Research
Abstract
Neoepitopes are the only truly tumor-specific antigens. Although potential neoepitopes can be readily identified using genomics, the neoepitopes that mediate tumor rejection constitute a small minority, and there is little consensus on how to identify them. Here, for the first time, we use a combination of genomics, unbiased discovery MS immunopeptidomics and targeted MS to directly identify neoepitopes that elicit actual tumor rejection in mice. We report that MS-identified neoepitopes are an astonishingly rich source of tumor rejection mediating neoepitopes. MS has also demonstrated unambiguously the presentation by MHC I, of confirmed tumor rejection neoepitopes which bind weakly to MHC I; this was done using DCs exogenously loaded with long peptides containing the weakly binding neoepitopes. Such weakly MHC I-binding neoepitopes are routinely excluded from analysis, and our demonstration of their presentation, and their activity in tumor rejection, reveals a broader universe of tumor-rejection neoepitopes than presently imagined. Modeling studies show that a mutation in the active neoepitope alters its conformation such that its T cell receptor-facing surface is significantly altered, increasing its exposed hydrophobicity. No such changes are observed in the inactive neoepitope. These results broaden our understanding of antigen presentation and help prioritize neoepitopes for personalized cancer immunotherapy.
Authors
Hakimeh Ebrahimi-Nik, Justine Michaux, William L. Corwin, Grant L.J. Keller, Tatiana Shcheglova, HuiSong Pak, George Coukos, Brian M. Baker, Ion I. Mandoiu, Michal Bassani-Sternberg, Pramod K. Srivastava
Abstract
Adeno-associated-viral (AAV) vector liver-directed gene therapy (GT) for hemophilia B (HB) is limited by a vector-dose-dependent hepatotoxicity. Recently, this obstacle has been partially circumvented by the use of a hyperactive factor IX (FIX) variant, R338L (Padua), which has an eightfold increased specific activity compared to FIX-WT. FIX-R338L has emerged as the standard for HB GT. However, the underlying mechanism of its hyperactivity is undefined; as such, safety concerns of unregulated coagulation and the potential for thrombotic complications have not been fully addressed. To this end, we evaluated the enzymatic and clotting activity as well as the activation, inactivation, and cofactor-dependence of FIX-R338L relative to FIX-WT. We observed that the high-specific-activity of FIX-R338L requires factor VIIIa (FVIIIa) cofactor. In a novel system utilizing emicizumab, a FVIII-mimicking bispecific antibody, the hyperactivity of both recombinant FIX-R338L and AAV-mediated-transgene-expressed FIX-R338L from HB GT subjects is ablated without FVIIIa activity. We conclude that the molecular regulation of activation, inactivation, and cofactor-dependence of FIX-R338L is similar to FIX-WT, but that the FVIIIa-dependent hyperactivity of FIX-R338L is the result of a faster rate of factor X activation. This mechanism helps mitigate safety concerns of unregulated coagulation and supports the expanded use of FIX-R338L in HB therapy.
Authors
Benjamin J. Samelson-Jones, Jonathan D. Finn, Lindsey A. George, Rodney M. Camire, Valder R. Arruda
Abstract
Heterozygous missense mutations in lysyl oxidase (LOX) are associated with thoracic aortic aneurysms and dissections. To assess how LOX mutations modify protein function and lead to aortic disease, we studied the factors that influence the onset and progression of vascular aneurysms in mice bearing a Lox mutation (p.M292R) linked to aortic dilation in humans. We show that mice heterozygous for the M292R mutation did not develop aneurysmal disease unless challenged with increased hemodynamic stress. Vessel dilation was confined to the ascending aorta although both the ascending and descending aortae showed changes in vessel wall structure, smooth muscle cell number and inflammatory cell recruitment that differed between wild-type and mutant animals. Studies with isolated cells found that M292R-mutant Lox is retained in the endoplasmic reticulum and ultimately cleared through an autophagy/proteasome pathway. Because the mutant protein does not transit to the Golgi where copper incorporation occurs, the protein is never catalytically active. These studies show that the M292R mutation results in LOX loss-of-function due to a secretion defect that predisposes the ascending aorta in mice (and by extension humans with similar mutations) to arterial dilation when exposed to risk factors that impart stress to the arterial wall.
