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1.
Cell ; 186(15): 3148-3165.e20, 2023 07 20.
Artículo en Inglés | MEDLINE | ID: mdl-37413990

RESUMEN

Chimeric antigen receptor (CAR) T cell therapy effectively treats human cancer, but the loss of the antigen recognized by the CAR poses a major obstacle. We found that in vivo vaccine boosting of CAR T cells triggers the engagement of the endogenous immune system to circumvent antigen-negative tumor escape. Vaccine-boosted CAR T promoted dendritic cell (DC) recruitment to tumors, increased tumor antigen uptake by DCs, and elicited the priming of endogenous anti-tumor T cells. This process was accompanied by shifts in CAR T metabolism toward oxidative phosphorylation (OXPHOS) and was critically dependent on CAR-T-derived IFN-γ. Antigen spreading (AS) induced by vaccine-boosted CAR T enabled a proportion of complete responses even when the initial tumor was 50% CAR antigen negative, and heterogeneous tumor control was further enhanced by the genetic amplification of CAR T IFN-γ expression. Thus, CAR-T-cell-derived IFN-γ plays a critical role in promoting AS, and vaccine boosting provides a clinically translatable strategy to drive such responses against solid tumors.


Asunto(s)
Vacunas contra el Cáncer , Neoplasias , Receptores Quiméricos de Antígenos , Humanos , Neoplasias/terapia , Linfocitos T , Inmunoterapia Adoptiva , Receptores de Antígenos de Linfocitos T/metabolismo
2.
Cell ; 174(2): 433-447.e19, 2018 07 12.
Artículo en Inglés | MEDLINE | ID: mdl-29909985

RESUMEN

Nearly all prostate cancer deaths are from metastatic castration-resistant prostate cancer (mCRPC), but there have been few whole-genome sequencing (WGS) studies of this disease state. We performed linked-read WGS on 23 mCRPC biopsy specimens and analyzed cell-free DNA sequencing data from 86 patients with mCRPC. In addition to frequent rearrangements affecting known prostate cancer genes, we observed complex rearrangements of the AR locus in most cases. Unexpectedly, these rearrangements include highly recurrent tandem duplications involving an upstream enhancer of AR in 70%-87% of cases compared with <2% of primary prostate cancers. A subset of cases displayed AR or MYC enhancer duplication in the context of a genome-wide tandem duplicator phenotype associated with CDK12 inactivation. Our findings highlight the complex genomic structure of mCRPC, nominate alterations that may inform prostate cancer treatment, and suggest that additional recurrent events in the non-coding mCRPC genome remain to be discovered.


Asunto(s)
Neoplasias de la Próstata Resistentes a la Castración/patología , Receptores Androgénicos/genética , Secuenciación Completa del Genoma , Anciano , Anilidas/uso terapéutico , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Elementos de Facilitación Genéticos/genética , Duplicación de Gen , Reordenamiento Génico , Genes myc , Sitios Genéticos , Haplotipos , Humanos , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Fosfohidrolasa PTEN/genética , Fenotipo , Antígeno Prostático Específico/sangre , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridinas/uso terapéutico
3.
Immunity ; 56(2): 386-405.e10, 2023 02 14.
Artículo en Inglés | MEDLINE | ID: mdl-36736322

RESUMEN

Local environmental factors influence CD8+ T cell priming in lymph nodes (LNs). Here, we sought to understand how factors unique to the tumor-draining mediastinal LN (mLN) impact CD8+ T cell responses toward lung cancer. Type 1 conventional dendritic cells (DC1s) showed a mLN-specific failure to induce robust cytotoxic T cells responses. Using regulatory T (Treg) cell depletion strategies, we found that Treg cells suppressed DC1s in a spatially coordinated manner within tissue-specific microniches within the mLN. Treg cell suppression required MHC II-dependent contact between DC1s and Treg cells. Elevated levels of IFN-γ drove differentiation Treg cells into Th1-like effector Treg cells in the mLN. In patients with cancer, Treg cell Th1 polarization, but not CD8+/Treg cell ratios, correlated with poor responses to checkpoint blockade immunotherapy. Thus, IFN-γ in the mLN skews Treg cells to be Th1-like effector Treg cells, driving their close interaction with DC1s and subsequent suppression of cytotoxic T cell responses.


