cells in blood-rich tissues spleen and lung have elevated expression of replicative senescence markers CD57 and KLRG1 relative to lung-associated lymph nodes (LLN), jejunum, and mesenteric lymph nodes (MLN). CD103+ TRM have the lowest expression of CD57 and KLRG1 across all tissue sites relative to both CD103− TRM and circulating TEM. CD8 T cells in lymphoid tissues spleen, LLN, and MLN exhibit the largest age-associated increases in expression of CD57, KLRG1, CD244, and PD-1. Together, these results demonstrate that age-associated phenotypic changes are most prominent in secondary lymphoid organs, while TRM and mucosal sites exhibit few senescent changes. Supported by the National Science Foundation (NSF) Graduate Research Fellowship Program (GRFP) and grants from NIH (P01 AI106697, U19 AI057266)

γδT cells are unconventional T lymphocytes which seed peripheral tissues in early life. They provide frontline anti-pathogen, anti-cancer and homeostatic responses, rapidly initiated in response to signals of cellular stress independent of MHC-recognition. However, the role of γδT cells in humans has been difficult to assess given their purported tissue residency. Using tissues obtained from organ donors, we conducted flow cytometry, TCR-sequencing, and stimulation assays on γδT cells isolated from blood, spleen, lung, intestines and lymph nodes (LN) to create an ‘atlas’ across the human body throughout life. γδT cells were enriched in the blood, spleen, lung, and jejunum in early life, whereas LN proportions were maintained at low levels. Vδ1 and Vδ2 γδT cell subsets also exhibited tissue- and age-specific phenotypes: Vδ1 cells predominated in all organs in infancy except the lung. They were ‘naïve-like’, acquiring a mature phenotype with age, whereas Vδ2 possessed an effector memory signature, even in donors a few days old. Functionally, Vδ2 cells produced IFNγ, TNFα, GzmB and perforin at high levels in childhood. By contrast, Vδ1 cells favored Areg production during infancy but acquired a cytotoxic profile into adulthood. TCR analysis revealed clonal expansion in mucosal organs, whereas lymphoid sites had increased diversity in early life. Despite this site-specific clonal expansion, V-chain usage was stably distributed across organs and did not show the tissue-specific biases seen in mice. Our analyses reveal that γδT cells may play an outsized role in pediatric tissue immune responses and identify potentially divergent roles of specific subsets that contribute to tissue immunity during the most vulnerable years of life.

Immunization with novel mRNA vaccines against SARS-CoV-2 administered worldwide protects against COVID-19 through the generation of neutralizing antibodies directed against the viral spike protein. While immune responses generated from vaccines have been evaluated in blood, the cellular stores of memory T and B cells are maintained in tissues. Using tissues from vaccinated, but previously uninfected organ donors, we surveyed lymphoid and barrier sites for the presence of SARS-CoV-2 vaccine specific memory B and T cells. Fluorescently labeled, full length Spike and RBD probes were used to identify vaccine specific memory B cells, while vaccine specific T cells were identified by expression of activation induced markers following stimulation with peptide pools spanning the entire spike protein. All donors were confirmed negative for anti-nucleocapsid antibodies and positive for anti-spike and anti-RBD antibodies via ELISA. Spike specific T cells were identified in the blood, spleen, lung, and lung associated lymph nodes (LLN), but not bone marrow (BM). In all tissues, these vaccine specific T cells were primarily CD4 and exhibited a mixed central- and effector-memory phenotype; in the LLN the majority (up to 55%) were T-follicular helper cells (CXCR5+/PD-1+). Spike and RBD specific memory B cells were detected in blood, lung, spleen, BM, and LLN. These vaccine specific B cells in the LLN specifically expressed the tissue residency marker CD69, but only a minority had features of germinal center B cells. These results indicate that memory B and T cells generated from mRNA vaccination localize primarily to lymphoid organs, including those draining the lung, which is important for understanding their longevity and protective capacity. Supported by grants from NIH (U19 AI128949, PO1 AI106697, T32 GM007367)

