Nanoscale or single-cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here, we report the development of a nanoPOTS (nanodroplet processing in one pot for trace samples) platform for small cell population proteomics analysis. NanoPOTS enhances the efficiency and recovery of sample processing by downscaling processing volumes to 3000 proteins are consistently identified from as few as 10 cells. Furthermore, we demonstrate quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.

T lymphocytes constitute a major effector cell population in autoimmune type 1 diabetes. Despite essential functions of mitochondria in regulating activation, proliferation, and apoptosis of T cells, little is known regarding T cell metabolism in the progression of human type 1 diabetes. In this study, we report, using two independent cohorts, that T cells from patients with type 1 diabetes exhibited mitochondrial inner-membrane hyperpolarization (MHP). Increased MHP was a general phenotype observed in T cell subsets irrespective of prior antigen exposure, and was not correlated with HbA1C levels, subject age, or duration of diabetes. Elevated T cell MHP was not detected in subjects with type 2 diabetes. T cell MHP was associated with increased activation-induced IFNγ production, and activation-induced IFNγ was linked to mitochondria-specific ROS production. T cells from subjects with type 1 diabetes also exhibited lower intracellular ATP levels. In conclusion, intrinsic mitochondrial dysfunction observed in type 1 diabetes alters mitochondrial ATP and IFNγ production; the latter is correlated with ROS generation. These changes impact T cell bioenergetics and function.

Aberrant mitochondrial function, including excessive reactive oxygen species (ROS) production, has been implicated in the pathogenesis of human diseases. The use of mitochondrial inhibitors to ascertain the sites in the electron transport chain (ETC) resulting in altered ROS production can be an important tool. However, the response of mouse mitochondria to ETC inhibitors has not been thoroughly assessed. Here we set out to characterize the differences in phenotypic response to ETC inhibitors between the more energetically demanding brain mitochondria and less energetically demanding liver mitochondria in commonly utilized C57BL/6J mice. We show that in contrast to brain mitochondria, inhibiting distally within complex I or within complex III does not increase liver mitochondrial ROS production supported by complex I substrates, and liver mitochondrial ROS production supported by complex II substrates occurred primarily independent of membrane potential. Complex I, II, and III enzymatic activities and membrane potential were equivalent between liver and brain and responded to ETC. inhibitors similarly. Brain mitochondria exhibited an approximately two-fold increase in complex I and II supported respiration compared with liver mitochondria while exhibiting similar responses to inhibitors. Elevated NADH transport and heightened complex II–III coupled activity accounted for increased complex I and II supported respiration, respectively in brain mitochondria. We conclude that important mechanistic differences exist between mouse liver and brain mitochondria and that mouse mitochondria exhibit phenotypic differences compared with mitochondria from other species.

Oxidants participate in lymphocyte activation and function. We previously demonstrated that eliminating the activity of NADPH oxidase 2 (NOX2) significantly impaired the effectiveness of autoreactive CD8+ CTLs. However, the molecular mechanisms impacting CD8+ T cell function remain unknown. In the present study, we examined the role of NOX2 in both NOD mouse and human CD8+ T cell function. Genetic ablation or chemical inhibition of NOX2 in CD8+ T cells significantly suppressed activation-induced expression of the transcription factor T-bet, the master transcription factor of the Tc1 cell lineage, and T-bet target effector genes such as IFN-γ and granzyme B. Inhibition of NOX2 in both human and mouse CD8+ T cells prevented target cell lysis. We identified that superoxide generated by NOX2 must be converted into hydrogen peroxide to transduce the redox signal in CD8+ T cells. Furthermore, we show that NOX2-generated oxidants deactivate the tumor suppressor complex leading to activation of RheB and subsequently mTOR complex 1. These results indicate that NOX2 plays a nonredundant role in TCR-mediated CD8+ T cell effector function.

NADPH oxidase (NOX) family members are major resources of intracellular reactive oxygen species (ROS). In the immune system, ROS derived from phagocytic NOX (NOX2) participate in both pathogen clearance and signaling transduction. The role of NOX2 in neutrophils and macrophages has been well studied as mutations in NOX2 subunits cause chronic granulomas disease (CGD). NOX2 is expressed across a wide range of immune cells and recent reports have demonstrated that NOX2-derived ROS play important roles in other immune cells during an immune response. In this review, we summarize current knowledge of functions of NADPH oxidase 2 in each subset of leukocytes, as well as associations of NOX2 deficiency with diseases associated specifically with autoimmunity and immune deficiency. We also discuss important knowledge gaps as well as potential future directions for NOX2 research.