Triple-negative breast cancer (TNBC) has few therapeutic options, and alternative approaches are urgently needed. Stimulator of IFN genes (STING) is becoming an exciting target for therapeutic adjuvants. However, STING resides inside the cell, and the intracellular delivery of CDNs, such as cGAMP, is required for the optimal activation of STING. We show that liposomal nanoparticle-delivered cGAMP (cGAMP-NP) activates STING more effectively than soluble cGAMP. These particles induce innate and adaptive host immune responses to preexisting tumors in both orthotopic and genetically engineered models of basal-like TNBC. cGAMP-NPs also reduce melanoma tumor load, with limited responsivity to anti–PD-L1. Within the tumor microenvironment, cGAMP-NPs direct both mouse and human macrophages (M), reprograming from protumorigenic M2-like phenotype toward M1-like phenotype; enhance MHC and costimulatory molecule expression; reduce M2 biomarkers; increase IFN-γ–producing T cells; augment tumor apoptosis; and increase CD4+ and CD8+ T cell infiltration. Activated T cells are required for tumor suppression, as their depletion reduces antitumor activity. Importantly, cGAMP-NPs prevent the formation of secondary tumors, and a single dose is sufficient to inhibit TNBC. These data suggest that a minimal system comprised of cGAMP-NP alone is sufficient to modulate the tumor microenvironment to effectively control PD-L1–insensitive TNBC.
Ning Cheng, Rebekah Watkins-Schulz, Robert D. Junkins, Clément N. David, Brandon M. Johnson, Stephanie A. Montgomery, Kevin J. Peine, David B. Darr, Hong Yuan, Karen P. McKinnon, Qi Liu, Lei Miao, Leaf Huang, Eric M. Bachelder, Kristy M. Ainslie, Jenny P-Y Ting
RBC alloimmunization represents a significant immunological challenge for patients requiring lifelong transfusion support. The majority of clinically relevant non-ABO(H) blood group antigens have been thought to drive antibody formation through T cell–dependent immune pathways. Thus, we initially sought to define the role of CD4+ T cells in formation of alloantibodies to KEL, one of the leading causes of hemolytic transfusion reactions. Unexpectedly, our findings demonstrated that KEL RBCs actually possess the ability to induce antibody formation independent of CD4+ T cells or complement component 3 (C3), two common regulators of antibody formation. However, despite the ability of KEL RBCs to induce anti-KEL antibodies in the absence of complement, removal of C3 or complement receptors 1 and 2 (CR1/2) rendered recipients completely reliant on CD4+ T cells for IgG anti-KEL antibody formation. Together, these findings suggest that C3 may serve as a novel molecular switch that regulates the type of immunological pathway engaged following RBC transfusion.
Amanda Mener, Seema R. Patel, Connie M. Arthur, Satheesh Chonat, Andreas Wieland, Manjula Santhanakrishnan, Jingchun Liu, Cheryl L. Maier, Ryan P. Jajosky, Kathryn Girard-Pierce, Ashley Bennett, Patricia E. Zerra, Nicole H. Smith, Jeanne E. Hendrickson, Sean R. Stowell
The mitochondrial Ca2+ uniporter (MCU) complex mediates acute mitochondrial Ca2+ influx. In skeletal muscle, MCU links Ca2+ signaling to energy production by directly enhancing the activity of key metabolic enzymes in the mitochondria. Here, we examined the role of MCU in skeletal muscle development and metabolic function by generating mouse models for the targeted deletion of Mcu in embryonic, postnatal, and adult skeletal muscle. Loss of Mcu did not affect muscle growth and maturation or otherwise cause pathology. Skeletal muscle–specific deletion of Mcu in mice also did not affect myofiber intracellular Ca2+ handling, but it did inhibit acute mitochondrial Ca2+ influx and mitochondrial respiration stimulated by Ca2+, resulting in reduced acute exercise performance in mice. However, loss of Mcu also resulted in enhanced muscle performance under conditions of fatigue, with a preferential shift toward fatty acid metabolism, resulting in reduced body fat with aging. Together, these results demonstrate that MCU-mediated mitochondrial Ca2+ regulation underlies skeletal muscle fuel selection at baseline and under enhanced physiological demands, which affects total homeostatic metabolism.
