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Guidelines for standardizing T-cell cytometry assays to link biomarkers, mechanisms, and disease outcomes in type 1 diabetes.

ABSTRACT: Cytometric immunophenotyping is a powerful tool to discover and implement T-cell biomarkers of type 1 diabetes (T1D) progression and response to clinical therapy. Although many discovery-based T-cell biomarkers have been described, to date, no such markers have been widely adopted in standard practice. The heterogeneous nature of T1D and lack of standardized assays and experimental design across studies is a major barrier to the broader adoption of T-cell immunophenotyping assays. There is an unmet need to harmonize the design of immunophenotyping assays, including those that measure antigen-agnostic cell populations, such that data collected from different clinical trial sites and T1D cohorts are comparable, yet account for cohort-specific features and different drug mechanisms of action. In these Guidelines, we aim to provide expert advice on how to unify aspects of study design and practice. We provide recommendations for defining cohorts, method implementation, as well as tools for data analysis and reporting by highlighting and building on selected successes. Harmonization of cytometry-based T-cell assays will allow researchers to better integrate findings across trials, ultimately enabling the identification and validation of biomarkers of disease progression and treatment response in T1D.

SUBMITTER: Yang JHM

PROVIDER: S-EPMC9006584 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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Guidelines for standardizing T-cell cytometry assays to link biomarkers, mechanisms, and disease outcomes in type 1 diabetes.

Yang Jennie H M JHM Ward-Hartstonge Kirsten A KA Perry Daniel J DJ Blanchfield J Lori JL Posgai Amanda L AL Wiedeman Alice E AE Diggins Kirsten K Rahman Adeeb A Tree Timothy I M TIM Brusko Todd M TM Levings Megan K MK James Eddie A EA Kent Sally C SC Speake Cate C Homann Dirk D Long S Alice SA

European journal of immunology 20220128 3

Cytometric immunophenotyping is a powerful tool to discover and implement T-cell biomarkers of type 1 diabetes (T1D) progression and response to clinical therapy. Although many discovery-based T-cell biomarkers have been described, to date, no such markers have been widely adopted in standard practice. The heterogeneous nature of T1D and lack of standardized assays and experimental design across studies is a major barrier to the broader adoption of T-cell immunophenotyping assays. There is an un ...[more]

PMID: 35025103

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Project description:BackgroundCytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online).ResultsThree sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC) were shipped to various sites, where ICS assays using cytomegalovirus (CMV) pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results ((CD4+)cytokine+ cells and (CD8+)cytokine+ cells) were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template. Mean inter-laboratory coefficient of variation (C.V.) ranged from 17-44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells (PBMC) yielded lower inter-lab C.V.'s than whole blood. Centralized analysis (using a dynamic gating template) reduced the inter-lab C.V. by 5-20%, depending upon the experiment. The inter-lab C.V. was lowest (18-24%) for samples with a mean of > 0.5% IFNgamma + T cells, and highest (57-82%) for samples with a mean of < 0.1% IFNgamma + cells.ConclusionICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. Cryopreserved PBMC may yield slightly more consistent results than shipped whole blood. Analysis, particularly gating, is a significant source of variability, and can be reduced by centralized analysis and/or use of a standardized dynamic gating template. Use of pre-aliquoted lyophilized reagents for stimulation and staining can provide further standardization to these assays.

Project description:BackgroundIntegrated care interventions for chronic conditions can lead to improved outcomes, but it is not clear when and why this is the case. This study aims to answer the following two research questions: First, what are the context, mechanisms and outcomes of integrated care for people with type 2 diabetes? Second, what are the relationships between context, mechanisms and outcomes of integrated care for people with type 2 diabetes?MethodsA systematic literature search was conducted for the period 2003-2013 in Cochrane and PubMed. Articles were included when they focussed on integrated care and type 2 diabetes, and concerned empirical research analysing the implementation of an intervention. Data extraction was performed using a common data extraction table. The quality of the studies was assessed with the Mixed Methods Appraisal Tool. The CMO model (context + mechanism = outcome) was used to study the relationship between context factors (described by the barriers and facilitators encountered in the implementation process and categorised at the six levels of the Implementation Model), mechanisms (defined as intervention types and described by their number of Chronic Care Model (sub-)components) and outcomes (the intentional and unintentional effects triggered by mechanism and context).ResultsThirty-two studies met the inclusion criteria. Most reported barriers to the implementation process were found at the organisational context level and most facilitators at the social context level. Due to the low number of articles reporting comparable quantitative outcome measures or in-depth qualitative information, it was not possible to make statements about the relationship between context, mechanisms and outcomes.ConclusionsEfficient resource allocation should entail increased investments at the organisational context level where most barriers are expected to occur. It is likely that investments at the social context level will also help to decrease the development of barriers at the organisational context level, especially by increasing staff involvement and satisfaction. If future research is to adequately inform practice and policy regarding the impact of these efforts on health outcomes, focus on the actual relationships between context, mechanisms and outcomes should be actively incorporated into study designs.

Project description:Type 2 diabetes mellitus (T2DM) has been confirmed as an independent risk factor for colorectal cancer (CRC) in many studies. However, the mechanisms behind T2DM's role in the progression of CRC remain unclear. This study aims to explore the potential biomarkers and molecular mechanisms involved in T2DM-promoted CRC progression. The limma package was used to identify differentially expressed genes in tumor tissue from CRC patients with or without T2DM. The key biological processes were screened by gene ontology and gene set enrichment analysis. A diagnostic model for co-morbidities was constructed by logistic regression model with least absolute shrinkage and selection operator (Lasso) regularization method. The diagnostic performance was assessed by supplementing external datasets to draw ROC curves on the diagnostic model. The diagnostic model was further screened for key genes by prognostic analysis. The relationship of key genes with immune cells and other cells was evaluated by immune infiltration algorithm and single-cell transcription analysis. Drug prediction was performed by cMAP and the obtained drugs were molecularly docked with the key genes. The differentially expressed genes of T2DM-promoted CRC progression were mainly enriched to O-linked glycosylation-related processes. The diagnostic model constructed based on Lasso logistic regression had good diagnostic performance (AUC > 0.8). COX11 was the key gene for co-morbidities: in tumor tissues, COX11 expression was significantly higher than that in normal colon tissues. However, COX11 gene expression was significantly lower in patients with comorbidities than in patients without T2DM in tumor tissue. External datasets confirmed from both mRNA and protein expression levels that low COX11 expression was significantly associated with poor CRC prognosis. Immune infiltration analysis suggested that its expression related to the proportion of M2 macrophages. Single-cell transcriptome analysis revealed a close association of COX11 expression with endothelial cells and macrophages. The top4 drugs predicted bound well to COX11. Our study revealed that the pathogenesis of T2DM-promoted CRC progression related to O-linked glycosylation. We constructed a diagnostic model for T2DM-CRC co-morbidity. Meanwhile, we identified COX11 as a potential immune-related molecular marker closely associated with T2DM-promoted CRC progression. These mechanisms and molecular markers may provide new ideas for further studies of T2DM-promoted CRC progression and contribute to drug discovery for the treatment of co-morbidities.

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Guidelines for standardizing T-cell cytometry assays to link biomarkers, mechanisms, and disease outcomes in type 1 diabetes.

Publications

Guidelines for standardizing T-cell cytometry assays to link biomarkers, mechanisms, and disease outcomes in type 1 diabetes.

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