The skin, gut, and lung are critical physical and immune barriers that protect from pathogen entry. Within these sites, T cell memory is largely maintained by populations of tissue resident memory T cells (TRMs), capable of rapid protective responses. The degree of interconnection between these TRMs from various sites of residence is poorly understood, particularly in humans. Here, we analyze how tissue specificity affects transcriptional heterogeneity and the T cell receptor (TCR) repertoire across 9 tissue sites within individual donors, including barrier sites (skin, gut, and lung) and associated lymph nodes, as well as blood and lymphoid organs (blood, bone marrow, spleen). Cytometry by time-of-flight (CyTOF) and single cell RNA sequencing reveal phenotypic and transcriptomic features unique to skin or gut T cells, which are distinct from T cells in lymphoid sites or blood. These results were reflected in both bulk sequencing of TRBV gene rearrangement and single cell TCR sequencing, where skin and gut T cells exhibited site-specific clonal expansions that were not present in other sites. Conversely, lung T cells showed clonal overlap and transcriptional similarity to T cells in lymphoid and blood rich sites. Site-specific expansions were mediated by TRMs, while clones disseminated across tissues exhibit large clonal expansions and a CD8 terminal effector (TEMRA) phenotype. Together, these results reveal that TRMs in barrier sites are maintained in situ, while memory T cells in the lung, blood-rich and lymphoid sites are more interconnected. Blood T cells are not representative of barrier T cell clonal space. These results have important implications for monitoring and promoting barrier immunity through site-specific targeting. Supported by grants from NIH (P01 AI106697)

Aging is associated with both overt and subclinical lung fibrosis, which increases risk for mortality from viruses and other respiratory pathogens. The molecular programs that induce fibrosis in the aging lung are not well understood. To overcome this knowledge gap, we undertook multimodal profiling of distal lung samples from healthy human donors across the lifespan. Telomere shortening, a cause of cell senescence and fibrosis, was progressive with age in a sample of 86 lungs and was associated with foci of DNA damage. Bulk RNA sequencing confirmed activation of cellular senescence and pro-fibrotic pathways as well as genes necessary for collagen processing with increasing age. These findings were validated in independent datasets for lung and sun-exposed skin, but not other organs including heart, liver and kidney. Cell type deconvolution analysis revealed a progressive loss of lung epithelial cells and an increasing proportion of fibroblasts. Consistent with the observed pro-fibrotic transcriptional profile, second harmonic imaging demonstrated increased density of interstitial collagen in aged human lungs. Furthermore, regions of parenchymal fibrosis were associated with decreased alveolar expansion and surfactant secretion. These findings reveal the transcriptional and structural features of fibrosis and associated physiologic impairments in normal lung aging.

Lung fibrosis is increasingly detected with aging and has been associated with poor outcomes in acute lung injury or infection. However, the molecular programs driving this pro-fibrotic evolution are unclear. Here we profile distal lung samples from healthy human donors across the lifespan. Gene expression profiling by bulk RNAseq reveals both increasing cellular senescence and pro-fibrotic pathway activation with age. Quantitation of telomere length shows progressive shortening with age, which is associated with DNA damage foci and cellular senescence. Cell type deconvolution analysis of the RNAseq data indicates a progressive loss of lung epithelial cells and an increasing proportion of fibroblasts with age. Consistent with this pro-fibrotic profile, second harmonic imaging of aged lungs demonstrates increased density of interstitial collagen as well as decreased alveolar expansion and surfactant secretion. In this work, we reveal the transcriptional and structural features of fibrosis and associated functional impairment in normal lung aging., Age is associated with increasing vulnerability to both acute and chronic lung diseases. Employing genomic analysis and live lung imaging, this study reveals a profile of increased cellular senescence, telomere shortening, and fibrosis-induced impaired alveolar function in the natural history of human lung aging.

