Deep Immune Profiling in Trauma and Sepsis: Flow Is the Way to Go!*

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High-content immune profiling technologies are transforming the field of predictive medicine. In the critical care unit, predicting which patient is at risk for developing sepsis after a major traumatic injury remains a clinical challenge. A precise understanding of the complex inflammatory response to traumatic injury is crucial for identification of immunologic dysfunction predictive of posttraumatic infection. In this issue of Critical Care Medicine, Seshadri et al (1) used a single-cell mass cytometry approach to characterize the phenotype and function of major innate and adaptive immune responses in patients who suffered from traumatic injury. This research breaks ground toward the larger goal of individualized risk stratification based on the deep immune profiling of critically ill patients.
Mass cytometry by time-of-flight (CyTOF) is a high-parameter flow cytometry platform that exploits the sensitivity, dynamic range, and minimal spectral overlap of mass spectrometry to enable the simultaneous interrogation of over 50 parameters on a cell-by-cell basis (2, 3). In their study, Seshadri et al (1) employed two parallel CyTOF antibody panels to analyze longitudinal peripheral blood mononuclear cell samples from 10 patients suffering from traumatic injury (Injury Severity Score > 20) and 10 age-matched controls. The first antibody panel allowed quantification of the abundance of immune cell subsets, and the second, characterization of intracellular expression of over 20 cytokines at the single-cell level.
Seshadri et al (1) should be lauded for the careful design of a complex mass cytometry assay and the use of state-of-the-art methods for sample processing and analysis. Samples were appropriately barcoded, using a mass-tag barcoding method known to significantly reduce experimental variability (4). Samples were also normalized to account for changes in instrument detection sensitivity (5). These methodological precautions are imperative when analyzing patient samples with mass cytometry, which, like any antibody-based multiplex platform, is vulnerable to batch effects due to antibody staining, as well as metal isotope- and instrument-specific considerations.
Several themes evolving from analysis by Seshadri et al (1) resonated with known hallmarks of the immune response to trauma (6, 7). For example, analysis of immune cell abundances recapitulated previously reported expansion of monocytes after injury and the concomitant decrease in CD4+ and CD8+ T-cell frequencies. Similarly, the expression of human leukocyte antigen - antigen D related in monocytes dramatically diminished after injury, consistent with clinically relevant phenotypic changes in these innate immune cells after traumatic injury (8).
The analysis also enabled intriguing discoveries, specifically the functional analysis of T helper 17 (Th17) cells and natural killer (NK) cell subsets. Th17 cells are a subset of CD4+ T cells that play a critical role at the interface of innate and adaptive immunity, particularly in the host’s pathogen defense against bacteria. However, evidences from animal and human studies have linked Th17-derived cytokines (such as interleukin [IL]-17 and IL-22) to worse outcomes after sepsis (9). Data by Seshadri et al (1) indicates that trauma rapidly induces Th17 differentiation, followed by the functional capacity to produce IL-17 5 days after injury. Further work will be necessary to determine whether the expansion of Th17 cells after trauma protects patients from posttraumatic infection or, in contrast, predominantly exacerbates an inflammatory process associated with poor clinical outcomes.
Seshadri et al (1) also find important alterations in NK cell function following traumatic injury. Biphasic changes in the expression of the transcription factor T-bet in NK cells were observed in response to trauma and paralleled the capacity of NK cells to produce tumor necrosis factor-β and interferon-γ. Seshadri et al (1) conclude that trauma induces the rapid recruitment of an immature NK cell population from the bone marrow within 1 day of injury, followed by differentiation into mature NK cells.

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