Abstract
To unequivocally address their unresolved intimate structures in blood, we scrutinized the size distribution of circulating cell-free DNA (cfDNA) using whole genome sequencing (WGS) from both double- and single-strand DNA library preparations (DSP and SSP), as well as using Q-PCR. The size profile in healthy individuals was remarkably homogenous when using either DSP sequencing (DSP-S) or SSP sequencing (SSP-S). Our findings also confirmed that cfDNA size profile shows a characteristic nucleosome fragmentation pattern. Overall, our data indicate that the proportion of cfDNA inserted in mono-nucleosomes, di-nucleosomes and chromatin of higher molecular size (>1,000bp) can be estimated as 67.5-80%, 9.4-11.5% and 8.5-21.0%, respectively. Thus, our data on WGS (N=7) and Q-PCR (N=116 taken together suggests that only a minor proportion of cfDNA is bigger than that existing in mono-nucleosome or transcription factor complexes circulating in blood. Although DNA on single chromatosomes or mono-nucleosomes is detectable, our data revealed that cfDNA is highly nicked (97-98%) on those structures, which appear to be subjected to continuous nuclease activity in the bloodstream. Fragments analysis allows the distinction of cfDNA of different origins: first, cfDNA size profile analysis may be useful in cfDNA extract quality control; second, subtle but reliable differences between healthy metastatic colorectal cancer (mCRC) patients and healthy individuals vary with the proportion of malignant cell-derived cfDNA in plasma extracts, pointing to a higher degree of cfDNA fragmentation and nuclease activity in samples with high malignant cell cfDNA content. Size profile analysis, or ‘fragmentomics’, has shown significant potential to improve diagnostics and cancer screening.
Authors
Cynthia Sanchez, Benoit Roch, Thilbault Mazard, Philippe Blache, Zahra Al Amir Dache, Brice Pastor, Ekaterina Pisareva, Rita Tanos, Alain R. Thierry
Abstract
Progress in our understanding of MR1-restricted Mucosa-associated Invariant T (MAIT) cells has raised an interest in harnessing these cells for immunotherapy. The innate-like response characteristics, abundance in the blood, donor-unrestricted nature, and tropism for tissues make MAIT cells suitable candidates for adoptive cell transfer therapies. However, reliable methods and tools to utilize MAIT cells in such approaches are lacking. Here, we established methodology for efficient expansion of human MAIT cells in culture with high purity and yield, preserved functional response toward their natural ligand, and with increased cytotoxic potential. The cultured MAIT cells retained their effector memory characteristics without signs of terminal differentiation, and expressed a more diverse set of chemokine receptors potentially widening their already broad tissue tropism. To investigate the potential of MAIT cells in a context outside their main role in controlling bacterial infection, we engineered cultured MAIT cells with a new TCR specificity to mediate effective antiviral HLA class I-restricted effector function. In summary, we developed robust and effective methodology for the expansion of human MAIT cells with enhanced cytolytic capacity, and for their engineering with a new specificity. These findings form a basis for the development of MAIT cells as a platform for adoptive immunotherapy.
Authors
Tiphaine Parrot, Katie Healy, Caroline Boulouis, Michał J. Sobkowiak, Edwin Leeansyah, Soo Aleman, Antonio Bertoletti, Margaret Sällberg Chen, Johan K. Sandberg
Abstract
MC4R mutations represent the largest monogenic cause of obesity, resulting mainly from receptor misfolding and intracellular retention by the cellular quality control system. The present study aimed at determining whether pharmacological chaperones (PC) that restore folding and plasma membrane trafficking by stabilizing near native protein conformation, may represent valid therapeutic avenues for the treatment of melanocortin type 4 receptor (MC4R) linked obesity.To test the therapeutic PC potential, we engineered humanized MC4R mouse models expressing either the wild type (WT) human MC4R or a prevalent obesity-causing mutant (R165W). Administration of a PC able to rescue cell surface expression and functional activity of R165W-hMC4R in cells, restored the anorexigenic response of the R165W-hMC4R obese mice to melanocortin agonist, providing a proof-of-principle for the therapeutic potential of MC4R-targetting PC in vivo. Interestingly, the expression of the WT-hMC4R in mice revealed lower sensitivity of the human receptor to alpha-melanocyte-stimulating hormone (α-MSH) but not β-MSH or MTII, resulting in a lower penetrance obese phenotype in the WT-hMC4R versus R165W-hMC4R mice. In conclusion, we created two new obesity models, one hypomorph highlighting species differences, and one amorphic that provides a pre-clinical model to test the therapeutic potential of PC to treat MC4R-linked obesity.