SUMMARY The persistence of anti-viral immunity is essential for protection and exhibits profound heterogeneity across individuals. Here, we elucidate the factors that shape maintenance and function of anti-viral T cell immunity in the body by comprehensive profiling of virus-specific T cells across blood, lymphoid organs, and mucosal tissues of organ donors. We use flow cytometry, T cell receptor sequencing, single-cell transcriptomics, and cytokine analysis to profile virus-specific CD8+ T cells recognizing the ubiquitous pathogens influenza and cytomegalovirus. Our results reveal that virus specificity determines overall magnitude, tissue distribution, differentiation, and clonal repertoire of virus-specific T cells. Age and sex influence T cell differentiation and dissemination in tissues, while T cell tissue residence and functionality are highly correlated with the site. Together, our results demonstrate how the covariates of virus, tissue, age, and sex impact the anti-viral immune response, which is important for targeting, monitoring, and predicting immune responses to existing and emerging viruses., In brief Through comprehensive cellular and molecular analysis of virus-specific T cells in circulation and across multiple lymphoid and mucosal tissues, Poon et al. elucidate how maintenance and function of the human anti-viral immune response against ubiquitous viruses influenza and cytomegalovirus are shaped by virus, tissue, age, and sex., Graphical Abstract

SUMMARYImmune responses to respiratory viruses like SARS-CoV-2 originate and function in the lung, yet assessments of human immunity are often limited to blood. Here, we conducted longitudinal, high-dimensional profiling of paired airway and blood samples from patients with severe COVID-19, revealing immune processes in the respiratory tract linked to disease pathogenesis. Survival from severe disease was associated with increased CD4+T cells and decreased monocyte/macrophage frequencies in the airway, but not in blood. Airway T cells and macrophages exhibited tissue-resident phenotypes and activation signatures, including high level expression and secretion of monocyte chemoattractants CCL2 and CCL3 by airway macrophages. By contrast, monocytes in blood expressed the CCL2-receptor CCR2 and aberrant CD163+and immature phenotypes. Extensive accumulation of CD163+monocyte/macrophages within alveolar spaces in COVID-19 lung autopsies suggested recruitment from circulation. Our findings provide evidence that COVID-19 pathogenesis is driven by respiratory immunity, and rationale for site-specific treatment and prevention strategies.

Clinical manifestations of COVID-19 caused by the novel coronavirus SARS-CoV-2 are associated with age. While children are largely spared from severe respiratory disease, they can present with a SARS-CoV-2-associated multisystem inflammatory syndrome (MIS-C) similar to Kawasaki’s disease. Here, we show distinct antibody (Ab) responses in children with MIS-C compared to adults with severe COVID-19 causing acute respiratory distress syndrome (ARDS), and those who recovered from mild disease. There was a reduced breadth and specificity of anti-SARS-CoV-2-specific antibodies in MIS-C patients compared to the COVID patient groups; MIS-C predominantly generated IgG Abs specific for the Spike (S) protein but not for the nucleocapsid (N) protein, while the COVID-19 cohorts had anti-S IgG, IgM and IgA Abs, as well as anti-N IgG Abs. Moreover, MIS-C patients had reduced neutralizing activity compared to both COVID-19 cohorts, indicating a reduced protective serological response. These results suggest a distinct infection course and immune response in children and adults who develop severe disease, with implications for optimizing treatments based on symptom and age.

