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Mass cytometry is capable of measuring more than 40 distinct proteins on individual cells making it a promising technology for innovating biomarker discovery. However, in order for this potential to be fully realized, best practices in panel design need to be further defined in order to achieve consistency and reproducibility in data analysis. Of particular importance are controls that reveal, and panel design principles that mitigate the effects of signal interference or overlap. We observed a disparity between the staining profiles of two noncompeting anti-integrin b7 mAbs and hypothesized that signal interference was responsible. A mass-minus-one (MMO) control was applied and demonstrated that signal overlap caused the perceived interclonal discrepancy in b7 expression. Panel redesign in consideration of mass-cytometry specific interference dynamics dramatically improved concordance between both mAbs by redistributing background signals caused by overlap. These studies visualize how signal overlap can complicate mass cytometry data interpretation and demonstrate how the rational distribution of interference can greatly improve panel design and data quality. V C 2016 International Society for Advancement of Cytometry

To better understand how innate and adaptive immune responses interact with each other, we combined 4-1BB T cell costimulation with specific adjuvants to determine whether these treatments would influence specific T cell expansion and function in vivo. In the presence of 4-1BB ligation and Toll-like receptor 3 (TLR)3 and͞or TLR4 triggering, CD8 T cell clonal expansion and survival was augmented profoundly. Specific T cells primed in vivo with TLR ligands responded normally to in vitro recall stimulus, but, surprisingly, copriming with 4-1BB costimulation significantly impaired the recall response even though many more specific effector T cells were rescued in vivo. Here, we demonstrate that the rescued CD8 T cells suppressed CD4 T cell proliferation via a type ␤ transforming growth factor-dependent mechanism. Thus, 4-1BB and TLR ligands induce survival of specific effector CD8 T cells with suppressive recall potential, which may explain the dual role that 4-1BB activation plays in mediating tumor clearance and prevention of autoimmune disease.T he activation-inducible T cell costimulatory receptor 4-1BB (also referred to as CD137) operates downstream of CD28 and is unique in its pattern of stimulation as compared with CD28. For example, 4-1BB is expressed earlier on activated CD8 than on CD4 T cells in vivo (1). Recently, it was shown that 4-1BB is expressed on dendritic cells but reported to be down-regulated after dendritic cell activation via CD40 ligation (2, 3). However, the function of 4-1BB on dendritic cells is not clear at this time (4). In contrast to CD28, 4-1BB is more effective at stimulating effector T cells than naïve T cells (5). Although it is clear that anti-4-1BB is very capable of stimulating CD4 T cells in vitro as well as CD8 T cells (6), it is evident that postactivation, 4-1BB-induced T cell survival preferentially occurs within the CD8 subpopulation (1, 7). Specifically, ligation of 4-1BB on activated CD8 T cells promotes in vivo long-term-specific T cell survival in the CD8 but not the CD4 compartment (1). This feature was not observed with other costimulatory molecules (8, 9). Thus, there appears to be specialized function unique to individual costimulatory molecules expressed on T cells.The data presented in this article suggest that ligands specific for 4-1BB could be used therapeutically to generate potent vaccines, and based on data from several other groups, it is clear that the 4-1BB-stimulated T cells can effectively carry out effector functions (10-12). For example, 4-1BB has been shown to mediate eradication of established tumors and heighten cytokine secretion, proliferative capacity, and cytotoxic T lymphocyte activity. Thus, 4-1BB-costimulated T cells may serve as excellent effector cells endowed with extended longevity in vivo.The view that 4-1BB costimulates T cells in a productive manner has been complicated in two recent observations. First, it was shown that 4-1BB-deficient T cells are hyperresponsive to mitogens compared with WT T cells (13). Second, it was demonstrat...