Dynamic establishment of recipient resident memory T cell repertoire after human intestinal transplantation.

BACKGROUND: Understanding formation of the human tissue resident memory T cell (TRM) repertoire requires longitudinal access to human non-lymphoid tissues. METHODS: By applying flow cytometry and next generation sequencing to serial blood, lymphoid tissue, and gut samples from 16 intestinal transplantation (ITx) patients, we assessed the origin, distribution, and specificity of human TRMs at phenotypic and clonal levels. FINDINGS: Donor age ≥1 year and blood T cell macrochimerism (peak level ≥4%) were associated with delayed establishment of stable recipient TRM repertoires in the transplanted ileum. T cell receptor (TCR) overlap between paired gut and blood repertoires from ITx patients was significantly greater than that in healthy controls, demonstrating increased gut-blood crosstalk after ITx. Crosstalk with the circulating pool remained high for years of follow-up. TCR sequences identifiable in pre-Tx recipient gut but not those in lymphoid tissues alone were more likely to populate post-Tx ileal allografts. Clones detected in both pre-Tx gut and lymphoid tissue had distinct transcriptional profiles from those identifiable in only one tissue. Recipient T cells were distributed widely throughout the gut, including allograft and native colon, which had substantial repertoire overlap. Both alloreactive and microbe-reactive recipient T cells persisted in transplanted ileum, contributing to the TRM repertoire. INTERPRETATION: Our studies reveal human intestinal TRM repertoire establishment from the circulation, preferentially involving lymphoid tissue counterparts of recipient intestinal T cell clones, including TRMs. We have described the temporal and spatial dynamics of this active crosstalk between the circulating pool and the intestinal TRM pool. FUNDING: This study was funded by the National Institute of Allergy and Infectious Diseases (NIAID) P01 grant AI106697.

Plasticity of intragraft alloreactive T cell clones in human gut correlates with transplant outcomes.

The site of transition between tissue-resident memory (TRM) and circulating phenotypes of T cells is unknown. We integrated clonotype, alloreactivity, and gene expression profiles of graft-repopulating recipient T cells in the intestinal mucosa at the single-cell level after human intestinal transplantation. Host-versus-graft (HvG)-reactive T cells were mainly distributed to TRM, effector T (Teff)/TRM, and T follicular helper compartments. RNA velocity analysis demonstrated a trajectory from TRM to Teff/TRM clusters in association with rejection. By integrating pre- and post-transplantation (Tx) mixed lymphocyte reaction-determined alloreactive repertoires, we observed that pre-existing HvG-reactive T cells that demonstrated tolerance in the circulation were dominated by TRM profiles in quiescent allografts. Putative de novo HvG-reactive clones showed a transcriptional profile skewed to cytotoxic effectors in rejecting grafts. Inferred protein regulon network analysis revealed upstream regulators that accounted for the effector and tolerant T cell states. We demonstrate Teff/TRM interchangeability for individual T cell clones with known (allo)recognition in the human gut, providing novel insight into TRM biology.

Dynamic establishment and maintenance of the human intestinal B cell population and repertoire following transplantation.

It is unknown how intestinal B cell populations and B cell receptor (BCR) repertoires are established and maintained over time in humans. Following intestinal transplantation (ITx), surveillance ileal mucosal biopsies provide a unique opportunity to map the dynamic establishment of gut lymphocyte populations. Using polychromatic flow cytometry that includes HLA allele group-specific mAbs distinguishing donor from recipient cells along with high throughput BCR sequencing, we tracked the establishment of recipient B cell populations and BCR repertoire in the allograft mucosa of ITx recipients. We confirm the early presence of naïve donor B cells in the circulation and, for the first time, document the establishment of recipient B cell populations, including B resident memory cells, in the intestinal allograft mucosa. Recipient B cell repopulation of the allograft was most rapid in infant (<1 year old)-derived allografts and, unlike T cell repopulation, did not correlate with rejection rates. While recipient memory B cell populations were increased in graft mucosa compared to circulation, naïve recipient B cells remained detectable in the graft mucosa for years. Comparisons of peripheral and intra-mucosal B cell repertoires in the absence of rejection revealed increased BCR mutation rates and clonal expansion in graft mucosa compared to circulating B cells, but these parameters did not increase markedly after the first year post-transplant. Furthermore, clonal mixing between the allograft mucosa and the circulation was significantly greater in ITx recipients, even years after transplantation, than in healthy control adults. Collectively, our data demonstrate intestinal mucosal B cell repertoire establishment from a circulating pool, a process that continues for years without evidence of establishment of a stable mucosal B cell repertoire.

