To purify alpha chain NC1 domains of type IV collagen [alpha (IV) NC1] from bovine kidney and to evaluate their application in ELISA for detecting anti-glomerular basement membrane (GBM) antibodies.Glomeruli were isolated by differential sieving from bovine kidney, and GBM was isolated by 40 g.L-1 deoxycholic acid extraction technique. Then the insoluble basement membrane material was digested using collagenase, and the non-collagenous domain (NC1) was isolated by Mono Q ion exchange chromatography. The purity and activity of the purified alpha (IV) NC1 technique were assessed using SDS-PAGE and Western blot analysis. An ELISA was established using purified bovine alpha (IV) NC1 as solid phase antigens to detect anti-GBM antibodies. Ninety sera from patients with known anti-GBM antibody positive were tested by alpha (IV) NC1-ELISA. One hundred sera from healthy blood donors and fifty sera from patients with other renal diseases were used as controls. The specificity and the sensitivity of the method were evaluated.Bovine alpha (IV) NC1 was purified with 25 x 10(3) and 50 x 10(3) on SDS-PAGE and could be blotted by known anti-GBM antibody positive sera. The specificity and the sensitivity of the alpha (IV) NC1-ELISA were 98% and 100% respectively.Purified bovine alpha (IV) NC1 could be used as a substitute for human alpha (IV) NC1 to detect anti-GBM antibodies.
1. Inosine monophosphate (IMP), is an essential component for meat flavour and microRNAs (miRNAs) play a vital role in its post-transcriptional regulation. However, the mechanism of how miRNA expression affects muscle-specific IMP deposition is unclear.2. The following study performed transcriptome sequencing and bioinformatics analysis of breast and leg muscle, which have significantly different IMP content in Jingyuan chicken. The differential miRNA-mRNAs were screened out and correlation analysis with IMP content was performed.3. A total of 39 differentially expressed miRNAs (DE miRNAs) and 666 differentially expressed mRNAs (DE mRNAs) were identified between breast muscles and leg muscles. Using miRNA-mRNA integrated analysis, 29 miRNA-target gene pairs were obtained, composed of 13 DE miRNAs and 28 DE mRNAs. Next, purine metabolism, glycolysis/gluconeogenesis, pyruvate metabolism and the biosynthesis of amino acid pathways as necessary for muscle IMP-specific deposition were identified. The differentially expressed gene PKM2, which was significantly enriched in all four pathways, is involved in IMP anabolism in the form of energy metabolism and enzyme activity regulation. The correlation analysis suggested that the gga-miR-107-3p-KLHDC2 negative interaction may be a key regulator in IMP deposition.4. This study explores the functional mechanism of IMP-specific deposition in Jingyuan chicken muscles at the miRNA and mRNA levels and highlights multiple candidate miRNAs and mRNAs for molecular-assisted breeding.