Jennifer Q. Kwong, Jiuzhou Huo, Michael J. Bround, Justin G. Boyer, Jennifer A. Schwanekamp, Nasab Ghazal, Joshua T. Maxwell, Young C. Jang, Zaza Khuchua, Kevin Shi, Donald M. Bers, Jennifer Davis, Jeffery D. Molkentin
Paramount to the efficacy of immune checkpoint inhibitors is proper selection of patients with adequate tumor immunogenicity and a robust but suppressed immune infiltrate. In colon cancer, immune-based therapies are approved for patients with DNA mismatch repair (MMR) deficiencies, in whom accumulation of genetic mutations results in increased neoantigen expression, triggering an immune response that is suppressed by the PD-L1/PD-1 pathway. Here, we report that characterization of the microenvironment of MMR-deficient metastatic colorectal cancer using multiplex fluorescent immunohistochemistry (mfIHC) identified increased infiltration of cytotoxic T lymphocytes (CTLs), which were more often engaged with epithelial cells (ECs) and improved overall survival. A subset of patients with intact MMR but a similar immune microenvironment to MMR-deficient patients was identified and found to universally express high levels of PD-L1, suggesting that they may represent a currently untreated, checkpoint inhibitor–responsive population. Further, PD-L1 expression on antigen-presenting cells (APCs) in the tumor microenvironment (TME) resulted in impaired CTL/EC engagement and enhanced infiltration and engagement of Tregs. Characterization of the TME by mfIHC highlights the interconnection between immunity and immunosuppression in metastatic colon cancer and may better stratify patients for receipt of immunotherapies.
Jenny Lazarus, Tomasz Maj, J. Joshua Smith, Mirna Perusina Lanfranca, Arvind Rao, Michael I. D’Angelica, Lawrence Delrosario, Alexander Girgis, Casey Schukow, Jinru Shia, Ilona Kryczek, Jiaqi Shi, Isaac Wasserman, Howard Crawford, Hari Nathan, Marina Pasca Di Magliano, Weiping Zou, Timothy L. Frankel
The mechanisms of J wave syndrome (JWS) are incompletely understood. Here, we showed that the concomitant activation of small-conductance calcium-activated potassium (SK) current (IKAS) and inhibition of sodium current by cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA) recapitulate the phenotypes of JWS in Langendorff-perfused rabbit hearts. CyPPA induced significant J wave elevation and frequent spontaneous ventricular fibrillation (SVF), as well as sinus bradycardia, atrioventricular block, and intraventricular conduction delay. IKAS activation by CyPPA resulted in heterogeneous shortening of action potential (AP) duration (APD) and repolarization alternans. CyPPA inhibited cardiac sodium current (INa) and decelerated AP upstroke and intracellular calcium transient. SVFs were typically triggered by short-coupled premature ventricular contractions, initiated with phase 2 reentry and originated more frequently from the right than the left ventricles. Subsequent IKAS blockade by apamin reduced J wave elevation and eliminated SVF. β-Adrenergic stimulation was antiarrhythmic in CyPPA-induced electrical storm. Like CyPPA, hypothermia (32.0°C) also induced J wave elevation and SVF. It facilitated negative calcium-voltage coupling and phase 2 repolarization alternans with spatial and electromechanical discordance, which were ameliorated by apamin. These findings suggest that IKAS activation contributes to the development of JWS in rabbit ventricles.