Immature and activated phenotype of blood NK cells is associated with acute rejection in adult liver transplant.

Natural killer (NK) cells contribute to liver transplant (LTx) rejection. However, the blood-circulating NK-cell dynamics of patients who experience acute rejection (AR) are unclear. Herein, we longitudinally profiled the total NK cells and their subsets, along with the expression of activating and inhibitory receptors in sequential peripheral blood mononuclear cell samples, spanning from before LTx to the first year after LTx of 32 patients with AR and 30 patients under a steady immune status. Before transplantation, patients with AR (rejectors) contained a significantly higher proportion of the immature CD56 bright CD16 - subset and a lower cytolytic CD56 dim CD16 + in the total blood-circulating NK cells than patients with steady immunity. Both subsets contained a high NKp30-positive population, and CD56 dim CD16 + additionally exhibited a high NKp46-positive ratio. The NKp30-positive ratio in CD56 dim CD16 + subset showed the most prominent AR predictive ability before LTx and was an independent risk factor of LTx AR. After transplantation, the blood-circulating NK cells in rejectors maintained a higher CD56 bright CD16 - and lower CD56 dim CD16 + composition than the controls throughout the first year after LTx. Moreover, both subsets maintained a high NKp30-positive ratio, and CD56 dim CD16 + retained a high NKp46-positive ratio. The blood-circulating NK cell subset composition was consistent during AR, while the expressions of NKp30 and NKp46 were augmented. Collectively, a more immature CD56 bright CD16 - subset composition and an activated phenotype of high NKp30 expression were the general properties of blood-circulating NK cells in rejected LTx recipients, and the NKp30-positive ratio in CD56 dim CD16 + NK subset before LTx possessed AR predictive potential.

Expansion kinetics of graft-versus-host T cell clones in patients with post-liver transplant graft-versus-host disease.

Graft-versus-host disease (GVHD) following liver transplantation is induced by the graft-versus-host (GVH) T cell that is transferred with the liver graft, but the dynamics remain poorly investigated in clinical liver transplantation GVHD. Here, we report that in two liver transplantation recipients who developed GVHD, both of whom showed donor T cell macrochimerism in the blood before clinical GVHD onset. Longitudinal tracking of GVH T cell clones in one of these recipients revealed that GVH T cell clonal expansion occurred before disease onset, and the dominant GVH T cells might also derive from non-hepatic tissue-resident memory T cells in the liver-graft. Additionally, a comparison of the inflammatory cytokine levels and TCR repertoire diversities in recipient pre-liver transplantation blood between 4 patients with GVHD and 12 non-GVHD patients showed that the levels of TNF-α and IL-8, and the overall TCR repertoire skewness in pre-transplant recipient blood samples may serve as potential independent risk factors for the disease.

T cell repertoire profiling in allografts and native tissues in recipients with COVID-19 after solid organ transplantation: Insight into T cell-mediated allograft protection from viral infection.

Introduction: The effects of the SARS-CoV-2 virus on the body, and why the effects are more severe in certain patients, remain incompletely understood. One population of special interest is transplant recipients because of their immunosuppressed state. Understanding the pathophysiology of graft dysfunction in transplant patients with the COVID-19 viral syndrome is important for prognosticating the risk to the graft as well as understanding how best to prevent and, if necessary, treat graft injury in these patients. Methods: We analyzed multiple types of solid organ transplant recipients (liver, kidney, heart or lung) at our institution who died from SARS-CoV-2 and underwent autopsy (n = 6) or whose grafts were biopsied during active SARS-CoV-2 infection (n = 8). Their serum inflammatory markers were examined together with the histological appearance, viral load, and TCR repertoire of their graft tissue and, for autopsy patients, several native tissues. Results: Histology and clinical lab results revealed a systemic inflammatory pattern that included elevated inflammatory markers and diffuse tissue damage regardless of graft rejection. Virus was detected throughout all tissues, although most abundant in lungs. The TCR repertoire was broadly similar throughout the tissues of each individual, with greater sharing of dominant clones associated with more rapid disease course. There was no difference in viral load or clonal distribution of overall, COVID-associated, or putative SARS-CoV-2-specific TCRs between allograft and native tissue. We further demonstrated that SARSCoV-2-specific TCR sequences in transplant patients lack a donor HLArestricted pattern, regardless of distribution in allograft or native tissues,suggesting that recognition of viral antigens on infiltrating recipient cells can effectively trigger host T cell anti-viral responses in both the host and graft. Discussion: Our findings suggest a systemic immune response to the SARS-CoV-2 virus in solid organ transplant patients that is not associated with rejection and consistent with a largely destructive effect of recipient HLA-restricted T cell clones that affects donor and native organs similarly.