Abstract During chronic viral infection and cancer, CD8 T-cells undergo a differentiation process commonly referred to as T-cell exhaustion. This process is traditionally defined by a stepwise loss of effector functions, eventually leading to cell death. Despite their inability to completely clear the infection, exhausted T-cells are still necessary for limiting viral replication during infection. Thus, it has been proposed that functional adaptation is a more appropriate term for T-cell exhaustion, as CD8 T-cells may be undergoing a multifaceted process of differentiation to better meet the needs of a chronic infection. In line with this hypothesis, it has recently been demonstrated that CD8 T-cells responding to chronic infection are non-homogenous and can be compartmentalized into at least two major subsets, with a TCF-1+ subset serving as a progenitor population that can give rise to a more terminally exhausted TCF-1- subset. However, whether additional heterogeneity exists among CD8 T-cells responding to persistent infection remains unclear. Here, we used ScRNA-seq to fully characterize the heterogeneity of CD8 T-cells during chronic LCMV Cl13 infection. We identified that several transcriptionally distinct subsets of CD8 T-cells develop during chronic LCMV infection, with 3 particular clusters, Slamf6, Pdcd1, and Cx3cr1 cell subsets dominating the antiviral CD8 T-cell response. Importantly both ScRNA-seq and flow cytometric analyses demonstrated that differential expression of cell surface receptors CX3CR1 and Ly108 (encoded by Slamf6) can distinguish these 3 major T-cell subsets. Notably, Ly108 cells shared similar characteristics to the previously described progenitor population and displayed elevated expression of TCF-1. Conversely, CX3CR1 CD8 T-cells displayed increased expression of killer cell lectin-like receptors Klre1 and Klra9, and the TFs T-bet and Zeb2, whereas CX3CR1-Ly108- (DN) cells exhibited elevated expression of multiple co-inhibitory receptors and the TFs Eomes and Nr4a2. Ex vivo functional analyses further indicated that Ly108 CD8 T-cells exhibit an enhanced capacity to co-produce IFN-γ and TNF-α upon GP33 peptide stimulation, whereas CX3CR1 CD8 T-cells display augmented cytotoxicity against peptide-pulsed targeT-cells. Sc trajectory modeling using Monocle analyses predicted that Ly108 CD8 T-cells give rise to both CX3CR1 and DN subsets, with the DN subset branch appearing closer in pseudotime to the Ly108 progenitor subset. To determine the in vivo differentiation trajectory, proliferative potential, and phenotypic stability of these 3 subsets, we performed adoptive transfer experiments using congenically marked CD8 T-cells. Importantly, and consistent with our Monocle predictions, our results demonstrate that Ly108 CD8 T-cells display robust secondary proliferation and give rise to both CX3CR1 and DN subsets. By contrast, CX3CR1 cells retained high CX3CR1 and T-bet expression and did not differentiate into Ly108 or DN CD8 T-cells. Intriguingly, although the DN subset appeared to be the most phenotypically and functionally exhausted subset, more than half of their progeny acquired high CX3CR1 and T-bet expression, indicating that this subset may not be as terminally differentiated as its CX3CR1 counterpart. Notably, our ScRNA-seq analyses also identified that DN cells displayed the highest levels of IL-21R expression, suggesting a potential role for CD4 help in regulating the differentiation of this subset. Strikingly, depletion of CD4 T-cells or deletion of IL-21R signaling in P14 transgenic CD8 T-cells abrogated the development of the CX3CR1 CD8 subset, indicating a critical role for CD4 help in facilitating the differentiation of exhausted CD8 T-cells into a potent cytotoxic CD8 subset. Collectively, our work supports a new model of CD8 T-cell differentiation during chronic viral infection and has important implications for T-cell-based immunotherapies aimed at treating persistent infections and/or cancer. Citation Format: Ryan Zander, David Schauder, Gang Xin, Christine Nguyen, Xiaopeng Wu, Weiguo Cui. Single-cell RNA-sequencing (ScRNA-seq) reveals broad heterogeneity among CD8 T-cells during chronic viral infection and identifies a critical role for CD4 help in promoting the differentiation of a potent cytotoxic CD8 T-cell subset [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B200.
It has been reported multiple times that exercise can prevent cancer development, help anti-tumor therapies, and lower relapse rate after successful cancer treatment. However, an underlying molecular mechanism for these beneficial effects remained elusive. Recent studies revealed that cytotoxic immune cell populations contribute to exercise mediated effects, and exercise induced hormones including insulin, cortisol, testosterone, and epinephrine have been studied in this context, especially related to CD8+ T cell function. Exercise also induces secretion of another myokine irisin, which was originally discovered in 2012. Role of irisin has been primarily studied in metabolic disease including obesity and diabetes. However, the role of irisin in tumor immunology has not been studied.
Methods
To address this point, multiple pre-clinical tumor models including colon, skin, bladder, and lung cancer were utilized. After tumor implantation, irisin was intraperitoneally injected for every 2 days starting at day 7. Tumor growth was monitored to check anti-tumoral effect of irisin. To address immunological changes within the tumor microenvironment (TME), high-dimensional flow cytometry panels were applied and analyzed. To confirm our findings, publicly available scRNA-seq data sets were re-analyzed and web-based TCGA database was utilized for further validation.
