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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.

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.

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)