Research
Human Tregs at the maternal-fetal interface show site-specific adaptation reminiscent of tumor Tregs
Abstract
Regulatory T cells (Tregs) are crucial for maintaining maternal immune-tolerance against the semi-allogeneic fetus. We investigated the elusive transcriptional profile and functional adaptation of human uterine Tregs (uTregs) during pregnancy. Uterine biopsies, from placental bed (=maternal-fetal interface) and incision site (=control), and blood were obtained from women with uneventful pregnancies undergoing Caesarean section. Tregs and CD4+ non-Tregs were isolated for transcriptomic profiling by Cel-Seq2. Results were validated on protein and single cell level by flow cytometry. Placental bed uterine Tregs (uTregs) showed elevated expression of Treg signature markers, including FOXP3, CTLA-4 and TIGIT. Their transcriptional profile was indicative of late-stage effector Treg differentiation and chronic activation, with increased expression of immune checkpoints GITR, TNFR2, OX-40, 4-1BB, genes associated with suppressive capacity (HAVCR2, IL10, LAYN, PDCD1), and transcription factors MAF, PRDM1, BATF, and VDR. uTregs mirrored non-Treg Th1 polarization and tissue-residency. The particular transcriptional signature of placental bed uTregs overlapped strongly with that of tumor-infiltrating Tregs, and was remarkably pronounced at the placental bed compared to uterine control site. Concluding, human uTregs acquire a differentiated effector Treg profile similar to tumor-infiltrating Tregs, specifically at the maternal-fetal interface. This introduces the novel concept of site-specific transcriptional adaptation of Tregs within one organ.
Authors
Judith Wienke, Laura Brouwers, Leone M. van der Burg, Michal Mokry, Rianne C. Scholman, Peter G. J. Nikkels, Bas B. van Rijn, Femke van Wijk
Abstract
Classical dynamins are large GTPases regulating membrane and cytoskeleton dynamics and are linked to different pathological conditions ranging from neuromuscular diseases to encephalopathy and cancer. Dominant DNM2 (dynamin 2) mutations lead to either mild adult onset or severe neonatal centronuclear myopathy (ADCNM). Our objectives were to better understand the pathomechanism of severe ADCNM and test a potential therapy. Here, we created the Dnm2SL/+ mouse line harboring the common S619L mutation found in patients with severe ADCNM and impairing the conformational switch regulating dynamin self-assembly and membrane remodeling. The Dnm2SL/+ mouse faithfully reproduces severe ADCNM hallmarks with early impaired muscle function and force together with myofibers hypotrophy. It revealed swollen mitochondria lacking cristae as the main ultrastructural defect and potential cause of the disease. Patient analysis confirmed this structural hallmark. In addition, DNM2 reduction with antisense oligonucleotides after disease onset efficiently reverted locomotor and force defects after only 3 weeks of treatment. Most histological defects including mitochondria alteration were partially or fully rescued. Overall, this study highlights an efficient approach to revert the severe form of dynamin-related centronuclear myopathy. These data also reveal that the dynamin conformational switch is key for muscle function and should be targeted for future therapeutic developments.
Authors
Xènia Massana Muñoz, Christine Kretz, Roberto Silva-Rojas, Julien Ochala, Alexia Menuet, Norma B. Romero, Belinda S. Cowling, Jocelyn Laporte
Abstract
Acute graft-versus-host disease (aGVHD) can occur after hematopoietic cell transplant in patients undergoing treatment for hematological malignancies or inborn errors. Although CD4 T helper (Th) cells play a major role in aGVHD, the mechanisms by which they contribute, particularly within the intestines, have remained elusive. We have identified a novel subset of Th cells that accumulated in the intestines and produced the serine protease granzyme A (GrA). GrA+ Th cells were distinct from other Th lineages and exhibited a non-cytolytic phenotype. In vitro, GrA+ Th cells differentiated in the presence of IL-4, IL-6, and IL-21 and were transcriptionally unique from cells cultured with either IL-4 or the IL-6/IL-21 combination alone. In vivo, both STAT3 and STAT6 were required for GrA+ Th cell differentiation and played roles in maintenance of the lineage identity. Importantly, GrA+ Th cells promoted aGVHD-associated morbidity and mortality and contributed to crypt destruction within intestines but were not required for the beneficial graft-versus-leukemia effect. Our data indicate that GrA+ Th cells represent a distinct Th subset and are critical mediators of aGVHD.