Mu Chen, Dong-Zhu Xu, Adonis Z. Wu, Shuai Guo, Juyi Wan, Dechun Yin, Shien-Fong Lin, Zhenhui Chen, Michael Rubart-von der Lohe, Thomas H. Everett IV, Zhilin Qu, James N. Weiss, Peng-Sheng Chen
Mutations in the ER chaperone calreticulin (CALR) are common in myeloproliferative neoplasm (MPN) patients, activate the thrombopoietin receptor (MPL), and mediate constitutive JAK/STAT signaling. The mechanisms by which CALR mutations cause myeloid transformation are incompletely defined. We used mass spectrometry proteomics to identify CALR-mutant interacting proteins. Mutant CALR caused mislocalization of binding partners and increased recruitment of FLI1, ERP57, and CALR to the MPL promoter to enhance transcription. Consistent with a critical role for CALR-mediated JAK/STAT activation, we confirmed the efficacy of JAK2 inhibition on CALR-mutant cells in vitro and in vivo. Due to the altered interactome induced by CALR mutations, we hypothesized that CALR-mutant MPNs may be vulnerable to disruption of aberrant CALR protein complexes. A synthetic peptide designed to competitively inhibit the carboxy terminal of CALR specifically abrogated MPL/JAK/STAT signaling in cell lines and primary samples and improved the efficacy of JAK kinase inhibitors. These findings reveal what to our knowledge is a novel potential therapeutic approach for patients with CALR-mutant MPN.
Elodie Pronier, Paolo Cifani, Tiffany R. Merlinsky, Katharine Barr Berman, Amritha Varshini Hanasoge Somasundara, Raajit K. Rampal, John LaCava, Karen E. Wei, Friederike Pastore, Jesper L.V. Maag, Jane Park, Richard Koche, Alex Kentsis, Ross L. Levine
BACKGROUND. Inflammation helps regulate normal growth and tissue repair. Although bone morphogenetic proteins (BMPs) and inflammation are known contributors to abnormal bone formation, how these pathways interact in ossification remains unclear. METHODS. We examined this potential link in patients with fibrodysplasia ossificans progressiva (FOP), a genetic condition of progressive heterotopic ossification caused by activating mutations in the Activin A type I receptor (ACVR1/ALK2). FOP patients show exquisite sensitivity to trauma, suggesting that BMP pathway activation may alter immune responses. We studied primary blood, monocyte, and macrophage samples from control and FOP subjects using multiplex cytokine, gene expression, and protein analyses; examined CD14+ primary monocyte and macrophage responses to TLR ligands; and assayed BMP, TGF-β activated kinase 1 (TAK1), and NF-κB pathways. RESULTS. FOP subjects at baseline without clinically evident heterotopic ossification showed increased serum IL-3, IL-7, IL-8, and IL-10. CD14+ primary monocytes treated with the TLR4 activator LPS showed increased CCL5, CCR7, and CXCL10; abnormal cytokine/chemokine secretion; and prolonged activation of the NF-κB pathway. FOP macrophages derived from primary monocytes also showed abnormal cytokine/chemokine secretion, increased TGF-β production, and p38MAPK activation. Surprisingly, SMAD phosphorylation was not significantly changed in the FOP monocytes/macrophages. CONCLUSIONS. Abnormal ACVR1 activity causes a proinflammatory state via increased NF-κB and p38MAPK activity. Similar changes may contribute to other types of heterotopic ossification, such as in scleroderma and dermatomyositis; after trauma; or with recombinant BMP-induced bone fusion. Our findings suggest that chronic antiinflammatory treatment may be useful for heterotopic ossification.
Emilie Barruet, Blanca M. Morales, Corey J. Cain, Amy N. Ton, Kelly L. Wentworth, Tea V. Chan, Tania A. Moody, Mariëlle C. Haks, Tom H.M. Ottenhoff, Judith Hellman, Mary C. Nakamura, Edward C. Hsiao
BACKGROUND. The molecular understanding of the progression from acute to chronic organ injury is limited. Ischemia/reperfusion injury (IRI) triggered during kidney transplantation can contribute to progressive allograft dysfunction. METHODS. Protocol biopsies (n = 163) were obtained from 42 kidney allografts at 4 time points after transplantation. RNA sequencing–mediated (RNA-seq–mediated) transcriptional profiling and machine learning computational approaches were employed to analyze the molecular responses to IRI and to identify shared and divergent transcriptional trajectories associated with distinct clinical outcomes. The data were compared with the response to IRI in a mouse model of the acute to chronic kidney injury transition. RESULTS. In the first hours after reperfusion, all patients exhibited a similar transcriptional program under the control of immediate-early response genes. In the following months, we identified 2 main transcriptional trajectories leading to kidney recovery or to sustained injury with associated fibrosis and renal dysfunction. The molecular map generated by this computational approach highlighted early markers of kidney disease progression and delineated transcriptional programs associated with the transition to chronic injury. The characterization of a similar process in a mouse IRI model extended the relevance of our findings beyond transplantation. CONCLUSIONS. The integration of multiple transcriptomes from serial biopsies with advanced computational algorithms overcame the analytical hurdles related to variability between individuals and identified shared transcriptional elements of kidney disease progression in humans, which may prove as useful predictors of disease progression following kidney transplantation and kidney injury. This generally applicable approach opens the way for an unbiased analysis of human disease progression. FUNDING. The study was supported by the California Institute for Regenerative Medicine and by the Swiss National Science Foundation.
