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Evaluation of a fit-for-purpose assay to monitor antigen-specific functional CD4+ T cell subpopulations in rheumatoid arthritis using flow-cytometry based peptide-MHC class-II tetramer staining.

ABSTRACT: Antigen-specific T cells can serve as a response biomarker in non-clinical or clinical immunotherapy studies in autoimmune disease. There are protocols with optimized multimer staining methods to detect peptide (p)MHCII+ CD4+ T cells, and some qualified and validated protocols for pMHCI+ CD8+ T cells. However, no protocol is fully or partially qualified to enumerate and characterise antigen-specific pMHCII+ CD4+ T cells from patient samples. Implementing such an assay requires a desired level of specificity and precision, in terms of assay repeatability and reproducibility. In transgenic type II collagen (CII)-immunised HLA-DR1/DR4 humanised mouse models of collagen-induced arthritis (CIA), CII259-273-specific T cells dominantly expand. Therefore antigen-specific T cells recognising this epitope presented by rheumatoid arthritis (RA)-associated risk HLA-DR allomorphs are of interest to understand disease progression and responses to immunotherapy in RA patients. Using HLA-DRB1*04:01 or *01:01-collagen type II (CII)259-273 tetramers, we evaluated parameters influencing precision and reproducibility of an optimized flow cytometry-based method for antigen-specific CD4+ T cells and eight specific subpopulations with and without tetramer positivity. We evaluated specificity, precision, and reproducibility for research environments and non-regulated laboratories. The assay has excellent overall precision with %CV<25% for intra-assay repeatability, inter-analyst precision, and inter-assay reproducibility. The precision of the assay correlated negatively with the cell viability after thawing, indicating that post-thaw viability is a critical parameter for reproducibility. This assay is suitable for longitudinal analysis of treatment response and disease activity outcome in RA patients, and adaptable for translational or immunotherapy clinical trial settings.

SUBMITTER: Patel S

PROVIDER: S-EPMC8802177 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Rationale, aims and objectivesThe field of implementation science is critical for embedding research evidence into healthcare practice, benefiting individuals, organizations, governments, and the broader community. Implementation science is messy and complex, underpinned by many theories and frameworks. Efficacious interventions for older people with multiple comorbidities exist, yet many lack effectiveness evaluation relevant to pragmatic implementation within aged care practice. This article outlines the conceptualization and development of an Implementation Framework for Aged Care (IFAC), fit-for-purpose for an aged care organization, Bolton Clarke, intent on embedding evidence into practice.MethodA four-stage process was adopted to (1) explore context and relevant literature to conceptualize the IFAC; (2) identify key elements for a draft IFAC; (3) expand elements and refine the draft in consultation with experts and (4) apply the IFAC to three existing projects, identifying key learnings. A checklist to operationalize the IFAC was then developed.ResultsThe IFAC is grounded in codesign principles and encapsulated by the implementation context, from a social, cultural and political perspective. The IFAC addresses the questions of (1) why do we need to change?; (2) what do we know?; (3) who will benefit?; (4) who will make the change?; (5) what strategies will be used?; and (6) what difference are we making? Three pilot projects: early adoption of a Wellness and Reablement approach; a care worker and virtual physiotherapist-led program to prevent falls; and a therapeutic horticulture program for residential communities, highlight learnings of applying the IFAC in practice.ConclusionThis fit-for-purpose IFAC was developed for a proactive and responsive aged care provider. The simplicity of the six-question IFAC is underpinned by substantial theoretical perspectives for its elements and their connections. This complexity is then consolidated into an 18-question checklist to operationalize the IFAC, necessary to advance the translation of evidence into clinical practice.