Asunto(s)
Neoplasias Pulmonares , Linfocitos T Reguladores , Humanos , Linfocitos T CD8-positivos , Interferón gamma , Linfocitos T Citotóxicos
4.
Nat Immunol ; 20(12): 1692-1699, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31745340

RESUMEN

High-throughput 3' single-cell RNA-sequencing (scRNA-seq) allows cost-effective, detailed characterization of individual immune cells from tissues. Current techniques, however, are limited in their ability to elucidate essential immune cell features, including variable sequences of T cell antigen receptors (TCRs) that confer antigen specificity. Here, we present a strategy that enables simultaneous analysis of TCR sequences and corresponding full transcriptomes from 3'-barcoded scRNA-seq samples. This approach is compatible with common 3' scRNA-seq methods, and adaptable to processed samples post hoc. We applied the technique to identify transcriptional signatures associated with T cells sharing common TCRs from immunized mice and from patients with food allergy. We observed preferential phenotypes among subsets of expanded clonotypes, including type 2 helper CD4+ T cell (TH2) states associated with food allergy. These results demonstrate the utility of our method when studying diseases in which clonotype-driven responses are critical to understanding the underlying biology.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Hipersensibilidad al Cacahuete/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Células Th2/inmunología , Albuminas 2S de Plantas/inmunología , Animales , Antígenos de Plantas/inmunología , Células Cultivadas , Regiones Determinantes de Complementariedad/genética , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunización , Inmunoglobulina E/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas E7 de Papillomavirus/inmunología , Análisis de la Célula Individual , Especificidad del Receptor de Antígeno de Linfocitos T/genética , Transcriptoma
5.
Immunity ; 55(5): 827-846.e10, 2022 05 10.
Artículo en Inglés | MEDLINE | ID: mdl-35483355

RESUMEN

Mycobacterium tuberculosis lung infection results in a complex multicellular structure: the granuloma. In some granulomas, immune activity promotes bacterial clearance, but in others, bacteria persist and grow. We identified correlates of bacterial control in cynomolgus macaque lung granulomas by co-registering longitudinal positron emission tomography and computed tomography imaging, single-cell RNA sequencing, and measures of bacterial clearance. Bacterial persistence occurred in granulomas enriched for mast, endothelial, fibroblast, and plasma cells, signaling amongst themselves via type 2 immunity and wound-healing pathways. Granulomas that drove bacterial control were characterized by cellular ecosystems enriched for type 1-type 17, stem-like, and cytotoxic T cells engaged in pro-inflammatory signaling networks involving diverse cell populations. Granulomas that arose later in infection displayed functional characteristics of restrictive granulomas and were more capable of killing Mtb. Our results define the complex multicellular ecosystems underlying (lack of) granuloma resolution and highlight host immune targets that can be leveraged to develop new vaccine and therapeutic strategies for TB.


Asunto(s)
Mycobacterium tuberculosis , Fibrosis Pulmonar , Tuberculosis , Animales , Ecosistema , Granuloma , Pulmón , Macaca fascicularis , Fibrosis Pulmonar/patología
6.
Immunity ; 53(4): 878-894.e7, 2020 10 13.
Artículo en Inglés | MEDLINE | ID: mdl-33053333

RESUMEN

High-throughput single-cell RNA-sequencing (scRNA-seq) methodologies enable characterization of complex biological samples by increasing the number of cells that can be profiled contemporaneously. Nevertheless, these approaches recover less information per cell than low-throughput strategies. To accurately report the expression of key phenotypic features of cells, scRNA-seq platforms are needed that are both high fidelity and high throughput. To address this need, we created Seq-Well S3 ("Second-Strand Synthesis"), a massively parallel scRNA-seq protocol that uses a randomly primed second-strand synthesis to recover complementary DNA (cDNA) molecules that were successfully reverse transcribed but to which a second oligonucleotide handle, necessary for subsequent whole transcriptome amplification, was not appended due to inefficient template switching. Seq-Well S3 increased the efficiency of transcript capture and gene detection compared with that of previous iterations by up to 10- and 5-fold, respectively. We used Seq-Well S3 to chart the transcriptional landscape of five human inflammatory skin diseases, thus providing a resource for the further study of human skin inflammation.


Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Inflamación/genética , ARN Citoplasmático Pequeño/genética , Piel/patología , Animales , Línea Celular , ADN Complementario/genética , Células HEK293 , Humanos , Ratones , Células 3T3 NIH , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Transcripción Genética/genética , Transcriptoma/genética
7.
Nat Immunol ; 15(2): 128-35, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24448570

RESUMEN

The complex heterogeneity of cells, and their interconnectedness with each other, are major challenges to identifying clinically relevant measurements that reflect the state and capability of the immune system. Highly multiplexed, single-cell technologies may be critical for identifying correlates of disease or immunological interventions as well as for elucidating the underlying mechanisms of immunity. Here we review limitations of bulk measurements and explore advances in single-cell technologies that overcome these problems by expanding the depth and breadth of functional and phenotypic analysis in space and time. The geometric increases in complexity of data make formidable hurdles for exploring, analyzing and presenting results. We summarize recent approaches to making such computations tractable and discuss challenges for integrating heterogeneous data obtained using these single-cell technologies.


Asunto(s)
Sistema Inmunológico/metabolismo , Técnicas Inmunológicas , Monitorización Inmunológica/métodos , Análisis de la Célula Individual/métodos , Animales , Biología Computacional , Humanos , Sistema Inmunológico/patología , Estadística como Asunto
8.
Microb Cell Fact ; 23(1): 217, 2024 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-39085844

RESUMEN

BACKGROUND: The yeast Komagataella phaffii is widely used for manufacturing recombinant proteins, but secreted titers of recombinant proteins could be improved by genetic engineering. In this study, we hypothesized that cellular resources could be redirected from production of endogenous proteins to production of recombinant proteins by deleting unneeded endogenous proteins. In non-model microorganisms such as K. phaffii, however, genetic engineering is limited by lack gene annotation and knowledge of gene essentiality. RESULTS: We identified a set of endogenous secreted proteins in K. phaffii by mass spectrometry and signal peptide prediction. Our efforts to disrupt these genes were hindered by limited annotation of essential genes. To predict essential genes, therefore, we designed, transformed, and sequenced a pooled library of guide RNAs for CRISPR-Cas9-mediated knockout of all endogenous secreted proteins. We then used predicted gene essentiality to guide iterative disruptions of up to 11 non-essential genes. Engineered strains exhibited a ~20× increase in the production of human serum albumin and a twofold increase in the production of a monoclonal antibody. CONCLUSIONS: We demonstrated that disruption of as few as six genes can increase production of recombinant proteins. Further reduction of the endogenous proteome of K. phaffii may further improve strain performance. The pooled library of secretome-targeted guides for CRISPR-Cas9 and knowledge of gene essentiality reported here will facilitate future efforts to engineer K. phaffii for production of other recombinant proteins and enzymes.


Asunto(s)
Sistemas CRISPR-Cas , Proteínas Recombinantes , Saccharomycetales , Saccharomycetales/genética , Saccharomycetales/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Humanos , Técnicas de Inactivación de Genes/métodos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteoma/metabolismo , Anticuerpos Monoclonales/biosíntesis , Albúmina Sérica Humana/genética , Albúmina Sérica Humana/metabolismo
9.
Child Dev ; 2024 Oct 02.
Artículo en Inglés | MEDLINE | ID: mdl-39359116

RESUMEN

This study examined long-term mediating effects of the smalltalk parenting intervention on children's effortful control at school age (7.5 years; 2016-2018). In 2010-2012, parents (96% female) of toddlers (N = 1201; aged 12-36 months; 52% female) were randomly assigned to either: standard playgroup, smalltalk playgroup (group-only), or smalltalk playgroup with additional home coaching (smalltalk plus). Multi-informant data indicated that smalltalk plus had unique indirect effects on children's effortful control, through parents' capacity to 'maintain and extend' children's focus during joint interactions. Possible mediating pathways via parent verbal responsivity, home learning activities, and descriptive language use were not supported. When parents received a structured playgroup program with additional home coaching, sustainable benefits were evident in children's self-regulation, assessed in the early school years.