Enhanced photocatalytic CO2-reduction activity to form CO and CH4 on S-scheme heterostructured ZnFe2O4/Bi2MoO6 photocatalyst.

A novel ZnFe2O4/Bi2MoO6 heterojunction photocatalyst was synthesized by a facile solvothermal route. The incorporation of a narrow bandgap ZnFe2O4 photocatalyst can efficiently improve the range of light response and light absorption capacity of the Bi2MoO6 via the formation of a hybrid structure at the interface. The formed hybrid interface facilitates the separation efficiency of photo-generated carriers at ZnFe2O4/Bi2MoO6 heterojunction significantly. The experimental results confirm that ZnFe2O4/Bi2MoO6-20% heterojunction showed the highest photocatalytic efficiency for CO2 reduction. Specifically, the total product yield of 47.1 µmol g-1 under 5 h simulated sunlight irradiation is measured in the counterparts of pure ZnFe2O4 (14.79 µmol g-1) and pure Bi2MoO6 (19.01 µmol g-1). Indeed, the formation of ZnFe2O4/Bi2MoO6 heterojunction improved the photocatalytic efficiency for CO2 reduction.

Down-Regulation of ZEB1 by miR-199a-3p Overexpression Restrains Tumor Stem-Like Properties and Mitochondrial Function of Non-Small Cell Lung Cancer.

OBJECTIVE: MicroRNA-199a-3p (miR-199a-3p or miR-199b-3p) targeting of 3'-UTR of ZEB1 was characterized as an important way to inhibit invasion and metastases in non-small cell lung cancer (NSCLC), one of the most common cancers around the world. Here we aimed to investigate the tumor-suppressive role of miR-199a-3p targeted ZEB1. MATERIALS AND METHODS: A549 cells were transfected with ZEB1 and/or miR-199a-3p. Then, tumor growth was investigated in xenograft mice. Stem-like property, proliferation and mitochondria injury were further validated in vitro. RESULTS: Overexpression of miR-199a-3p with premiRNAs significantly reduced tumor growth inhibited CD44 and Ki67 and increased Caspase-3 in A549 xenograft mice. Sphere formation and protein expression of stem-like markers showed that miR-199a-3p inhibited stemness of A549 cell. miR-199a-3p reduced proliferation of A549 cells, as showed with EdU staining and reduced expression of Ki67. Transfection of miR-199a-3p also promoted apoptosis, as indicated with increased apoptotic cells with flow cytometry, and increased cleaved Caspase-3/Caspase3 and Bcl-2/Bax. Apoptosis was further validated to be induced with mitochondria dysfunction, which indicated with JC-1 labeled loss of mitochondrial membrane potential, reduced activity of SOD, and increased MDA and LDH. All these effects were inverted with overexpression of ZEB1. CONCLUSION: Altogether, the findings suggested that the up-regulation of miR-199a-3p significantly inhibited NSCLC growth in vivo, and reduced A549 cell proliferation and promoted mitochondrial-mediated apoptosis, through down-regulation of ZEB1. The findings supported ZEB1 down-expression with miR-199a-3p as a novel therapeutic target for NSCLC treatment.

MIER3 suppresses the progression of non-small cell lung cancer by inhibiting Wnt/ß-Catenin pathway and histone acetyltransferase activity.