AimTo compare the clinical outcomes of posterior chamber phakic intraocular lens implantation with a central hole (Hole Implantable Collamer Lens (ICL), STAAR Surgical) for low-to-moderate myopia and for high myopia.MethodsThis multicentre retrospective case series comprised 351 eyes of 351 consecutive patients undergoing ICL implantation. Eyes were divided into groups based on preoperative degree of myopia: group 1; 57 eyes, manifest spherical equivalent less than −6 dioptres (D), and group 2; 294 eyes, −6 D or more. Safety, efficacy, predictability, stability and adverse events were compared preoperatively; and at 1 day, 1 week and 1, 3, 6 and 12 months postoperatively,ResultsUncorrected and corrected visual acuities were −0.17±0.14 and −0.21±0.10 logMAR in group 1, and −0.16±0.09 and −0.21±0.08 logMAR in group 2, 1 year postoperatively. In groups 1 and 2, 98% and 99% of eyes were within 1.0 D of the targeted correction. Manifest refraction changes of −0.12±0.34 D (group 1) and −0.18±0.43 D (group 2) occurred from 1 day to 1 year. ICL exchanges were necessary in two eyes (0.7%) in group 2. No vision-threatening complications occurred at any time.ConclusionsThe ICL performed well for the correction of both low-to-moderate myopia and high myopia throughout the 1-year observation period. The clinical outcomes of ICL implantation for low-to-moderate myopia are essentially equivalent to those for high myopia.

Generation and maintenance of memory T cells within tissue sites of infection is critical for long-term antiviral protection. Current knowledge of human antiviral responses is largely derived from studies sampling peripheral blood. Yet, little is known about human tissue-localized antiviral immunity. Utilizing our human donor tissue resource established in collaboration with LiveOnNY, we investigated how tissue, virus, and age shape T cell maintenance across diverse tissues and their function when presented with antigens derived from human cytomegalovirus (HCMV) or influenza A virus (flu). Using flow cytometry, we show that virus-specific CD8 T cells are maintained in diverse tissues with subset differentiation and distribution shaped by virus specificity and age. Compared to T cells recognizing HCMV, flu-specific T cells display higher levels of residency markers—CD69 and CD103—particularly in adult compared to pediatric donors. In contrast, HCMV-specific T cells are maintained in greater abundance as terminally-differentiated effector T cells. Sequencing of the T cell receptor CDR3b chain reveals that, while both HCMV- and flu-specific T cells demonstrate clonal overlap across multiple tissues, HCMV-specific T cells display greater clonality and less diversity. When presented with viral antigens, HCMV- and flu-specific T cells are polyfunctional. Additionally, single-cell transcriptome profiling reveals tissue localization is the primary determinant of antigen-driven responses. Together, these studies demonstrate the dynamics of T cell differentiation and maintenance throughout the human body, which is primarily driven by virus specificity, and the role of tissue localization in shaping antiviral response.

Natural killer (NK) cells are innate immune cells with the inherent ability to directly kill tumor and virus infected cells. Due to their ability to kill cancer cells without any prior priming, and their role in preventing metastasis NK cells have since long been the choice for autologous adoptive cell transfer therapy in cancer. However, poor expansion potential of PBMC derived NK cells in vitro is a major roadblock preventing the widespread use of NK cells in immunotherapy. We found that human NK cells isolated from lymph nodes (LNs) express higher levels of genes encoding for stem-like transcription factors (TCF7, LEF1, MYC) compared to NK cells from blood, spleen, bone marrow (BM) and lung. Therefore, we hypothesized that NK cells isolated from LNs will show superior expansion potential in vitro. Flow cytometric analysis shows that LN derived NK cells express high levels of TCF1 protein ex vivo, and show greater proliferation compared to NK cells isolated from blood, spleen and BM following stimulation with IL-2 and IL-15 in vitro. We also observed that a significant frequency of LN NK cells expressed TCF1 even after expansion, suggesting preserved proliferation potential of these cells. Additionally, the expanded NK cells from LN acquired properties of mature, highly functional NK cells such as increased expression of CD16, CD57 and higher Granzyme B expression. Lastly, LN derived NK cells also demonstrate enhanced cytolytic activity in vitro after expansion. Taken together our results suggest that in vitro expanded NK cells from LNs are potentially efficacious anti-tumor agents and could be leveraged for the development of future generation of NK cell directed immunotherapies.