Pietro E. Cippà, Bo Sun, Jing Liu, Liang Chen, Maarten Naesens, Andrew P. McMahon
Juvenile dermatomyositis (JDM) is a debilitating pediatric autoimmune disease manifesting with characteristic rash and muscle weakness. To delineate signaling abnormalities in JDM, mass cytometry was performed with PBMCs from treatment-naive JDM patients and controls. NK cell percentages were lower while frequencies of naive B cells and naive CD4+ T cells were higher in JDM patients than in controls. These cell frequency differences were attenuated with cessation of active disease. A large number of signaling differences were identified in treatment-naive JDM patients compared with controls. Classification models incorporating feature selection demonstrated that differences in phospholipase Cγ2 (PLCγ2) phosphorylation comprised 10 of 12 features (i.e., phosphoprotein in a specific immune cell subset) distinguishing the 2 groups. Because NK cells represented 5 of these 12 features, further studies focused on the PLCγ2 pathway in NK cells, which is responsible for stimulating calcium flux and cytotoxic granule movement. No differences were detected in upstream signaling or total PLCγ2 protein levels. Hypophosphorylation of PLCγ2 and downstream mitogen-activated protein kinase-activated protein kinase 2 were partially attenuated with cessation of active disease. PLCγ2 hypophosphorylation in treatment-naive JDM patients resulted in decreased calcium flux. The identification of dysregulation of PLCγ2 phosphorylation and decreased calcium flux in NK cells provides potential mechanistic insight into JDM pathogenesis.
Allison A. Throm, Joshua B. Alinger, Jeanette T. Pingel, Allyssa L. Daugherty, Lauren M. Pachman, Anthony R. French
Our previous work demonstrated a protective role of protein S in early diabetic kidney disease (DKD). Protein S exerts antiinflammatory and antiapoptotic effects through the activation of TYRO3, AXL, and MER (TAM) receptors. Among the 3 TAM receptors, we showed that the biological effects of protein S were mediated largely by TYRO3 in diabetic kidneys. Our data now show that TYRO3 mRNA expression is highly enriched in human glomeruli and that TYRO3 protein is expressed in podocytes. Interestingly, glomerular TYRO3 mRNA expression increased in mild DKD but was suppressed in progressive DKD, as well as in focal segmental glomerulosclerosis (FSGS). Functionally, morpholino-mediated knockdown of tyro3 altered glomerular filtration barrier development in zebrafish larvae, and genetic ablation of Tyro3 in murine models of DKD and Adriamycin-induced nephropathy (ADRN) worsened albuminuria and glomerular injury. Conversely, the induction of TYRO3 overexpression specifically in podocytes significantly attenuated albuminuria and kidney injury in mice with DKD, ADRN, and HIV-associated nephropathy (HIVAN). Mechanistically, TYRO3 expression was suppressed by activation of TNF-α/NF-κB pathway, which may contribute to decreased TYRO3 expression in progressive DKD and FSGS, and TYRO3 signaling conferred antiapoptotic effects through the activation of AKT in podocytes. In conclusion, TYRO3 plays a critical role in maintaining normal podocyte function and may be a potential new drug target to treat glomerular diseases.