10.
Proc Natl Acad Sci U S A ; 118(38)2021 09 21.
Artículo en Inglés | MEDLINE | ID: mdl-34493582

RESUMEN

Global containment of COVID-19 still requires accessible and affordable vaccines for low- and middle-income countries (LMICs). Recently approved vaccines provide needed interventions, albeit at prices that may limit their global access. Subunit vaccines based on recombinant proteins are suited for large-volume microbial manufacturing to yield billions of doses annually, minimizing their manufacturing cost. These types of vaccines are well-established, proven interventions with multiple safe and efficacious commercial examples. Many vaccine candidates of this type for SARS-CoV-2 rely on sequences containing the receptor-binding domain (RBD), which mediates viral entry to cells via ACE2. Here we report an engineered sequence variant of RBD that exhibits high-yield manufacturability, high-affinity binding to ACE2, and enhanced immunogenicity after a single dose in mice compared to the Wuhan-Hu-1 variant used in current vaccines. Antibodies raised against the engineered protein exhibited heterotypic binding to the RBD from two recently reported SARS-CoV-2 variants of concern (501Y.V1/V2). Presentation of the engineered RBD on a designed virus-like particle (VLP) also reduced weight loss in hamsters upon viral challenge.


Asunto(s)
Vacunas contra la COVID-19/inmunología , COVID-19/prevención & control , Ingeniería de Proteínas/métodos , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/genética , Animales , Anticuerpos Antivirales/inmunología , Antígenos Virales , Sitios de Unión , COVID-19/virología , Vacunas contra la COVID-19/economía , Humanos , Inmunogenicidad Vacunal , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Unión Proteica , Conformación Proteica , Saccharomycetales/metabolismo , Vacunas de Subunidad
11.
Bioinformatics ; 38(14): 3645-3647, 2022 07 11.
Artículo en Inglés | MEDLINE | ID: mdl-35674381

RESUMEN

SUMMARY: Diversity of the T-cell receptor (TCR) repertoire is central to adaptive immunity. The TCR is composed of α and ß chains, encoded by the TRA and TRB genes, of which the variable regions determine antigen specificity. To generate novel biological insights into the complex functioning of immune cells, combined capture of variable regions and single-cell transcriptomes provides a compelling approach. Recent developments enable the enrichment of TRA and TRB variable regions from widely used technologies for 3'-based single-cell RNA-sequencing (scRNA-seq). However, a comprehensive computational pipeline to process TCR-enriched data from 3' scRNA-seq is not available. Here, we present an analysis pipeline to process TCR variable regions enriched from 3' scRNA-seq cDNA. The tool reports TRA and TRB nucleotide and amino acid sequences linked to cell barcodes, enabling the reconstruction of T-cell clonotypes with associated transcriptomes. We demonstrate the software using peripheral blood mononuclear cells from a healthy donor and detect TCR sequences in a high proportion of single T cells. Detection of TCR sequences is low in non-T-cell populations, demonstrating specificity. Finally, we show that TCR clones are larger in CD8 Memory T cells than in other T-cell types, indicating an association between T-cell clonotypes and differentiation states. AVAILABILITY AND IMPLEMENTATION: The Workflow for Association of T-cell receptors from 3' single-cell RNA-seq (WAT3R), including test data, is available on GitHub (https://github.com/mainciburu/WAT3R), Docker Hub (https://hub.docker.com/r/mainciburu/wat3r) and a workflow on the Terra platform (https://app.terra.bio). The test dataset is available on GEO (accession number GSE195956). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Asunto(s)
Leucocitos Mononucleares , Receptores de Antígenos de Linfocitos T , Leucocitos Mononucleares/metabolismo , Receptores de Antígenos de Linfocitos T/química , Programas Informáticos , Células Clonales/metabolismo , ARN , Análisis de la Célula Individual , Receptores de Antígenos de Linfocitos T alfa-beta/genética
12.
Biotechnol Bioeng ; 2023 Mar 17.
Artículo en Inglés | MEDLINE | ID: mdl-36929469