Infants exhibit increased susceptibility to viral and bacterial pathogens with pulmonary infections representing one of the top causes of infant mortality worldwide. Optimal protection from pathogens is mediated by coordinated functions of different T cell subsets, with tissue resident populations maintaining site-specific immunity in the respiratory tract. Currently, little is known of how tissue localized T cells populate and mediate mucosal immune responses in early life in humans. Here we analyzed T cell populations in the lungs obtained from infant and pediatric organ donors aged 8 days postnatal (PN) to 8 years by imaging and flow cytometry. In the earliest stage of life at 8 days PN, few αβ T cells were present in the lung, while innate-type γδ T cells were present around the airways. During the first two years of life, αβ T cells exhibiting memory phenotypes preferentially accumulated around the central airways and expressed canonical markers of tissue residency (CD69 and CD103), consistent with flow cytometry data. By contrast, the total number of γδ T cells in the lung did not change significantly with age for up to 8 years of life; for example, the ratio of αβ:γδ T cells in neonatal donors was 1.5:1, which increased to 16:1 in lungs of a 3 year old donor. Consistent with the imaging data, the frequency of γδ T cells by flow cytometry was significantly reduced after the early neonatal period. These results show that γδ T cells populate the lung during the early neonatal period and are maintained in relatively constant numbers over childhood, while tissue resident αβ T cells accumulate over first 2 years of life and predominate thereafter. Our results demonstrate the dynamic nature of lung-localized T cell immune responses in a mucosal site.

Generation of memory T cells specific for conserved viral epitopes provides heterosubtypic protection to influenza infection. Previous mouse studies have shown that tissue-resident memory T cells (TRM) are critical for optimal protection. However, knowledge of human antiviral response is largely derived from blood. With our unique human donor tissue resource established in collaboration with LiveOnNY, we investigate the maintenance of influenza-specific T cells across diverse tissue sites and their response when presented with viral antigens. Using multimer staining and flow cytometry, we show that influenza-specific CD8 T cells are maintained in the lung tissue site of infection, as well as in blood, bone marrow, multiple lymph nodes, and small intestines. Compared to T cells recognizing persistent cytomegalovirus infection, influenza-specific T cells display higher levels of canonical residency markers—CD69 and CD103. Additionally, deep sequencing of the T cell receptor CDR3b repertoire reveals that influenza-specific T cells isolated from human tissue donors without active infection are clonally expanded and display a high degree of clonal overlap across tissue sites. When presented with viral antigens, influenza-specific T cells in all sites are polyfunctional and rapidly respond through cytokine production. Together, these studies reveal the dynamics of immune memory maintenance and antiviral response across tissues in the human body in response to influenza A virus.

The gut, lung, and skin are physical and immunological barriers that serve as the first line of defense against pathogens and foreign substances. Due to sampling limitations, previous studies have been limited to studying human barrier sites in isolation and often in the context of specific diseases. Our donor tissue resource established in collaboration with LiveOnNY offers a unique opportunity to examine human barrier T cell immunity as a network and elucidate how barrier sites are connected to each other and to lymphoid sites (lymph nodes, bone marrow, spleen). Here, using cytometry by time-of-flight (CyTOF) and T cell receptor (TCR) CDR3b sequencing, we investigate the unique defining characteristics and connectivity of T cells in barrier tissue – gut, lung, and skin – to lymphoid sites and blood. Through high-dimensional marker analysis by CyTOF, our results show that T cell subset composition and phenotype across 8 different sites comprise 33 clusters that are highly conserved between donors of different ages (22–70 yrs). Skin and gut T cells are distinct from each other and from lung, lymphoid sites, and blood, while lung T cells share features with lymphoid sites and blood. TCR repertoire analysis shows that, though majority of gut and skin T cell clones are unique to the tissue, expanded clones within these barrier sites share clonal overlap with other barrier and lymphoid sites. Moreover, T cells in the lung exhibit increased sharing with lymphoid sites and blood. Together, these results reveal how human T cells are compartmentalized in the skin and gut through site-specific adaptations while demonstrating that gut, lung, and skin barrier T cells are connected through shared specificities in a network of immune protection across the body.