Fang Zhong, Zhaohong Chen, Liwen Zhang, Yifan Xie, Viji Nair, Wenjun Ju, Matthias Kretzler, Robert G. Nelson, Zhengzhe Li, Hongyu Chen, Yongjun Wang, Aihua Zhang, Kyung Lee, Zhihong Liu, John Cijiang He
Obesity is characterized by accumulation of adipose tissue and is one the most important risk factors in the development of insulin resistance. Carbon monoxide–releasing (CO-releasing) molecules (CO-RMs) have been reported to improve the metabolic profile of obese mice, but the underlying mechanism remains poorly defined. Here, we show that oral administration of CORM-401 to obese mice fed a high-fat diet (HFD) resulted in a significant reduction in body weight gain, accompanied by a marked improvement in glucose homeostasis. We further unmasked an action we believe to be novel, by which CO accumulates in visceral adipose tissue and uncouples mitochondrial respiration in adipocytes, ultimately leading to a concomitant switch toward glycolysis. This was accompanied by enhanced systemic and adipose tissue insulin sensitivity, as indicated by a lower blood glucose and increased Akt phosphorylation. Our findings indicate that the transient uncoupling activity of CO elicited by repetitive administration of CORM-401 is associated with lower weight gain and increased insulin sensitivity during HFD. Thus, prototypic compounds that release CO could be investigated for developing promising insulin-sensitizing agents.
Laura Braud, Maria Pini, Lucie Muchova, Sylvie Manin, Hiroaki Kitagishi, Daigo Sawaki, Gabor Czibik, Julien Ternacle, Geneviève Derumeaux, Roberta Foresti, Roberto Motterlini
Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant or digenic disorder linked to derepression of the toxic DUX4 gene in muscle. There is currently no pharmacological treatment. The emergence of DUX4 enabled development of cell and animal models that could be used for basic and translational research. Since DUX4 is toxic, animal model development has been challenging, but progress has been made, revealing that tight regulation of DUX4 expression is critical for creating viable animals that develop myopathy. Here, we report such a model — the tamoxifen-inducible FSHD mouse model called TIC-DUX4. Uninduced animals are viable, born in Mendelian ratios, and overtly indistinguishable from WT animals. Induced animals display significant DUX4-dependent myopathic phenotypes at the molecular, histological, and functional levels. To demonstrate the utility of TIC-DUX4 mice for therapeutic development, we tested a gene therapy approach aimed at improving muscle strength in DUX4-expressing muscles using adeno-associated virus serotype 1.Follistatin (AAV1.Follistatin), a natural myostatin antagonist. This strategy was not designed to modulate DUX4 but could offer a mechanism to improve muscle weakness caused by DUX4-induced damage. AAV1.Follistatin significantly increased TIC-DUX4 muscle mass and strength even in the presence of DUX4 expression, suggesting that myostatin inhibition may be a promising approach to treat FSHD-associated weakness. We conclude that TIC-DUX4 mice are a relevant model to study DUX4 toxicity and, importantly, are useful in therapeutic development studies for FSHD.
Carlee R. Giesige, Lindsay M. Wallace, Kristin N. Heller, Jocelyn O. Eidahl, Nizar Y. Saad, Allison M. Fowler, Nettie K. Pyne, Mustafa Al-Kharsan, Afrooz Rashnonejad, Gholamhossein Amini Chermahini, Jacqueline S. Domire, Diana Mukweyi, Sara E. Garwick-Coppens, Susan M. Guckes, K. John McLaughlin, Kathrin Meyer, Louise R. Rodino-Klapac, Scott Q. Harper
Tissue-resident memory T cells (TRMs) accelerate pathogen clearance through rapid and enhanced functional responses in situ. TRMs are prevalent in diverse anatomic sites throughout the human lifespan, yet their phenotypic and functional diversity has not been fully described. Here, we identify subpopulations of human TRMs based on the ability to efflux fluorescent dyes [efflux(+) TRMs] located within mucosal and lymphoid sites with distinct transcriptional profiles, turnover, and functional capacities. Compared with efflux(–) TRMs, efflux(+) TRMs showed transcriptional and phenotypic features of quiescence including reduced turnover, decreased expression of exhaustion markers, and increased proliferative capacity and signaling in response to homeostatic cytokines. Moreover, upon activation, efflux(+) TRMs secreted lower levels of inflammatory cytokines such as IFN-γ and IL-2 and underwent reduced degranulation. Interestingly, analysis of TRM subsets following activation revealed that both efflux(+) and efflux(–) TRMs undergo extensive transcriptional changes following TCR ligation but retain core TRM transcriptional properties including retention markers, suggesting that TRMs carry out effector function in situ. Overall, our results suggest a model for tissue-resident immunity wherein heterogeneous subsets have differential capacities for longevity and effector function.