RESUMEN

Analytical characterization of proteins is a critical task for developing therapeutics and subunit vaccine candidates. Assessing candidates with a battery of biophysical assays can inform the selection of one that exhibits properties consistent with a given target product profile (TPP). Such assessments, however, require several milligrams of purified protein, and ideal assessments of the physicochemical attributes of the proteins should not include unnatural modifications like peptide tags for purification. Here, we describe a fast two-stage minimal purification process for recombinant proteins secreted by the yeast host Komagataella phaffii from a 20 mL culture supernatant. This method comprises a buffer exchange and filtration with a Q-membrane filter and we demonstrate sufficient removal of key supernatant impurities including host-cell proteins (HCPs) and DNA with yields of 1-2 mg and >60% purity. This degree of purity enables characterizing the resulting proteins using affinity binding, mass spectrometry, and differential scanning calorimetry. We first evaluated this method to purify an engineered SARS-CoV-2 subunit protein antigen and compared the purified protein to a conventional two-step chromatographic process. We then applied this method to compare several SARS-CoV-2 RBD sequences. Finally, we show this simple process can be applied to a range of other proteins, including a single-domain antibody, a rotavirus protein subunit, and a human growth hormone. This simple and fast developability methodology obviates the need for genetic tagging or full chromatographic development when assessing and comparing early-stage protein therapeutics and vaccine candidates produced in K. phaffii.

13.
J Allergy Clin Immunol ; 150(4): 748-755, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-36205449

RESUMEN

Single-cell RNA sequencing (scRNA-seq) offers the ability to resolve whole transcriptomes of single cells with substantial throughput, and it has revolutionized studies of gene expression. The transcriptional resolution available can uncover fine structures of biologic heterogeneity that are manifest among cell populations. Here, we review the applications of scRNA-seq to profile the phenotypes and clonotypes of CD4+ T cells. First, we describe challenges inherent to scRNA-seq that are important for analysis of CD4+ T cells, as well as the technical solutions that are emerging to address these challenges. We then consider major themes of the application of scRNA-seq to CD4+ T cells, including investigation of CD4+ T-cell heterogeneity in model systems, analysis of populations from the peripheral blood, and the profiling of tissue-resident populations. We place emphasis on capabilities unique to scRNA-seq, such as the ability to obtain paired T-cell receptor and transcriptome information from single T cells and the potential to elucidate interactions between CD4+ T cells and other cells in their environment. Finally, we conclude by considering future areas of technologic advancement and innovation through which scRNA-seq may further shape our understanding of the roles of CD4+ T cells in health and disease.


Asunto(s)
Productos Biológicos , Transcriptoma , Linfocitos T CD4-Positivos , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Linfocitos T
14.
Biotechnol Bioeng ; 119(1): 59-71, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34596238

RESUMEN

Developing media to sustain cell growth and production is an essential and ongoing activity in bioprocess development. Modifications to media can often address host or product-specific challenges, such as low productivity or poor product quality. For other applications, systematic design of new media can facilitate the adoption of new industrially relevant alternative hosts. Despite manifold existing methods, common approaches for optimization often remain time and labor-intensive. We present here a novel approach to conventional media blending that leverages stable, simple, concentrated stock solutions to enable rapid improvement of measurable phenotypes of interest. We applied this modular methodology to generate high-performing media for two phenotypes of interest: biomass accumulation and heterologous protein production, using high-throughput, milliliter-scale batch fermentations of Pichia pastoris as a model system. In addition to these examples, we also created a flexible open-source package for modular blending automation on a low-cost liquid handling system to facilitate wide use of this method. Our modular blending method enables rapid, flexible media development, requiring minimal labor investment and prior knowledge of the host organism, and should enable developing improved media for other hosts and phenotypes of interest.