Authors
Vivian S. Lee, Carmen M. Halabi, Thomas J. Broekelmann, Philip C. Trackman, Nathan O. Stitziel, Robert P. Mecham
Abstract
The lung is a relatively quiescent organ during homeostasis, but has a remarkable capacity for repair after injury. Alveolar epithelial type I cells (AEC1s) line airspaces and mediate gas exchange. After injury, they are regenerated by differentiation from their progenitors — alveolar epithelial type II cells (AEC2s) — which also secrete surfactant to maintain surface tension and alveolar patency. While recent studies showed that the maintenance of AEC2 stemness is Wnt dependent, the molecular mechanisms underlying AEC2-AEC1 differentiation in adult lung repair are still incompletely understood. Here we show that WWTR1 (TAZ) plays a crucial role in AEC differentiation. Using an in vitro organoid culture system, we found that tankyrase inhibition can efficiently block AEC2-AEC1 differentiation, and this effect was due to the inhibition of TAZ. In a bleomycin induced lung injury model, conditional deletion of TAZ in AEC2s dramatically reduced AEC1 regeneration during recovery, leading to exacerbated alveolar lesions and fibrosis. In patients with idiopathic pulmonary fibrosis (IPF), decreased blood levels of RAGE, a biomarker of AEC1 health, were associated with more rapid disease progression. Our findings implicate TAZ as a critical factor involved in AEC2 to AEC1 differentiation, and hence the maintenance of alveolar integrity after injury.
Authors
Tianhe Sun, Zhiyu Huang, Hua Zhang, Clara Posner, Guiquan Jia, Thirumalai R. Ramalingam, Min Xu, Hans D. Brightbill, Jackson G. Egen, Anwesha Dey, Joseph R. Arron
Abstract
Non-integrative AAV-mediated gene therapy in the liver is effective in adult patients, but faces limitations in pediatric settings due to episomal DNA loss during hepatocyte proliferation. Gene targeting is a promising approach by permanently modifying the genome. We previously rescued neonatal lethality in Crigler-Najjar mice by inserting a promoterless human uridine glucuronosyl transferase A1 (UGT1A1) cDNA in exon 14 of the albumin gene, without the use of nucleases. To increase recombination rate and therapeutic efficacy, here we used CRISPR/SaCas9. Neonatal mice were transduced with two AAVs: one expressing the SaCas9 and sgRNA, and one containing a promoterless cDNA flanked by albumin homology regions. Targeting efficiency increased ~26-fold with an eGFP reporter cDNA, reaching up to 24% of eGFP-positive hepatocytes. Next, we fully corrected the diseased phenotype of Crigler-Najjar mice by targeting the hUGT1A1 cDNA. Treated mice had normal plasma bilirubin up to 10 months after administration, hUGT1A1 protein levels were ~6-fold higher than in WT liver, with a 90-fold increase in recombination rate. Liver histology, inflammatory markers, and plasma albumin were normal in treated mice, with no off-targets in predicted sites. Thus, the improved efficacy and reassuring safety profile support the potential application of the proposed approach to other liver diseases.