Brahma V. Kumar, Radomir Kratchmarov, Michelle Miron, Dustin J. Carpenter, Takashi Senda, Harvey Lerner, Amy Friedman, Steven L. Reiner, Donna L. Farber
BACKGROUND. Matrix metalloprotease 9 (MMP-9) is associated with inflammation and lung remodeling in chronic obstructive pulmonary disease (COPD). We hypothesized that elevated circulating MMP-9 represents a potentially novel biomarker that identifies a subset of individuals with COPD with an inflammatory phenotype who are at increased risk for acute exacerbation (AECOPD). METHODS. We analyzed Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS) and Genetic Epidemiology of COPD (COPDGene) cohorts for which baseline and prospective data were available. Elevated MMP-9 was defined based on >95th percentile plasma values from control (non-COPD) sample in SPIROMICS. COPD subjects were classified as having elevated or nonelevated MMP-9. Logistic, Poisson, and Kaplan-Meier analyses were used to identify associations with prospective AECOPD in both cohorts. RESULTS. Elevated MMP-9 was present in 95/1,053 (9%) of SPIROMICS and 41/140 (29%) of COPDGene participants with COPD. COPD subjects with elevated MMP-9 had a 13%–16% increased absolute risk for AECOPD and a higher median (interquartile range; IQR) annual AECOPD rate (0.33 [0–0.74] versus 0 [0–0.80] events/year and 0.9 [0.5–2] versus 0.5 [0–1.4] events/year for SPIROMICS and COPDGene, respectively). In adjusted models within each cohort, elevated MMP-9 was associated with increased odds (odds ratio [OR], 1.71; 95%CI, 1.00–2.90; and OR, 3.03; 95%CI, 1.02–9.01), frequency (incidence rate ratio [IRR], 1.45; 95%CI, 1.23–1.7; and IRR, 1.24; 95%CI, 1.03–1.49), and shorter time-to-first AECOPD (21.7 versus 31.7 months and 14 versus 21 months) in SPIROMICS and COPDGene, respectively. CONCLUSIONS. Elevated MMP-9 was independently associated with AECOPD risk in 2 well-characterized COPD cohorts. These findings provide evidence for MMP-9 as a prognostic biomarker and potential therapeutic target in COPD. TRIAL REGISTRATION. ClinicalTrials.gov: NCT01969344 (SPIROMICS) and NCT00608764 (COPDGene). FUNDING. This work was funded by K08 HL123940 to JMW; R01HL124233 to PJC; Merit Review I01 CX000911 to JLC; R01 (R01HL102371, R01HL126596) and VA Merit (I01BX001756) to AG. SPIROMICS (Subpopulations and Intermediate Outcomes in COPD Study) is funded by contracts from the NHLBI (HHSN268200900013C, HHSN268200900014C,HHSN268200900015C HHSN268200900016C, HHSN268200900017C, HHSN268200900018C, HHSN268200900019C, and HHSN268200900020C) and a grant from the NIH/NHLBI (U01 HL137880), and supplemented by contributions made through the Foundation for the NIH and the COPD Foundation from AstraZeneca/MedImmune; Bayer; Bellerophon Therapeutics; Boehringer-Ingelheim Pharmaceuticals Inc.; Chiesi Farmaceutici; Forest Research Institute Inc.; GlaxoSmithKline; Grifols Therapeutics Inc.; Ikaria Inc.; Novartis Pharmaceuticals Corporation; Nycomed GmbH; ProterixBio; Regeneron Pharmaceuticals Inc.; Sanofi; Sunovion; Takeda Pharmaceutical Company; and Theravance Biopharma and Mylan. COPDGene is funded by the NHLBI (R01 HL089897 and R01 HL089856) and by the COPD Foundation through contributions made to an Industry Advisory Board composed of AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, Novartis, Pfizer, Siemens, and Sunovion.