Authors
Sungtae Park, Brad Griesenauer, Hua Jiang, Djamilatou Adom, Pegah Mehrpouya-Bahrami, Srishti Chakravorty, Majid Kazemian, Tanbeena Imam, Rajneesh Srivastava, Tristan A. Hayes, Julian Pardo, Sarath Chandra Janga, Sophie Paczesny, Mark H. Kaplan, Matthew R. Olson
Abstract
Evaluation of potential immunity against the novel severe acute respiratory syndrome (SARS) coronavirus that emerged in 2019 (SARS-CoV-2) is essential for health, as well as social and economic recovery. Generation of antibody response to SARS-CoV-2 (seroconversion) may inform on acquired immunity from prior exposure, and antibodies to the SARS-CoV-2 spike protein receptor binding domain (S-RBD) are speculated to neutralize virus infection. Some serology assays rely solely on SARS-CoV-2 nucleocapsid protein (N-protein) as the antibody detection antigen; however, whether such immune responses correlate with S-RBD response and COVID-19 immunity remains unknown. Here, we generated a quantitative serological enzyme-linked immunosorbent assay (ELISA) using recombinant S-RBD and N-protein for the detection of circulating antibodies in 138 serial serum samples from 30 RT-PCR confirmed SARS-CoV-2 hospitalized patients, as well as 464 healthy and non-COVID-19 serum samples that were collected between June 2017 and June 2020. Quantitative detection of IgG antibodies to the two different viral proteins showed a moderate correlation. Antibodies to N-protein were detected at a rate of 3.6% in healthy and non-COVID-19 sera collected during the pandemic in 2020, whereas 1.6% of these sera were positive for S-RBD. Approximately 86% of individuals positive for S-RBD binding antibodies exhibited neutralizing capacity, but only 74% of N-protein positive individuals exhibited neutralizing capacity. Collectively, our studies show that detection of N-protein binding antibodies does not always correlate with presence of S-RBD neutralizing antibodies, and cautions against the extensive use of N-protein based serology testing for determination of potential COVID-19 immunity.
Authors
Kathleen M. McAndrews, Dara P. Dowlatshahi, Jianli Dai, Lisa M. Becker, Janine Hensel, Laura M. Snowden, Jennifer M. Leveille, Michael R. Brunner, Kylie Holden, Nikolas S. Hopkins, Alexandria Harris, Jerusha J. Kumpati, Michael A. Whitt, J. Jack Lee, Luis Ostrosky-Zeichner, Ramesha Papanna, Valerie LeBleu, James Allison, Raghu Kalluri
Abstract
Angiogenesis is essential for cardiac functional recovery after myocardial infarction (MI). HSPA12B is predominately expressed in endothelial cells and required for angiogenesis. Yes-associated protein (YAP) plays an important role in tumor angiogenesis. This study investigated the cooperative role of HSPA12B and YAP in angiogenesis post-MI. Silencing of either HSPA12B or YAP impairs hypoxia-promoted endothelial cell proliferation and angiogenesis. Deficiency of HSPA12B suppresses YAP expression and nuclear translocation following hypoxia. Knockdown of YAP attenuates hypoxia-stimulated HSPA12B nuclear translocation and abrogates HSPA12B-promoted endothelial cell angiogenesis. Mechanistically, hypoxia induced an interaction between endothelial HSPA12B and YAP. ChIP assay shows that HSPA12B is a target gene of YAP/transcriptional enhanced associated domain4 (TEAD4) and a co-activator in YAP-associated angiogenesis. In vivo studies using the MI model show that endothelial specific deficiency of HSPA12B (eHspa12b-/-) or YAP (eYap-/-) impairs angiogenesis and exacerbates cardiac dysfunction, when compared with wild type (WT) mice. MI increased YAP expression and nuclear translocation in WT hearts, but not in eHspa12b-/- hearts. HSPA12B expression and nuclear translocation were up-regulated in WT MI hearts, but not in eYap-/- MI myocardium. Our data demonstrated that endothelial HSPA12B is a novel target and co-activator for YAP/TEAD4 and cooperates with YAP to regulate endothelial angiogenesis post-MI.