The majority of T cells throughout the human body persist as non-circulating tissue-resident memory T cells (TRM) in lymphoid and mucosal sites, and are vital for orchestrating protective immune responses. However, given the difficulty of sampling healthy human tissues, a full understanding of TRM identity and function across tissue sites remains lacking. Here, we utilize a unique human tissue resource where tissues are obtained from organ donors to dissect CD4+ and CD8+ TRM heterogeneity using high-dimensional flow cytometry and single cell RNA-sequencing (scRNA-seq). We identify remarkable heterogeneity in expression of key surface markers that define TRM, namely CD103, CD101, CD49a, PD-1 and CXCR6, particularly in the lung and the intestines. These phenotypic groups of TRM denote different functional subsets after activation: CD103+ and CD101+ TRM preferentially produce IL-17A, CD49a+ and CXCR6+ TRM show enhanced IFNγ production, and PD-1+ TRM produce granzyme-B. To investigate the transcriptional programming underlying TRM, we profile ~20,000 resting and activated T cells from the lungs and intestines of human organ donors with scRNA-seq and identify TRM based on our previously defined tissue signature. In both tissues, we detect multiple clusters of TRM enriched for expression of individual or a combination of TRM marker genes. Importantly, these clusters show variable expression of genes coding for cytokines (IFNG, IL23A, IL26), transcription factors (TBX21, GATA3, BATF), and cytotoxic molecules (GNLY, GZMs), suggesting differentially regulated TRM subsets. Our results therefore highlight the heterogeneity of TRM across human tissue sites and define the transcriptional programming underlying TRM function.

Immune responses in diverse tissue sites are critical for protective immunity and homeostasis. Here, we investigate how tissue localization regulates the development and function of human natural killer (NK) cells, innate lymphocytes important for anti-viral and tumor immunity. Integrating high-dimensional analysis of NK cells from blood, lymphoid organs, and mucosal tissue sites from 60 individuals, we identify tissue-specific patterns of NK cell subset distribution, maturation, and function maintained across age and between individuals. Mature and terminally differentiated NK cells with enhanced effector function predominate in blood, bone marrow, spleen, and lungs and exhibit shared transcriptional programs across sites. By contrast, precursor and immature NK cells with reduced effector capacity populate lymph nodes and intestines and exhibit tissue-resident signatures and site-specific adaptations. Together, our results reveal anatomic control of NK cell development and maintenance as tissue-resident populations, whereas mature, terminally differentiated subsets mediate immunosurveillance through diverse peripheral sites. VIDEO ABSTRACT.

Seven reticulated giraffes were hand-reared at Nogeyama Zoological Gardens, because the dam had agalactia. Six of the 7 calves exhibited polyarthritis and/or phlegmon in the lower legs. However, the cause of the disorder was unclear. The present study reviewed the clinical records of the 7 giraffes, including the type and amount of colostrum ingested during the first 72 hr. The disorder involved the fetlocks and carpal and tarsal joints in 6 of the 7 calves within an average of 8 days of birth. The average amount of fed bovine or powdered colostrum was 0–2.4 l in the first 24 hr and 2.0–6.2 l during the first 72 hr. Insufficient colostrum quantity might be a factor in polyarthritis and/or phlegmon.

蚊媒介性感染症のベクターとなる蚊の生息状況解明を目的として，2005年5月から5か月間，横浜市立よこはま動物園内においてドライアイストラップ，グラビッドトラップおよびスウィーピング法を用いた捕集調査を行った。その結果，アカイエカ種群蚊（Culex pipiens group），ヒトスジシマカ（Aedes albopictus），トラフカクイカ（Lutzia vorax）など計9属14種2,623個体が捕集された。アカイエカ種群蚊およびヒトスジシマカが捕集蚊全体の約85％を占め，これらの蚊が園内における優占種であると考えられた。神奈川県内で生息が確認されている蚊26種のうち53.8％にあたる種が捕集されたことから，本動物園およびその周辺地域は，各種蚊が選好する多様な環境で構成されていると考えられた。なお，捕集蚊の10.6％で吸血が認められ，動物園動物を吸血源としている可能性が示唆された。動物園動物の蚊媒介性感染症を制御し，希少種の生息域外保全を行うためには，蚊種の生態に応じた防除対策が必要である。