J. Michael Wells, Margaret M. Parker, Robert A. Oster, Russ P. Bowler, Mark T. Dransfield, Surya P. Bhatt, Michael H. Cho, Victor Kim, Jeffrey L. Curtis, Fernando J. Martinez, Robert Paine III, Wanda O’Neal, Wassim W. Labaki, Robert J. Kaner, Igor Barjaktarevic, MeiLan K. Han, Edwin K. Silverman, James D. Crapo, R. Graham Barr, Prescott Woodruff, Peter J. Castaldi, Amit Gaggar, the SPIROMICS and COPDGene Investigators
Allograft tolerance, in which a graft is accepted without long-term immunosuppression, could overcome numerous obstacles in transplantation. Human allograft tolerance has been intentionally induced across HLA barriers via combined kidney and bone marrow transplantation (CKBMT) with a regimen that induces only transient chimerism. Tregs are enriched early after CKBMT. While deletional tolerance contributes to long-term tolerance, the role of Tregs remains unclear. We have optimized a method for identifying the donor-specific Treg repertoire and used it to interrogate the fate of donor-specific Tregs after CKBMT. We expanded Tregs with several different protocols. Using functional analyses and T cell receptor sequencing, we found that expanding sorted Tregs with activated donor B cells identified the broadest Treg repertoire with the greatest potency and donor specificity of suppression. This method outperformed both alloantigen stimulation with CTLA4Ig and sequencing of CFSElo cells from the primary mixed lymphocyte reaction. In 3 tolerant and 1 nontolerant CKBMT recipients, we sequenced donor-specific Tregs before transplant and tracked them after transplant. Preexisting donor-specific Tregs were expanded at 6 months after CKBMT in tolerant patients and were reduced in the nontolerant patient. These results suggest that early expansion of donor-specific Tregs is involved in tolerance induction following CKBMT.
Thomas M. Savage, Brittany A. Shonts, Aleksandar Obradovic, Susan Dewolf, Saiping Lau, Julien Zuber, Michael T. Simpson, Erik Berglund, Jianing Fu, Suxiao Yang, Siu-Hong Ho, Qizhi Tang, Laurence A. Turka, Yufeng Shen, Megan Sykes
BACKGROUND. Increasing evidence indicates a role for EBV in the pathogenesis of multiple sclerosis (MS). EBV-infected autoreactive B cells might accumulate in the CNS because of defective cytotoxic CD8+ T cell immunity. We sought to determine the feasibility and safety of treating progressive MS patients with autologous EBV-specific T cell therapy. METHODS. An open-label phase I trial was designed to treat 5 patients with secondary progressive MS and 5 patients with primary progressive MS with 4 escalating doses of in vitro–expanded autologous EBV-specific T cells targeting EBV nuclear antigen 1, latent membrane protein 1 (LMP1), and LMP2A. Following adoptive immunotherapy, we monitored the patients for safety and clinical responses. RESULTS. Of the 13 recruited participants, 10 received the full course of T cell therapy. There were no serious adverse events. Seven patients showed improvement, with 6 experiencing both symptomatic and objective neurological improvement, together with a reduction in fatigue, improved quality of life, and, in 3 patients, reduced intrathecal IgG production. All 6 patients receiving T cells with strong EBV reactivity showed clinical improvement, whereas only 1 of the 4 patients receiving T cells with weak EBV reactivity showed improvement (P = 0.033, Fisher’s exact test). CONCLUSION. EBV-specific adoptive T cell therapy was well tolerated. Clinical improvement following treatment was associated with the potency of EBV-specific reactivity of the administered T cells. Further clinical trials are warranted to determine the efficacy of EBV-specific T cell therapy in MS. TRIAL REGISTRATION. Australian New Zealand Clinical Trials Registry, ACTRN12615000422527. FUNDING. MS Queensland, MS Research Australia, Perpetual Trustee Company Ltd., and donations from private individuals who wish to remain anonymous.