Authors
Min Fan, Kun Yang, Xiaohui Wang, Yana Wang, Fei Tu, Tuanzhu Ha, Li Liu, David L. Williams, Chuanfu Li
Abstract
Age-associated systemic, chronic inflammation is partially attributed to increased self (auto)-reactivity, resulting from disruption of central tolerance in the aged, involuted thymus. This involution causally results from gradually decreased expression of the transcription factor FOXN1 in thymic epithelial cells (TECs), while exogenous FOXN1 in TECs can partially rescue age-related thymic involution. Given the findings that TECs induced from FOXN1-overexpressing embryonic fibroblasts can generate an ectopic de novo thymus under the kidney capsule and intra-thymically injected naturally young TECs can lead to middle-aged thymus regrowth, we attempted to extend these two findings by combining them as a novel thymic rejuvenation strategy with two types of promoter-driven (Rosa26CreERT and FoxN1Cre) Cre-mediated FOXN1-reprogrammed embryonic fibroblasts (FREFs). We engrafted these two-types of FREFs directly into the aged murine thymus. We found significant regrowth of the native aged thymus with rejuvenated architecture and function in both males and females, exhibiting increased thymopoiesis and reinforced thymocyte negative selection, along with reduced senescent T cells and auto-reactive T cell-mediated inflammation in old mice. Therefore, this strategy has preclinical significance and presents a strategy to potentially rescue decreased thymopoiesis and perturbed negative selection to significantly, albeit partially, restore defective central tolerance and reduce subclinical autoimmune symptoms in the elderly.
Authors
Jiyoung Oh, Weikan Wang, Rachel Thomas, Dong-Ming Su
Abstract
Abstract: S-Nitroso-L-cysteine (L-CSNO) behaves as a ligand. Its soluble guanylate cyclase (sGC)-independent effects are stereoselective - that is, not recapitulated by S-nitroso-D-cysteine (D-CSNO) – and are inhibited by chemical cogeners. However, candidate L-CSNO receptors have never been identified. Here, we have used two complementary affinity chromatography assays - followed by unbiased proteomic analysis - to identify voltage-gated K+ channel (Kv) proteins as binding partners for L-CSNO. Stereoselective L-CSNO-Kv interaction was confirmed structurally and functionally using surface plasmon resonance spectroscopy, hydrogen deuterium exchange and, in Kv1.1/Kv1.2/Kvβ2 overexpressing cells, patch clamp assays. Remarkably, these sGC-independent L-CSNO effects did not involve S-nitrosylation of Kv proteins. In isolated rat and mouse respiratory control (petrosyl) ganglia, L-CSNO stereoselectively inhibited Kv channel function. Genetic ablation of Kv 1.1 prevented this effect. In intact animals, L-CSNO injection at level of the carotid body (CB) dramatically and stereoselectively increased minute ventilation while having no effect on blood pressure; this effect was inhibited by the L-CSNO cogener S-methyl-L-cysteine. Kv proteins are physiologically relevant targets of endogenous L-CSNO. This may be a signaling pathway of broad relevance.
Authors
Benjamin Gaston, Laura A. Smith, Jürgen Bosch, James M. Seckler, Diana L. Kunze, Janna Kiselar, Nadzeya Marozkina, Craig Hodges, Patrick Wintrode, Kellen McGee, Tatiana Morozkina, Spencer T. Burton, Tristan Lewis, Timothy Strassmaier, Paulina Getsy, James N. Bates, Stephen J. Lewis
Abstract
New strategies are needed to enhance the efficacy of anti-programmed cell death protein (PD-1) antibody (Ab) in cancer. Here, we report that inhibiting palmitoyl-protein thioesterase 1 (PPT1), a target of CQ derivatives like hydroxychloroquine (HCQ), enhances the antitumor efficacy of anti-PD-1 Ab in melanoma. The combination resulted tumor growth impairment and improved survival in mouse models. Genetic suppression of core autophagy genes, but not Ppt1, in cancer cells reduced priming and cytotoxic capacity of primed T cells. Exposure of antigen primed T cells to macrophage conditioned medium derived from macrophages treated with PPT1 inhibitors enhanced melanoma specific killing. Genetic or chemical PPT1 inhibition resulted an M2 to M1 phenotype switching in macrophages. The combination was associated with a reduction in myeloid-derived suppressor cells (MDSCs) in the tumor. Ppt1 inhibition by HCQ, or DC661, induced cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), tank-binding kinase 1 (TBK1) pathway activation and the secretion of interferon β (IFN-β) in macrophages which was a key component for augmented T cell-mediated cytotoxicity. Genetic Ppt1 inhibition produced similar findings. These data provide the rationale for a melanoma clinical trial testing this new immunotherapy combination and may also be effective in other cancers.