Project description:Exosomes are defined as submicron (30-150 nm), lipid bilayer-enclosed extracellular vesicles (EVs), specifically generated by the late endosomal compartment through fusion of multivesicular bodies with the plasma membrane. Produced by almost all cells, exosomes were originally considered to represent just a mechanism for jettisoning unwanted cellular moieties. Although this may be a major function in most cells, evolution has recruited the endosomal membrane-sorting pathway to duties beyond mere garbage disposal, one of the most notable examples being its cooption by retroviruses for the generation of Trojan virions. It is, therefore, tempting to speculate that certain cell types have evolved an exosome subclass active in intracellular communication. We term this EV subclass "signalosomes" and define them as exosomes that are produced by the "signaling" cells upon specific physiological or environmental cues and harbor cargo capable of modulating the programming of recipient cells. Our recent studies have established that signalosomes released by mesenchymal stem/stromal cells (MSCs) represent the main vector of MSC immunomodulation and therapeutic action in animal models of lung disease. The efficacy of MSC-exosome treatments in a number of preclinical models of cardiovascular and pulmonary disease supports the promise of application of exosome-based therapeutics across a wide range of pathologies within the near future. However, the full realization of exosome therapeutic potential has been hampered by the absence of standardization in EV isolation, and procedures for purification of signalosomes from the main exosome population. This is mainly due to immature methodologies for exosome isolation and characterization and our incomplete understanding of the specific characteristics and molecular composition of signalosomes. In addition, difficulties in defining metrics for potency of exosome preparations and the challenges of industrial scale-up and good manufacturing practice compliance have complicated smooth and timely transition to clinical development. In this manuscript, we focus on cell culture conditions, exosome harvesting, dosage, and exosome potency, providing some empirical guidance and perspectives on the challenges in bringing exosome-based therapies to clinic.

Project description:BackgroundQualified or validated assays are essential in clinical trials. Short-term stimulation of whole blood and intracellular cytokine staining assay is commonly used to measure immunogenicity in tuberculosis vaccine clinical trials. Previously, the short-term stimulation process of whole blood with BCG was optimized. We aimed to qualify the intracellular cytokine staining process and assess the effects of long-term cryopreservation. Our hypotheses were that the assay is robust in the measurement of the mycobacteria-specific T cells, and long-term cryopreservation of fixed cells from stimulated whole blood would not compromise reliable measurement of mycobacteria induced CD4 T cell immunity.MethodsWhole blood from healthy adults was collected in sodium heparinized tubes. The blood was left unstimulated or stimulated with mycobacterial antigens or mitogens for 12h. Cells were harvested, fixed and multiple aliquots from each participant cryopreserved. Later, mycobacteria-specific CD4 and CD8 T cells expressing IFN-γ, TNF-α, IL-2 and IL-17 were quantitated by flow cytometry. Assay performance characteristics evaluated included limit of quantification and detection, reproducibility, precision, robustness, specificity and sensitivity. To assess the effects of long-term cryopreservation, fixed cells from the stimulated bloods were analysed one week post-cryopreservation and at 3-month intervals over a 3-year period.ResultsThe limit of quantification for the different cytokines was variable: 0.04% for frequencies of IFN-γ- and IL-2-expressing T cells and less than 0.01% for TNF-α- and IL-17-expressing T cells. When measurement of the mycobacteria-specific T cells was assessed at levels above the detection limit, the whole blood intracellular cytokine assay showed high precision that was operator-independent. The assay was also robust: variation in staining conditions including temperature (4 °C or 20-23 °C) and time (45, 60 or 90 min) did not markedly affect quantification of specific T cells. Finally, prolonged periods of cryopreservation also did not significantly influence quantification of mycobacteria-specific CD4 T cells.ConclusionsThe whole blood intracellular cytokine assay is robust and reliable in quantification of the mycobacteria-specific T cells and is not significantly affected by cryopreservation of fixed cells.

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Evaluation of a fit-for-purpose assay to monitor antigen-specific functional CD4+ T cell subpopulations in rheumatoid arthritis using flow-cytometry based peptide-MHC class-II tetramer staining.

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