BACKGROUND: The mesoderm induction early response 1, family member 3 (MIER3) gene has been recognized as potentially being associated with cancer. However, in relation to the development of non-small cell lung cancer (NSCLC), the expression pattern and the role of MIER3 are yet to be reported. The aim of this research was to investigate the rate of expression of MIER3 in NSCLC cells and tissues and to investigate the role of MIER3 in NSCLC. METHODS: Seventeen patients received NSCLC tissues and corresponding healthy tissues. MTT assay was used to determine cell proliferation. For detecting mRNA and protein expression, we used both quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot method. To measure cell apoptosis and cell cycle distribution, we applied the flow cytometry technique. We used a wound-healing assay and a Transwell invasion assay to study cell migration and invasion. RESULTS: In comparison with adjacent normal tissues, the expression of MIER3 was down-regulated in NSCLC tissues. In addition, the level of MIER3 in NSCLC cell lines was also lower than in pulmonary epithelial cell BEAS-2B. Moreover, when MIER3 was overexpressed, cell proliferation, migration, and invasion were significantly inhibited, apoptosis increased, and cell cycle arrest was induced in A549 and H460 cells. MIER3 overexpression also suppressed tumor growth in NSCLC xenograft mouse models. Furthermore, our study demonstrated that MIER3 down-regulated the Wnt/ß-catenin signaling pathway in NSCLC cells. More importantly, MIER3 decreased the activity of histone acetyltransferase (HAT) p300, which may have contributed to its regulation on ß-catenin and tumorigenesis. CONCLUSIONS: The data suggests MIER3 takes on the tumor-suppressor role in the progression of NSCLC and, therefore, could prove to be a valuable clinical marker in the prognosis of the disease.

Erratum to MIER3 suppresses the progression of non-small cell lung cancer by inhibiting Wnt/ß-Catenin pathway and histone acetyltransferase activity.

[This corrects the article DOI: 10.21037/tcr.2020.01.07.].

Resolution of inflammation in neuromyelitis optica spectrum disorders.

BACKGROUND: Neuromyelitis optica spectrum disorders (NMOSD) are a spectrum of neuroinflammatory disorders associated with autoimmune antibodies against aquaporin-4 (AQP4). Accumulating evidence suggests that inflammation is involved in NMOSD pathogenesis. Resolution of inflammation, which is a highly regulated process mediated by specialized pro-resolving lipid mediators (SPMs) is important to prevent over-responsive inflammation. Deficiency in resolution of inflammation may lead to or accelerates inflammatory diseases. However, whether resolution of inflammation is impaired in NMOSD is not known. The objective of this study was to analyze the levels of SPMs in the serum and cerebrospinal fluid (CSF) of NMOSD patients, and to explore the roles of SPMs in clinical features of NMOSD. METHODS: Thirty-five patients with NMOSD, 34 patients with multiple sclerosis, and 36 patients with non-inflammatory neurological diseases were enrolled in this study. Pro-resolving mediators including Annexin A1 (ANXA1) and resolvin D1 (RvD1), as well as pro-inflammatory lipid mediator leukotriene B4 (LTB4) levels were analyzed by enzyme-linked immunosorbent assay. Pro- and anti-inflammatory cytokines as well as chemokine levels were analyzed using cytometric beads array (CBA). RESULTS: Our results showed RvD1 levels were significantly decreased, whereas LTB4 levels were significantly increased in the CSF of NMOSD patients. AQP4-IgG titer was negatively correlated with RvD1 levels in the CSF of NMOSD patients. CONCLUSIONS: Decreased RvD1 levels indicate impaired resolution of inflammation in NMOSD patients. AQP4-IgG may contribute to increased inflammation and lead to unresolved inflammation in NMOSD.

CART peptide activates the Nrf2/HO-1 antioxidant pathway and protects hippocampal neurons in a rat model of Alzheimer's disease.

The accumulation of amyloid-beta (Aß) and oxidative stress damage in the brain are recognized as early features of Alzheimer's disease (AD). The cocaine- and amphetamine-regulated transcript (CART) peptide may possibly play an antioxidative role in neurons. The aim of this study was to investigate the potential antioxidant mechanism of CART peptide in a rat model of AD. We microinjected of Aß1-42 (2µl/4µg/hemisphere) into rat hippocampus to set a rat model of AD. A pre-microinjection of CART peptide (1µl/0.02µg/hemisphere) into rat hippocampus was administered for five consecutive days before Aß1-42 treatment. We found that Aß1-42 microinjection led to reduction of endogenous CART level in rat hippocampus. CART pretreatment improved the spatial memory and locomotor ability of AD rats. CART peptide decreased the Aß1-42 and Aß production-associated enzyme BACE1 levels. Moreover, CART peptide attenuated the oxidative stress damage with a concrete manifestation of increased MDA as well as decreased T-SOD, GSH and ATP levels in the hippocampus of Aß1-42-treated rat, which may be causatively implicated the activating of Nrf2/HO-1 signaling pathway. Furthermore, CART peptide attenuated neuronal apoptosis with decreased Bax, caspase-9 and caspase-3 levels and increased Bcl-2 level in rat hippocampus. Our results therefore indicate that CART peptide could serve as an antioxidant in early therapy for AD.