Authors
Gaurav Sharma, Rani Ojha, Estela Noguera-Ortega, Vito W. Rebecca, John Attanasio, Shujing Liu, Shengfu Piao, Jennifer J. Lee, Michael C. Nicastri, Sandra L. Harper, Amruta Ronghe, Vaibhav Jain, Jeffrey D. Winkler, David W. Speicher, Jerome Mastio, Phyllis A Gimotty, Xiaowei Xu, E. John Wherry, Dmitry I. Gabrilovich, Ravi K. Amaravadi
Abstract
Tumor-associated macrophages (TAMs) affect cancer progression and therapy. Ovarian carcinoma often metastasizes to the peritoneal cavity. Here, we found two peritoneal macrophage subsets in mice bearing ID8 ovarian cancer based on the Tim-4 (T-cell immunoglobulin and mucin domain containing 4) expression. Tim-4+ TAMs were embryonically originated and locally sustained while Tim-4- TAMs were replenished from circulating monocytes. Tim-4+ TAMs, but not Tim-4- TAMs, promoted tumor growth in vivo. Relative to Tim-4- TAMs, Tim-4+ TAMs manifested high oxidative phosphorylation and adapted mitophagy to alleviate oxidative stress. High levels of arginase-1 in Tim-4+ TAMs contributed to potent mitophagy activities via weakened mTORC1 activation due to low arginine resultant from arginase-1-mediated metabolism. Furthermore, genetic deficiency of autophagy element FIP200 resulted in Tim-4+ TAM loss via ROS-mediated apoptosis, and elevated T cell-immunity and ID8 tumor inhibition in vivo. Moreover, human ovarian cancer-associated CRIg (complement receptor of the Immunoglobulin superfamily) positive macrophages were transcriptionally, metabolically, and functionally similar to murine Tim-4+ TAMs. Thus, targeting CRIg+ (Tim-4+) TAMs may potentially treat ovarian cancer patients with peritoneal metastasis.
Authors
Houjun Xia, Shasha Li, Xiong Li, Weichao Wang, Yingjie Bian, Shuang Wei, Sara Grove, Weimin Wang, Linda Vatan, J. Rebecca Liu, Karen McLean, Ramandeep Rattan, Adnan R. Munkarah, Jun-Lin Guan, Ilona Kryczek, Weiping Zou
Abstract
Electroconvulsive therapy is highly effective in neuropsychiatric disorders by unknown mechanisms. Microglial toxicity plays key role in neuroinflammatory and degenerative diseases, where there is critical shortage in therapies. This study examined the effects of electroconvulsive seizures (ECS) on chronic neuroinflammation and microglial neurotoxicity.Electric brain stimulation inducing full tonic-clonic seizures during chronic relapsing-progressive experimental autoimmune encephalomyelitis (EAE) reduced spinal immune cell infiltration, reduced myelin and axonal loss, and prevented clinical deterioration. Using the transfer EAE model we examined the effect of ECS on systemic immune response in donor mice versus ECS effect on CNS innate immune activity in recipient mice. ECS did not affect encephalitogenicity of systemic T cells, but targeted the CNS directly to inhibit T-cell induced neuroinflammation. In vivo and ex-vivo assays indicated that ECS suppressed microglial neurotoxicity, by reducing iNOS expression, nitric oxide and reactive oxygen species (ROS) production, and by reducing CNS oxidative stress. Microglia from ECS treated EAE mice expressed less T cell stimulatory and chemoattractant factors. Our finding indicate that Electroconvulsive therapy targets the CNS innate immune system to reduce neuroinflammation by attenuating microglial neurotoxicity. These findings signify a novel therapeutic approach for chronic neuroinflammatory, neuropsychiatric and neurodegenerative diseases.
Authors
Smadar Goldfarb, Nina Fainstein, Tamir Ben-Hur
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