Authors
Alessia De Caneva, Fabiola Porro, Giulia Bortolussi, Riccardo Sola, Michela Lisjak, Adi Barzel, Mauro Giacca, Mark A. Kay, Kristian Vlahoviček, Lorena Zentilin, Andrés F. Muro
Abstract
Genetic susceptibility to chronic pancreatitis in humans is frequently associated with mutations that increase activation of the digestive protease trypsin. Intrapancreatic trypsin activation is an early event in experimental acute pancreatitis in rodents, suggesting that trypsin is a key driver of pathology. In contrast to trypsin, the pancreatic protease chymotrypsin serves a protective function by mitigating trypsin activation through degradation. In humans, loss-of-function mutations in chymotrypsin C (CTRC) are common risk factors for chronic pancreatitis; however, the pathogenic effect of CTRC deficiency has not been corroborated in animal models yet. Here we report that C57BL/6 mice that are widely used for genetic manipulations do not express functional CTRC due to a single-nucleotide deletion in exon 2 of the Ctrc gene. We restored a functional Ctrc locus in C57BL/6N mice and demonstrated that in the novel Ctrc+ strain the severity of cerulein-induced experimental acute and chronic pancreatitis was significantly ameliorated. Improved disease parameters were associated with reduced intrapancreatic trypsin activation suggesting a causal link between CTRC-mediated trypsinogen degradation and protection against pancreatitis. Taken together with prior human genetic and biochemical studies, the observations provide conclusive evidence for the protective role of CTRC against pancreatitis.
Authors
Andrea Geisz, Zsanett Jancsó, Balázs Csaba Németh, Eszter Hegyi, Miklós Sahin-Tóth
Abstract
NK cell exhaustion (NCE) due to sustained proliferation results in impaired NK cell function with loss of cytokine production and lytic activity. Using murine models of chronic NK cell stimulation, we have identified a phenotypic signature of NCE characterized by up-regulation of the terminal differentiation marker KLRG1 and by down-regulation of eomesodermin and the activating receptor NKG2D. Chronic stimulation of mice lacking NKG2D resulted in minimized NCE compared to control mice, thus identifying NKG2D as a crucial mediator of NCE. NKG2D internalization and downregulations on NK cells has been previously observed in the presence of tumor cells with high expression of NKG2D ligands (NKG2DL) due to the activation of the DNA damage repair pathways. Interestingly, our study revealed that during NK cell activation there is an increase of MULT1, and NKG2DL, that correlates with an induction of DNA damage. Treatment with the ATM DNA damage repair pathway inhibitor KU55933 (KU) during activation reduced NCE by improving expression of activation markers and genes involved in cell survival, by sustaining NKG2D expression and by preserving cell functionality. Importantly, NK cells expanded ex vivo in the presence of KU displayed increased anti-tumor efficacy in both NKG2D-dependent and -independent mouse models. Collectively, these data demonstrate that NCE is caused by DNA damage and regulated, at least in part, by NKG2D. Further, the prevention of NCE is a promising strategy to improve NK cell-based immunotherapy.
Authors
Maite Alvarez, Federico Simonetta, Jeanette Baker, Antonio Pierini, Arielle S. Wenokur, Alyssa R. Morrison, William J. Murphy, Robert S. Negrin
Abstract
Arrhythmogenic cardiomyopathy (ACM) is an inherited disorder with variable genetic etiologies. Here we focused on understanding the precise molecular pathology of a single clinical variant in DSP, the gene encoding desmoplakin. We initially identified a novel missense desmoplakin variant (p.R451G) in a patient diagnosed with biventricular ACM. An extensive single-family ACM cohort was assembled, revealing a pattern of coinheritance for R451G desmoplakin and the ACM phenotype. An in vitro model system using patient-derived induced pluripotent stem cell lines showed depressed levels of desmoplakin in the absence of abnormal electrical propagation. Molecular dynamics simulations of desmoplakin R451G revealed no overt structural changes, but a significant loss of intramolecular interactions surrounding a putative calpain target site was observed. Protein degradation assays of recombinant desmoplakin R451G confirmed increased calpain vulnerability. In silico screening identified a subset of 3 additional ACM-linked desmoplakin missense mutations with apparent enhanced calpain susceptibility, predictions that were confirmed experimentally. Like R451G, these mutations are found in families with biventricular ACM. We conclude that augmented calpain-mediated degradation of desmoplakin represents a shared pathological mechanism for select ACM-linked missense variants. This approach for identifying variants with shared molecular pathologies may represent a powerful new strategy for understanding and treating inherited cardiomyopathies.