Authors
Patricia René, Damien Lanfray, Denis Richard, Michel Bouvier
Abstract
Recent in vivo tracer studies demonstrated that targeted mass spectrometry (MS) on the Q Exactive Orbitrap could determine the metabolism of HDL proteins 100s-fold less abundant than APOA1. In this study, we demonstrate that the Orbitrap Lumos can measure tracer in proteins whose abundances are 1000s-fold less than APOA1, specifically the lipid transfer proteins PLTP, CETP, and LCAT. Relative to the Q Exactive, the Lumos improved tracer detection by reducing tracer enrichment compression, thereby providing consistent enrichment data across multiple HDL sizes from six participants. We determined by compartmental modeling that PLTP is secreted in medium and large HDL (alpha2, 1, and 0), and is transferred from medium to larger sizes during circulation from where it is catabolized. CETP is secreted mainly in alpha1 and alpha2, and remains in these sizes during circulation. LCAT is secreted mainly in medium and small HDL (alpha2, 3, prebeta). Unlike PLTP and CETP, LCAT appearance on HDL is markedly delayed compared to APOA1 and the other transfer proteins, indicating that LCAT may reside for a time outside of systemic circulation before attaching to HDL in plasma. The determination of these lipid transfer proteins’ unique metabolic structures was possible due to advances in MS technologies.
Authors
Sasha A. Singh, Allison B. Andraski, Hideyuki Higashi, Lang Ho Lee, Ashisha Ramsaroop, Frank M. Sacks, Masanori Aikawa
Abstract
Human lung adenocarcinoma (LUAD) in current or former smokers exhibits a high tumor mutational burden (TMB) and distinct mutational signatures. Syngeneic mouse models of clinically relevant smoking-related LUAD are lacking. We established and characterized a tobacco-associated transplantable murine LUAD cell line, designated FVBW-17, from a LUAD induced by the tobacco carcinogen 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) in the FVB/N mouse strain. Whole exome sequencing of FVBW-17 cells identified tobacco-associated KrasG12D and Trp53 mutations and a similar mutation profile to that of classic alkylating agents with a TMB >500. FVBW-17 cells transplanted subcutaneously, via tail vein and orthotopically generated tumors in FVB/N mice that were histologically similar to human LUAD. FVBW-17 tumors expressed PD-L1, were infiltrated with CD8+ T cells, and responsive to anti-PD-L1 therapy. FVBW-17 cells were also engineered to express green fluorescent protein and luciferase to facilitate the detection and quantification of tumor growth. Distant metastases to lung, spleen, liver, and kidney were observed from subcutaneously transplanted tumors. This novel cell line is a robust representation of human smoking-related LUAD biology and provides a much needed pre-clinical model in which to test promising new agents and combinations including immune-based therapies.
