Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry.

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of "intrinsic" patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native "intrinsic pathogenic" disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Reference values for CD4+ and CD8+ T lymphocytes with naïve or memory phenotype and their association with mortality in the elderly.

BACKGROUND: Well-established reference values which take into account the influence of age on immune cell phenotype, and the impact of naïve or memory T cells on mortality have not been well defined in the elderly. OBJECTIVE: The aim of this study was to evaluate the reference values for the peripheral number of total and naïve or memory CD4 and CD8 T cells in a healthy population in Italy, and to analyze whether the immune phenotype was associated with an increased risk of death among older adults. METHODS: The number of total or naïve and memory CD4+ or CD8+ T cells was evaluated in the peripheral blood of 288 healthy people ranging in age from 20 to 107 years. Furthermore, to correlate peripheral immune phenotype with mortality rate after a 3-years follow-up, a retrospective analysis was performed on the results from those individuals aged >65 years at the time of the enrollment in the study. RESULTS: The absolute number of total and naïve T cells was progressively reduced with increasing age in both the CD4+ and CD8+ T cell populations. The decrease was particularly evident for cells with naïve phenotype, since CD4-naïve and CD8-naïve T cells respectively showed a 4- and a 2- to 3-fold reduction in 70- to >90-year-old subjects in comparison with young adults. The number of CD4 memory T cells significantly increased with age. No significant age-related change was observed in the number of CD8+ memory T cells. Of the 194 subjects included in the study of association of immune phenotype with mortality, 121 were alive and 73 deceased during the 3-year follow-up. The impact of immune parameters on survival demonstrated that only the absolute number of CD8 memory T cells, after adjustment for age, correlated with increased mortality (OR 1.007, 95% CI 1.002-1.012, p = 0.01). The correlation was significant in female but not in male subjects. CONCLUSION: We provide reference values for total and naïve or memory CD4 and CD8 T cell populations, and demonstrate that the absolute number of CD8 memory T cells, after adjustment for age, correlates with increased mortality.