Authors
Ronald Ng, Heather R Manring, Nikolaos Papoutsidakis, Taylor Albertelli, Nicole Tsai, Claudia See, Xia Li, Jinkyu Park, Tyler L. Stevens, Prameela J. Bobbili, Muhammad Riaz, Yongming Ren, Christopher E. Stoddard, Paul M.L. Janssen, T. Jared Bunch, Stephen P. Hall, Ying-Chun Lo, Daniel L. Jacoby, Yibing Qyang, Nathan Wright, Maegen A. Ackermann, Stuart G. Campbell
Abstract
Immunotherapy holds promise for multiple myeloma (MM) patients but little is known about how MM-induced immunosuppression influences response to therapy. Here, we investigated the impact of disease progression on immunotherapy efficacy in the Vk*MYC mouse model. Treatment with agonistic anti-CD137 (4-1BB) mAbs efficiently protected mice when administered early but failed to contain MM growth when delayed more than three weeks after Vk*MYC tumor cell challenge. The quality of CD8+ T cell response to CD137 stimulation was not altered by the presence of MM, but CD8+ T cell numbers were profoundly reduced at the time of treatment. Our data suggest that an insufficient ratio of CD8+ T cells over MM cells (CD8/MM) accounts for the loss of anti-CD137 mAb efficacy. We established serum M-protein levels prior to therapy as a predictive factor of response. Moreover, we developed an in silico model to capture the dynamic interactions between CD8+ T cells and MM cells. Finally, we explored two methods to improve the CD8/MM ratio: anti-CD137 mAb immunotherapy combined with Treg-depletion or administered after chemotherapy treatment with cyclophosphamide or melphalan efficiently reduced MM burden and prolonged survival. Altogether, our data indicate that consolidation treatment with anti-CD137 mAbs might prevent MM relapse.
Authors
Camille Guillerey, Kyohei Nakamura, Andrea C. Pichler, Deborah Barkauskas, Sophie Krumeich, Kimberley Stannard, Kim Miles, Heidi Harjunpää, Yuan Yu, Mika Casey, Alina I. Doban, Mircea Lazar, Gunter Hartel, David Smith, Slavica Vuckovic, Michele W.L. Teng, P. Leif Bergsagel, Marta Chesi, Geoffrey R. Hill, Ludovic Martinet, Mark J. Smyth
Abstract
Cellular senescence is a tumor suppressive mechanism that can paradoxically contribute to aging pathologies. Despite evidence of immune clearance in mouse models, it is not known how senescent cells (SnCs) persist and accumulate with age or in tumors in individuals. Here, we identify cooperative mechanisms that orchestrate the immunoevasion and persistence of normal and cancer human SnCs through extracellular targeting of natural killer receptor signaling. Damaged SnCs avoid immune recognition through MMPs-dependent shedding of NKG2D-ligands reinforced via paracrine suppression of NKG2D receptor-mediated immunosurveillance. These coordinated immunoediting processes are evident in residual, drug-resistant tumors from cohorts of >700 prostate and breast cancer patients treated with senescence-inducing genotoxic chemotherapies. Unlike in mice, these reversible senescence-subversion mechanisms are independent of p53/p16 and exacerbated in oncogenic RAS-induced senescence. Critically, the p16INK4A tumor suppressor can disengage the senescence growth arrest from the damage-associated immune senescence program, which is manifest in benign nevi lesions where indolent SnCs accumulate over time and preserve a non-pro-inflammatory tissue microenvironment maintaining NKG2D-mediated immunosurveillance. Our study shows how subpopulations of SnCs elude immunosurveillance, and reveals secretome-targeted therapeutic strategies to selectively eliminate –and restore the clearance of– the detrimental SnCs that actively persist after chemotherapy and accumulate at sites of aging pathologies.
Authors
Denise P. Muñoz, Steve M. Yannone, Anneleen Daemen, Yu Sun, Funda Vakar-Lopez, Misako Kawahara, Adam M. Freund, Francis Rodier, Jennifer D. Wu, Pierre-Yves Desprez, David H. Raulet, Peter S. Nelson, Laura J. van 't Veer, Judith Campisi, Jean-Philippe Coppé
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