Results
Irisin displayed anti-tumoral activity when tested in multiple pre-clinical models. High-dimensional flow cytometry analysis revealed that irisin treatment reduced accumulation of regulatory T cells (Tregs) in TME. Suppressive marker expressions in Tregs were also decreased and led to better CD8+ T cell function with evidence of less dysfunctional signature. Integrin αv-TGF-β axis was identified for responsible mechanism, and myeloid cell specific knockout of integrin αv further confirmed importance of this pathway. Our findings were validated using publicly available scRNA-seq, spatial-transcriptomics, and TCGA data sets. Integrin αv was specifically expressed in myeloid cells, and TGF-β signaling pathway was higher in integrin av adjacent spots. Also, overall survival rate and immunotherapy response rate was lower in patients with high expression of integrin complex and TGF-β. This was experimentally validated in pre-clinical model by combining irisin with PD-1 blocking antibody; the combination group exhibited better tumor control.
Conclusions
We conclude that irisin treatment reduced tumor growth in multiple tumor models and this outcome was mediated by T cells. Integrin αv-TGF-β axis in the TME was responsible for this effect, and blocking this pathway exhibited better tumor control. Re-analysis of publicly available data sets further validated importance of this signaling pathway, implying therapeutic potential of irisin in treating cancer.
Manipulation of host cellular pathways is a strategy employed by gammaherpesviruses, including mouse gammaherpesvirus 68 (MHV68), in order to negotiate a chronic infection. Ataxia-telangiectasia mutated (ATM) plays a unique yet incompletely understood role in gammaherpesvirus infection, as it has both proviral and antiviral effects. Chronic gammaherpesvirus infection is poorly controlled in a host with global ATM insufficiency, whether the host is a mouse or a human. In contrast, ATM facilitates replication, reactivation, and latency establishment of several gammaherpesviruses
Targeting negative regulators downstream of the T cell receptor (TCR) represents a novel strategy to improve cancer immunotherapy. Two proteins that serve as critical inhibitory regulators downstream of the TCR are diacylglycerol kinase ζ (DGKζ), a regulator of Ras and PKC-θ signaling, and Casitas b-lineage proto-oncogene b (Cbl-b), an E3 ubiquitin ligase that predominantly regulates PI(3)K signaling. We sought to compare the signaling and functional effects that result from deletion of DGKζ, Cbl-b, or both (double knockout, DKO) in T cells, and to evaluate tumor responses generated in a clinically relevant orthotopic pancreatic tumor model. We found that whereas deletion of Cbl-b primarily served to enhance NF-κB signaling, deletion of DGKζ enhanced TCR-mediated signal transduction downstream of Ras/Erk and NF-κB. Deletion of DGKζ or Cbl-b comparably enhanced CD8+ T cell functional responses, such as proliferation, production of IFNγ, and generation of granzyme B when compared with WT T cells. DKO T cells demonstrated enhanced function above that observed with single knockout T cells after weak, but not strong, stimulation. Deletion of DGKζ, but not Cbl-b, however, resulted in significant increases in numbers of activated (CD44hi) CD8+ T cells in both non-treated and tumor-bearing mice. DGKζ-deficient mice also had enhanced control of pancreatic tumor cell growth compared to Cbl-b-deficient mice. This represents the first direct comparison between mice of these genotypes and suggests that T cell immunotherapies may be better improved by targeting TCR signaling molecules that are regulated by DGKζ as opposed to molecules regulated by Cbl-b.