Authors
Laura P. Stabile, Vinod Kumar, Autumn Gaither-Davis, Eric H.B. Huang, Frank P. Vendetti, Princey Devadassan, Sanja Dacic, Riyue Bao, Richard A. Steinman, Timothy F. Burns, Christopher J. Bakkenist
Abstract
Adoptive cell therapy involves the infusion of tumor-reactive T cells into patients with cancer to provide antitumor immunity. The ex vivo expansion and differentiation of such T cells are key parameters that affect their therapeutic potential. Human T cells are presently expanded in culture through the use of anti-CD3 and anti-CD28 mAbs immobilized on beads, expressed on cells, or assembled in the context of soluble antibody complexes. Here we report the design of a small, bispecific single-chain variable fragment construct agonizing both CD3 and CD28 pathways. This soluble T cell expansion protein, termed T-CEP, activates, expands, and differentiates human T cells ex vivo at concentrations in the femtomolar range. Importantly, T-CEP promotes the preferential growth of human CD8+ T cells over the course of 12 days in comparison with methods involving immobilized anti-CD3 mAb/soluble anti-CD28 mAb or soluble anti-CD3/CD28 mAb complexes. The differentiation profile of the resulting human T cell population is also singularly affected by T-CEP, favoring the expansion of a preferred CD8+CD27+ T cell phenotype. The activity profile of T-CEP on human T cells ex vivo suggests its use in generating human T cell populations that are more suited for adoptive cell therapy.
Authors
Esther I. Matus, Amanda Sparkes, Jean Gariépy
Abstract
The challenge of discovering a completely new human tumor virus of unknown phylogeny or sequence depends on detecting viral molecules and differentiating them from host molecules in the virus-associated neoplasm. We developed differential peptide subtraction (DPS) using differential mass-spectrometry (dMS) followed by targeted analysis to facilitate this discovery. We validated this approach by analyzing Merkel cell carcinoma (MCC), an aggressive human neoplasm, in which ~80% of cases are caused by the human Merkel cell polyomavirus (MCV). Approximately 20% of MCC have a high mutational burden and are negative for MCV, but are microscopically indistinguishable from virus positive cases. Using 23 (12 MCV positive, 11 MCV negative) formalin-fixed MCC, DPS identified both viral and human biomarkers (MCV Large T antigen, CDKN2AIP, SERPINB5 and TRIM29) that discriminates MCV positive and negative MCC. Statistical analysis of 498,131 dMS features not matching the human proteome by DPS revealed 562 (0.11%) to be up-regulated in virus-infected samples. Remarkably, four (20%) of the top 20 candidate MS spectra originated from MCV T oncoprotein peptides and confirmed by reverse translation degenerate oligonucleotide sequencing. DPS is a robust proteomic approach to identify novel viral sequences in infectious tumors when nucleic acid-based methods are not feasible.
Authors
Tuna Toptan, Pamela S. Cantrell, Xuemei Zeng, Yang Liu, Mai Sun, Nathan A. Yates, Yuan Chang, Patrick S. Moore
Abstract
Infections caused by multi-drug resistant Staphylococcus aureus, especially MRSA, are responsible for high mortality and morbidity worldwide. Resistant lineages were previously confined to hospitals, but are now also causing infections among healthy individuals in the community. It is therefore imperative to explore therapeutic avenues that are less prone to raise drug resistance compared to today’s antibiotics. An opportunity to achieve this ambitious goal could be provided by targeted antimicrobial photodynamic therapy (aPDT), which relies on the combination of a bacteria-specific targeting agent and light-induced generation of reactive oxygen species by an appropriate photosensitizer. Here we conjugated the near-infrared photosensitizer IRDye700DX to a fully human monoclonal antibody, specific for the invariantly expressed staphylococcal antigen IsaA. The resulting immunoconjugate 1D9-700DX was characterized biochemically and in preclinical infection models. As demonstrated in vitro, in vivo, and in a human post-mortem orthopedic implant infection model, targeted aPDT with 1D9-700DX is highly effective. Importantly, combined with the non-toxic aPDT-enhancing agent potassium iodide, 1D9-700DX overcomes the antioxidant properties of human plasma and fully eradicates high titers of MRSA. We show that the developed immunoconjugate 1D9-700DX targets MRSA and kills it upon illumination with red light, without causing collateral damage to human cells.
