Flow cytometry is a widely applied approach for exploratory immune profiling and biomarker discovery in cancer and other diseases. However, flow cytometry is limited by the number of parameters that can be simultaneously analyzed, severely restricting its utility. Recently, the advent of mass cytometry (CyTOF) has enabled high dimensional and unbiased examination of the immune system, allowing simultaneous interrogation of a large number of parameters. This is important for deep interrogation of immune responses and particularly when sample sizes are limited (such as in tumors). Our goal was to compare the accuracy and reproducibility of CyTOF against flow cytometry as a reliable analytic tool for human PBMC and tumor tissues for cancer clinical trials. We developed a 40+ parameter CyTOF panel and demonstrate that compared to flow cytometry, CyTOF yields analogous quantification of cell lineages in conjunction with markers of cell differentiation, function, activation, and exhaustion for use with fresh and viably frozen PBMC or tumor tissues. Further, we provide a protocol that enables reliable quantification by CyTOF down to low numbers of input human cells, an approach that is particularly important when cell numbers are limiting. Thus, we validate CyTOF as an accurate approach to perform high dimensional analysis in human tumor tissue and to utilize low cell numbers for subsequent immunologic studies and cancer clinical trials.
BackgroundElevated levels of type I interferons (IFNs) are a characteristic feature of the systemic autoimmune rheumatic diseases (SARDs) and are thought to play an important pathogenic role. However, it is unknown whether these elevations are seen in anti-nuclear antibody–positive (ANA+) individuals who lack sufficient criteria for a SARD diagnosis. We examined IFN-induced gene expression in asymptomatic ANA+ individuals and patients with undifferentiated connective tissue disease (UCTD) to address this question.MethodsHealthy ANA− control subjects and ANA+ titre (≥1:160 by immunofluorescence) participants meeting no criteria, meeting at least one criterion (UCTD) or meeting SARD classification criteria were recruited. Whole peripheral blood IFN-induced and BAFF gene expression were quantified using NanoString technology. The normalized levels of five IFN-induced genes were summed to produce an IFN5 score.ResultsThe mean IFN5 scores were increased in all ANA+ participant subsets as compared with healthy control subjects. We found that 36.8% of asymptomatic ANA+ and 50% of UCTD participants had IFN5 scores >2 SD above the mean for healthy control subjects. In all ANA+ subsets, the IFN5 score correlated with the presence of anti-Ro/La antibodies. In the asymptomatic ANA+ subset, this score also correlated with the ANA titre, whereas in the other ANA+ subsets, it correlated with the number of different ANA specificities. Development of new SARD criteria was seen in individuals with normal and high IFN5 scores.ConclusionsAn IFN signature is seen in a significant proportion of ANA+ individuals and appears to be associated with ANA titre and type of autoantibodies, rather than with the presence or development of clinical SARD symptoms.
BackgroundDiagnosis of systemic autoimmune rheumatic diseases (SARD) relies on the presence of hallmark anti-nuclear antibodies (ANA), many of which can be detected years before clinical manifestations. However, ANAs are also seen in healthy individuals, most of whom will not develop SARD. Here, we examined a unique cohort of asymptomatic ANA+ individuals to determine whether they share any of the cellular immunologic features seen in SARD.MethodsHealthy ANA− controls and ANA+ (ANA ≥1:160 by immunofluorescence) participants with no SARD criteria, with at least one criterion (undifferentiated connective tissue disease (UCTD)), or meeting SARD classification criteria were recruited. Peripheral blood cellular immunological changes were assessed by flow cytometry and transcript levels of BAFF, interferon (IFN)-induced and plasma cell-expressed genes were quantified by NanoString.ResultsA number of the immunologic abnormalities seen in SARD, including changes in peripheral B (switched memory) and T (iNKT, T regulatory, activated memory T follicular helper) subsets and B cell activation, were also seen in asymptomatic ANA+ subjects and those with UCTD. The extent of these immunologic changes correlated with ANA titer or the number of different specific ANAs produced. Principal component analysis of the cellular data indicated that a significant proportion of asymptomatic ANA+ subjects and subjects with UCTD clustered with patients with early SARD, rather than ANA− healthy controls.ConclusionsANA production is associated with altered T and B cell activation even in asymptomatic individuals. Some of the currently accepted cellular features of SARD may be associated with ANA production rather than the immunologic events that cause symptoms in SARD.Electronic supplementary materialThe online version of this article (10.1186/s13075-018-1752-3) contains supplementary material, which is available to authorized users.
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