Early exhaustion of tumor infiltrating CD8 T cells is a major obstacle that restricts the effectiveness of immunotherapy in a broader range of cancer patients.1 Mitochondrial health is a crucial factor determining the performance and endurance of CD8 T cells within the immunosuppressive tumor microenvironment (TME).2 Apart from encountering immunosuppressive signals, CD8 T cells also face metabolic challenges such as hypoxia and nutrient deprivation in the TME.3 4 These metabolic stresses disrupt mitochondrial function, leading to diminished CD8 T cell activity and T cell exhaustion.3 4 Recent studies have shown promising results by employing strategies that enhance mitochondrial function to rejuvenate exhausted tumor-infiltrating lymphocytes (TILs).1 2 Mitophagy, an intrinsic process for removing dysfunctional mitochondria, plays a critical role in maintaining mitochondrial quality and metabolic homeostasis.5 Inhibiting USP30, a mitochondrial deubiquitinase, has been demonstrated to enhance mitophagy and improve mitochondrial function.6 7 We propose that the inhibition of USP30 in CD8 T cells could enhance their performance and endurance within the TME by preserving mitochondrial function.
Methods
In this study, we utilized mitophagy reporter (mt-Keima) mice to investigate the mitophagy activity of tumor-infiltrating CD8 T cells based on their exhaustion statuses. We genetically and pharmacologically inhibited USP30 in CD8 T cells and assessed their mitophagy activity, mitochondrial functions, T cell functions, and exhaustion levels within the TME. Additionally, we treated tumor xenograft mice with a USP30 inhibitor and adoptive CD8 T cells with USP30 deletion to evaluate the potential benefits of targeting USP30 in immunotherapies.
Results
Our findings revealed that mitophagy activity, indicated by the mt-Keima fluorescence, is intrinsically reduced in activated CD8 T cells and further compromised in exhausted CD8 T cells expressing PD1 and TIM3 markers (figure 1). Inhibiting USP30 effectively increased mitophagy activity in exhausted CD8 T cells, thereby enhancing mitochondrial efficiency (figure 2). Furthermore, treating mice with a USP30 inhibitor or transferring USP30 knockout adoptive CD8 T cells resulted in reduced T cell exhaustion and improved CD8 T cell functionality, ultimately enhancing anti-tumor immunity (figure 3).
Conclusions
Mitophagy plays a critical role in maintaining mitochondrial health in CD8 T cells within the TME. However, the exhaustion of CD8 T cells can result in the suppression of mitophagy and impaired mitochondrial function. By promoting mitophagy, exhausted CD8 T cells can be rejuvenated, leading to improved function and endurance within the TME. Targeting USP30 is a promising approach to promote mitophagy activity in CD8 T cells and develop more effective immunotherapy strategies.
Acknowledgements
I am offering my sincere appreciation to all the lab members in Dr. Nuo Sun and Dr. Gang Xin's lab. This work couldn't be finished without the help from Dr. Sun in the field of mitochondrial metabolism and Dr. Xin in the field of cancer immunology. Former lab manager, now Ph.D. student, Jianying Li, offered me many helps in using FACS machines.
References
Huang Y, et al. Rewiring mitochondrial metabolism to counteract exhaustion of CAR-T cells. J Hematol Oncol, 2022;15(1):38. Scharping NE, et al. The Tumor Microenvironment Represses T Cell Mitochondrial Biogenesis to Drive Intratumoral T Cell Metabolic Insufficiency and Dysfunction. Immunity, 2016;45(3):701–703. Scharping NE, et al. Mitochondrial stress induced by continuous stimulation under hypoxia rapidly drives T cell exhaustion. Nat Immunol, 2021;22(2):205–215. Yu YR, et al. Disturbed mitochondrial dynamics in CD8(+) TILs reinforce T cell exhaustion. Nat Immunol, 2020;21(12):1540–1551. Palikaras K, E Lionaki, N Tavernarakis. Mechanisms of mitophagy in cellular homeostasis, physiology and pathology. Nat Cell Biol, 2018;20(9):1013–1022. Luo H, et al. Pharmacological inhibition of USP30 activates tissue-specific mitophagy. Acta Physiol (Oxf), 2021;232(3):e13666. Tsefou E, et al. Investigation of USP30 inhibition to enhance Parkin-mediated mitophagy: tools and approaches. Biochem J, 2021;478(23):4099–4118.
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