Michael P. Pender, Peter A. Csurhes, Corey Smith, Nanette L. Douglas, Michelle A. Neller, Katherine K. Matthews, Leone Beagley, Sweera Rehan, Pauline Crooks, Tracey J. Hopkins, Stefan Blum, Kerryn A. Green, Zara A. Ioannides, Andrew Swayne, Blake T. Aftab, Kaye D. Hooper, Scott R. Burrows, Kate M. Thompson, Alan Coulthard, Rajiv Khanna
Anemia is a major complication of malaria, driven largely by loss of uninfected RBCs during infection. RBC clearance through loss of complement regulatory proteins (CRPs) is a significant contributor to anemia in Plasmodium falciparum infection, but its role in Plasmodium vivax infection is unknown. CRP loss increases RBC susceptibility to macrophage clearance, a process that is also regulated by CD47. We compared CRPs and CD47 expression on infected and uninfected RBCs in adult patients with vivax and falciparum malaria and different anemia severities from Papua, Indonesia. Complement activation and parasite-specific complement-fixing antibodies were measured by ELISA. Levels of CR1 and CD55 were reduced in severe anemia in both falciparum and vivax malaria. Loss of CRPs and CD47 was restricted to uninfected RBCs, with infected RBCs having higher expression. There was no association among complement-fixing antibodies, complement activation, and CRP loss. Our findings demonstrate that CRP loss is a pan-species, age-independent mechanism of malarial anemia. Higher levels of CRP and CD47 expression on infected RBCs suggest that parasites are protected from complement-mediated destruction and macrophage clearance. Lack of associations between protective antibodies and CRP loss highlight that complement pathogenic and protective pathways are distinct mechanisms during infection.
Damian A. Oyong, Enny Kenangalem, Jeanne R. Poespoprodjo, James G. Beeson, Nicholas M. Anstey, Ric N. Price, Michelle J. Boyle
Mono-ADP-ribosylation of an (arginine) protein catalyzed by ADP-ribosyltransferase 1 (ART1) — i.e., transfer of ADP-ribose from NAD to arginine — is reversed by ADP-ribosylarginine hydrolase 1 (ARH1) cleavage of the ADP-ribose–arginine bond. ARH1-deficient mice developed cardiomyopathy with myocardial fibrosis, decreased myocardial function under dobutamine stress, and increased susceptibility to ischemia/reperfusion injury. The membrane repair protein TRIM72 was identified as a substrate for ART1 and ARH1; ADP-ribosylated TRIM72 levels were greater in ARH1-deficient mice following ischemia/reperfusion injury. To understand better the role of TRIM72 and ADP-ribosylation, we used C2C12 myocytes. ARH1 knockdown in C2C12 myocytes increased ADP-ribosylation of TRIM72 and delayed wound healing in a scratch assay. Mutant TRIM72 (R207K, R260K) that is not ADP-ribosylated interfered with assembly of TRIM72 repair complexes at a site of laser-induced injury. The regulatory enzymes ART1 and ARH1 and their substrate TRIM72 were found in multiple complexes, which were coimmunoprecipitated from mouse heart lysates. In addition, the mono-ADP-ribosylation inhibitors vitamin K1 and novobiocin inhibited oligomerization of TRIM72, the mechanism by which TRIM72 is recruited to the site of injury. We propose that a mono-ADP-ribosylation cycle involving recruitment of TRIM72 and other regulatory factors to sites of membrane damage is critical for membrane repair and wound healing following myocardial injury.
Hiroko Ishiwata-Endo, Jiro Kato, Akihiko Tonouchi, Youn Wook Chung, Junhui Sun, Linda A. Stevens, Jianfeng Zhu, Angel M. Aponte, Danielle A. Springer, Hong San, Kazuyo Takeda, Zu-Xi Yu, Victoria Hoffmann, Elizabeth Murphy, Joel Moss
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