Several species of captive birds at zoological gardens of Japan were found to be infected with avian Plasmodium. However, incriminated vector mosquito species have not been identified yet. To indicate the competent vectors of avian malaria parasite, we collected mosquitoes at a zoological garden in Japan and examined for the avian malaria parasite DNA. Totally, 1,361 mosquitoes of 11 species were collected in the zoological garden of Kanagawa, the south of Tokyo in Japan in 2005. Captured mosquitoes were pooled by each species, date collected, and location and used for DNA extraction. Eight out of 169 DNA samples were positive for the nested PCR of avian Plasmodium cyt b gene. Estimated minimum infection rates of mosquitoes were 5.9 per 1,000. The PCR positive mosquito species were Culex pipiens group and Lutzia vorax. Some DNA sequences amplified from collected mosquitoes were identical to avian Plasmodium lineages detected from captive birds in the same zoological garden studied. Our results suggest that C. pipiens group and L. vorax could be incriminated vectors of avian malaria parasite transmitting in captive birds kept in the zoological garden in Japan.

Treatment of bis(N,N-dimethylcar- bamoylseleno)methanes with SnCl4 afforded β-1,3,5-triselenanes in moderate to high yields, and the key intermediates of the reactions, i.e., acylselonium ions and selenoaldehydes, were successfully trapped by using allyltrimethylsilane or 2,3-dimethyl-1,3-butadiene to obtain the allylation products or the cycloadducts, respectively. © 2006 Wiley Periodicals, Inc. Heteroatom Chem 17:125–135, 2006; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/hc.20190

Treatment of a benzene or a CH2Cl2 solution of bis(N,N-dimethylcarbamoylseleno)methanes with SnCl4 afforded β-1,3,5-triselenanes, and the key intermediates, acylselonium ions and selenoaldehydes, were successfully trapped by using allyltrimethylsilane or 2,3-dimethyl-1,3-butadiene to obtain the allylation products or the cycloadducts, respectively.

In the chicken proventricular mucosa, aggregations of lymphocytes were localized in three different sites of the lamina propria, namely, underneath the surface epithelium, near the duct orifice of the deep proventricular gland, and in the gland tissue itself. In the lymphoid masses underneath the surface epithelium and in those near the duct orifice, CD4+ T lymphocytes and TCR2+ T lymphocytes occupied their central part, and B lymphocytes were localized in the periphery. CD8+ T lymphocytes and TCR1+ lymphocytes were evenly distributed in the masses. Infiltration of lymphocytes into these sites was first observed on the 20th embryonic day. At 1 week after hatching, CD3+ lymphocytes began to occupy the central area of the masses and His-C1+ B lymphocytes tended to be located in the periphery. Ultrastructurally, M cells were found neither in the epithelium of the mucosa nor in that of the excretory duct close to the lymphoid masses. In the deep proventricular gland, the lymphoid masses had a germinal center consisting of B lymphocytes, surrounded by the T lymphocyte-rich periphery. These masses were first recognized at the 3rd post-hatching week, presumably being formed against possible antigens invading into the lumen of the proventricular gland. On the other hand, the lymphoid masses beneath the surface epithelium and those near the duct orifice existing before the hatching period were considered to be prepared to establish the local mucosal immune barriers against the expectant antigenic invasion.

An adult male white eared-pheasant (Crossoptilon crossoptilon) at a Japanese zoo exhibited lethargy and emaciation. Microscopic examination of a blood smear revealed a haemosporidian parasitic infection. Based on the morphological characteristics and molecular analysis of the parasite, it was identified as Plasmodium (Bennettinia) juxtanucleare. This is the first report of P. juxtanucleare infection in bird species belonging to the genus Crossoptilon. Caution against avian malaria infection is required for the conservation of endangered bird species in zoos.