Asunto(s)
Automatización de Laboratorios/métodos , Reactores Biológicos , Medios de Cultivo , Fermentación/fisiología , Biomasa , Medios de Cultivo/análisis , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Pichia/genética , Pichia/metabolismo
15.
Biotechnol Bioeng ; 119(2): 657-662, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-34780057

RESUMEN

Prevention of COVID-19 on a global scale will require the continued development of high-volume, low-cost platforms for the manufacturing of vaccines to supply ongoing demand. Vaccine candidates based on recombinant protein subunits remain important because they can be manufactured at low costs in existing large-scale production facilities that use microbial hosts like Komagataella phaffii (Pichia pastoris). Here, we report an improved and scalable manufacturing approach for the SARS-CoV-2 spike protein receptor-binding domain (RBD); this protein is a key antigen for several reported vaccine candidates. We genetically engineered a manufacturing strain of K. phaffii to obviate the requirement for methanol induction of the recombinant gene. Methanol-free production improved the secreted titer of the RBD protein by >5X by alleviating protein folding stress. Removal of methanol from the production process enabled to scale up to a 1200 L pre-existing production facility. This engineered strain is now used to produce an RBD-based vaccine antigen that is currently in clinical trials and could be used to produce other variants of RBD as needed for future vaccines.

16.
Microb Cell Fact ; 21(1): 180, 2022 Sep 05.
Artículo en Inglés | MEDLINE | ID: mdl-36064410

RESUMEN

BACKGROUND: Komagataella phaffii is a commonly used alternative host for manufacturing therapeutic proteins, in part because of its ability to secrete recombinant proteins into the extracellular space. Incorrect processing of secreted proteins by cells can, however, cause non-functional product-related variants, which are expensive to remove in purification and lower overall process yields. The secretion signal peptide, attached to the N-terminus of the recombinant protein, is a major determinant of the quality of the protein sequence and yield. In K. phaffii, the signal peptide from the Saccharomyces cerevisiae alpha mating factor often yields the highest secreted titer of recombinant proteins, but the quality of secreted protein can vary highly. RESULTS: We determined that an aggregated product-related variant of the SARS-CoV-2 receptor binding domain is caused by N-terminal extension from incomplete cleavage of the signal peptide. We eliminated this variant and improved secreted protein titer up to 76% by extension of the N-terminus with a short, functional peptide moiety or with the EAEA residues from the native signal peptide. We then applied this strategy to three other recombinant subunit vaccine antigens and observed consistent elimination of the same aggregated product-related variant. Finally, we demonstrated that this benefit in quality and secreted titer can be achieved with addition of a single amino acid to the N-terminus of the recombinant protein. CONCLUSIONS: Our observations suggest that steric hindrance of proteases in the Golgi that cleave the signal peptide can cause unwanted N-terminal extension and related product variants. We demonstrated that this phenomenon occurs for multiple recombinant proteins, and can be addressed by minimal modification of the N-terminus to improve steric accessibility. This strategy may enable consistent secretion of a broad range of recombinant proteins with the highly productive alpha mating factor secretion signal peptide.


Asunto(s)
COVID-19 , Humanos , Factor de Apareamiento , Señales de Clasificación de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SARS-CoV-2 , Saccharomyces cerevisiae/metabolismo , Saccharomycetales
17.
Stroke ; 52(5): 1798-1808, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33840225

RESUMEN

Background and Purpose: Brain tissue-resident microglia and monocyte-derived macrophages (MDMs) are innate immune cells that contribute to the inflammatory response, phagocytosis of debris, and tissue repair after injury. We have previously reported that both microglia and MDMs transition from proinflammatory to reparative phenotypes over days after an intracerebral hemorrhage (ICH). However, their individual functional properties in the brain remain largely unknown. Here we characterized the differences between microglia and MDMs and further elucidate their distinct activation states and functional contributions to the pathophysiology and recovery after ICH. Methods: Autologous blood injection was used to model ICH in mice. Longitudinal transcriptomic analyses on isolated microglia and MDMs from mice at days 1, 3, 7 and 10 after ICH and naive controls identified core transcriptional programs that distinguish these cells. Imaging flow cytometry and in vivo phagocytosis assays were used to study phagocytic ability of microglia and MDMs. Antigen presentation was evaluated by ovalbumin-OTII CD4 T-cell proliferation assays with bone marrow­derived macrophages and primary microglia cultures. Results: MDMs had higher phagocytic activity and higher erythrophagocytosis in the ICH brain. Differential gene expression revealed distinct transcriptional signatures in the MDMs and microglia after ICH. MDMs had higher expression of MHCII (major histocompatibility complex class II) genes than microglia at all time points and greater ability to induce antigen-specific T-cell proliferation. Conclusions: The different ontogeny of microglia and MDMs lead to divergent responses and functions in the inflamed brain as these 2 cell populations differ in phagocytic functions and antigen-presenting capabilities in the brain after ICH.