Authors
Mafalda Bispo, Andrea Anaya-Sanchez, Sabrina Suhani, Elisa J.M. Raineri, Marina López-Álvarez, Marjolein Heuker, Wiktor Szymański, Francisco Romero Pastrana, Girbe Buist, Alexander R. Horswill, Kevin P. Francis, Gooitzen M. van Dam, Marleen van Oosten, Jan Maarten van Dijl
Abstract
Plasma antimalarial antibody can mediate anti-parasite immunity but has not previously been characterized at the molecular level. Here, we develop an innovative strategy to characterize humoral responses by integrating profiles of plasma immunoglobulins (IG) or antibodies with those expressed on B cells as part of BcR. We applied this strategy to define plasma IG and determine variable V gene usage after vaccination with the Plasmodium falciparum zygote antigen Pfs25. First, using proteomic tools coupled with bulk immunosequencing data, we determined human F(ab′)2 peptide sequences from plasma IG of adults who received four doses of Pfs25-EPA/Alhydrogel. Specifically, Pfs25 antigen-specific F(ab′)2 peptides (Pfs25-IG) were aligned to cDNA sequences of IGH complementarity determining region 3 (CDR3) from a dataset generated by total peripheral B cell immunosequencing of the entire vaccinated population. IGHV4 was the most commonly identified IGHV subgroup of Pfs25-IG, a pattern that was corroborated by VH/VL sequencing of Pfs25-specific single B cells from five vaccinees and by matching plasma Pfs25-IG peptides and V-(D)-J sequences of Pfs25-specific single B cells from the same donor. Among 13 recombinant human mAbs generated from IG sequences of Pfs25-specific single B cells, a single IGHV4 mAb displayed strong neutralizing activity, reducing the number of P. falciparum oocysts in infected mosquitoes by more than 80% at 100 μg/mL. Our approach characterizes the human plasma antibody repertoire in response to the Pfs25-EPA/Alhydrogel vaccine and will be useful to study circulating antibodies in response to other vaccines as well as those induced during infections or autoimmune disorders.
Authors
Camila H. Coelho, Steven T. Nadakal, Patricia A. Gonzales Hurtado, Robert Morrison, Jacob D. Galson, Jillian Neal, Yimin Wu, C. Richter King, Virginia Price, Kazutoyo Miura, Sharon Wong-Madden, Justin Y.A. Dortichamou, David L. Narum, Nicholas J. MacDonald, Maryonne Snow-Smith, Marissa Vignali, Justin J. Taylor, Marie-Paule Lefranc, Johannes Trück, Carole A. Long, Issaka Sagara, Michal Fried, Patrick E. Duffy
Abstract
Rapid and specific antibody testing is crucial for improved understanding, control, and treatment of COVID-19 pathogenesis. Herein, we describe and apply a rapid, sensitive, and accurate virus neutralization assay for SARS-CoV-2 antibodies. The new assay is based on an HIV-1 lentiviral vector that contains a secreted intron Gaussia luciferase or secreted Nano-luciferase reporter cassette, pseudotyped with the SARS-CoV-2 spike (S) glycoprotein, and is validated with a plaque reduction assay using an authentic, infectious SARS-CoV-2 strain. The new assay was used to evaluate SARS-CoV-2 antibodies in serum from individuals with a broad range of COVID-19 symptoms, including intensive care unit (ICU) patients, health care workers (HCWs), and convalescent plasma donors. The highest neutralizing antibody titers were observed among ICU patients, followed by general hospitalized patients, HCWs and convalescent plasma donors. Our study highlights a wide phenotypic variation in human antibody responses against SARS-CoV-2, and demonstrates the efficacy of a novel lentivirus pseudotype assay for high-throughput serological surveys of neutralizing antibody titers in large cohorts.
Authors
Cong Zeng, John P. Evans, Rebecca Pearson, Panke Qu, Yi-Min Zheng, Richard T. Robinson, Luanne Hall-Stoodley, Jacob S. Yount, Sonal Pannu, Rama K. Mallampalli, Linda Saif, Eugene Oltz, Gerard Lozanski, Shan-Lu Liu
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