Asunto(s)
Encéfalo/metabolismo , Hemorragias Intracraneales/metabolismo , Macrófagos/metabolismo , Fagocitosis/fisiología , Animales , Proliferación Celular/fisiología , Modelos Animales de Enfermedad , Ratones , Microglía/metabolismo
18.
Retrovirology ; 18(1): 35, 2021 10 30.
Artículo en Inglés | MEDLINE | ID: mdl-34717659

RESUMEN

BACKGROUND: The critical role of antibody Fc-mediated effector functions in immune defense has been widely reported in various viral infections. These effector functions confer cellular responses through engagement with innate immune cells. The precise mechanism(s) by which immunoglobulin G (IgG) Fc domain and cognate receptors may afford protection are poorly understood, however, in the context of HIV/SHIV infections. Many different in vitro assays have been developed and utilized to measure effector functions, but the extent to which these assays capture distinct antibody activities has not been fully elucidated. RESULTS: In this study, six Fc-mediated effector function assays and two biophysical antibody profiling assays were performed on a common set of samples from HIV-1 infected and vaccinated subjects. Biophysical antibody profiles supported robust prediction of diverse IgG effector functions across distinct Fc-mediated effector function assays. While a number of assays showed correlated activities, supervised machine learning models indicated unique antibody features as primary contributing factors to the associated effector functions. Additional experiments established the mechanistic relevance of relationships discovered using this unbiased approach. CONCLUSIONS: In sum, this study provides better resolution on the diversity and complexity of effector function assays, offering a clearer perspective into this family of antibody mechanisms of action to inform future HIV-1 treatment and vaccination strategies.


Asunto(s)
Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/virología , VIH-1/inmunología , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/química , Inmunoglobulina G/inmunología , Infecciones por VIH/inmunología , Humanos
19.
Biotechnol Bioeng ; 118(9): 3435-3446, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-33782945

RESUMEN

Straight-through chromatography, wherein the eluate from one column passes directly onto another column without adjustment, is one strategy to integrate and intensify manufacturing processes for biologics. Development and optimization of such straight-through chromatographic processes is a challenge, however. Conventional high-throughput screening methods optimize each chromatographic step independently, with limited consideration for the connectivity of steps. Here, we demonstrate a method for the development and optimization of fully integrated, multi-column processes for straight-through purification. Selection of resins was performed using an in silico tool for the prediction of processes for straight-through purification based on a one-time characterization of host-cell proteins combined with the chromatographic behavior of the product. A two-step optimization was then conducted to determine the buffer conditions that maximized yield while minimizing process- and product-related impurities. This optimization of buffer conditions included a series of range-finding experiments on each individual column, similar to conventional screening, followed by the development of a statistical model for the fully integrated, multi-column process using design of experiments. We used this methodology to develop and optimize integrated purification processes for a single-domain antibody and a cytokine, obtaining yields of 88% and 86%, respectively, with process- and product-related variants reduced to phase-appropriate levels for nonclinical material.


Asunto(s)
Productos Biológicos/química , Productos Biológicos/aislamiento & purificación , Modelos Teóricos , Cromatografía , Ensayos Analíticos de Alto Rendimiento
20.
Biotechnol Bioeng ; 118(5): 1832-1839, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33527350

RESUMEN

Development of continuous biopharmaceutical manufacturing processes is an area of active research. This study considers the long-term transgene copy number stability of Pichia pastoris in continuous bioreactors. We propose a model of copy number loss that quantifies population heterogeneity. An analytical solution is derived and compared with existing experimental data. The model is then used to provide guidance for stable operating timescales. The model is extended to consider copy number dependent growth such as in the case of Zeocin supplementation. The model is also extended to analyze a continuous seeding strategy. This study is a critical step towards understanding the impact of continuous processing on the stability of Pichia pastoris and the resultant products.


Asunto(s)
Reactores Biológicos/microbiología , Variaciones en el Número de Copia de ADN/genética , Inestabilidad Genómica/genética , Proteínas Recombinantes , Saccharomycetales , ADN de Hongos/genética , Modelos Genéticos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo
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