Reactive oxygen intermediates (ROI) generated in response to receptor stimulation play an important role in mediating cellular responses. We have examined the importance of reversible cysteine sulfenic acid formation in naive CD8⁺ T cell activation and proliferation. We observed that, within minutes of T cell activation, naive CD8⁺ T cells increased ROI levels in a manner dependent upon Ag concentration. Increased ROI resulted in elevated levels of cysteine sulfenic acid in the total proteome. Analysis of specific proteins revealed that the protein tyrosine phosphatases SHP-1 and SHP-2, as well as actin, underwent increased sulfenic acid modification following stimulation. To examine the contribution of reversible cysteine sulfenic acid formation to T cell activation, increasing concentrations of 5,5-dimethyl-1,3-cyclohexanedione (dimedone), which covalently binds to cysteine sulfenic acid, were added to cultures. Subsequent experiments demonstrated that the reversible formation of cysteine sulfenic acid was critical for ERK1/2 phosphorylation, calcium flux, cell growth, and proliferation of naive CD8⁺ and CD4⁺ T cells. We also found that TNF-α production by effector and memory CD8⁺ T cells was more sensitive to the inhibition of reversible cysteine sulfenic acid formation than IFN-γ. Together, these results demonstrate that reversible cysteine sulfenic acid formation is an important regulatory mechanism by which CD8⁺ T cells are able to modulate signaling, proliferation, and function.

Purpose: Immunotherapy with checkpoint inhibitors is improving the outcomes of several cancers. However, only a subset of patients respond. Therefore, predictive biomarkers are critically needed to guide treatment decisions and develop approaches to the treatment of therapeutic resistance. Experimental Design: We compared bioenergetics of circulating immune cells and metabolomic profiles of plasma obtained at baseline from patients with melanoma treated with anti–PD-1 therapy. We also performed single-cell RNA sequencing (scRNAseq) to correlate transcriptional changes associated with metabolic changes observed in peripheral blood mononuclear cells (PBMC) and patient plasma. Results: Pretreatment PBMC from responders had a higher reserve respiratory capacity and higher basal glycolytic activity compared with nonresponders. Metabolomic analysis revealed that responder and nonresponder patient samples cluster differently, suggesting differences in metabolic signatures at baseline. Differential levels of specific lipid, amino acid, and glycolytic pathway metabolites were observed by response. Further, scRNAseq analysis revealed upregulation of T-cell genes regulating glycolysis. Our analysis showed that SLC2A14 (Glut-14; a glucose transporter) was the most significant gene upregulated in responder patients' T-cell population. Flow cytometry analysis confirmed significantly elevated cell surface expression of the Glut-14 in CD3+, CD8+, and CD4+ circulating populations in responder patients. Moreover, LDHC was also upregulated in the responder population. Conclusions: Our results suggest a glycolytic signature characterizes checkpoint inhibitor responders; consistently, both ECAR and lactate-to-pyruvate ratio were significantly associated with overall survival. Together, these findings support the use of blood bioenergetics and metabolomics as predictive biomarkers of patient response to immune checkpoint inhibitor therapy.

After Ag encounter, CD8⁺ T cells become activated and begin to proliferate. Early during infection, when Ag-specific effector CD8⁺ T cells are proliferating, producing cytokines, and lysing infected cells in vivo, their mitochondrial potential is increased. The purpose of the experiments presented here was to determine whether mitochondrial function was required for CD8⁺ T cell function. To block mitochondrial function, transgenic CD8⁺ T cells were incubated with increasing doses of rotenone, an inhibitor of electron transport complex I. Within minutes of T cell activation, rotenone incubation decreased the production of H₂O₂, calcium flux, and ERK1/2 phosphorylation. Failure to undergo signal transduction resulted in a decrease in T cell division initiated by peptide-coated cells, CD3/CD28 Abs, and PMA/ionomycin stimulation. Decreased function following rotenone incubation was not restricted to naive cells, as effector and memory CD8⁺ T cells isolated directly ex vivo from lymphocytic choriomeningitis virus-infected mice displayed decreased production of IFN-γ and TNF-α production after peptide stimulation. Furthermore, incubation with rotenone decreased degranulation of effector and memory cells, a critical step in the cytolysis of infected cells. These data suggest that electron transport complex I is required for CD8⁺ T cell signal transduction, proliferation, cytokine production, and degranulation.

Apoptosis is critical for the development and maintenance of the immune system. The proapoptotic Bcl-2 family member Bim is important for normal immune system homeostasis. Although previous experiments have shown that Bim is critical for the apoptosis of antigen-specific CD8⁺T cells during acute viral infection, the role of Bim during chronic viral infection is unclear. Using lymphocytic choriomeningitis virus clone 13 infection of mice, we demonstrate a role for Bim in CD8⁺T-cell apoptosis during chronic viral infection. Enumeration of antigen-specific CD8⁺T cells by major histocompatibility complex class I tetramer staining revealed that CD8⁺D^bNP396-404⁺T cells, which undergo extensive deletion in wild-type mice, exhibited almost no decrease in Bim mutant mice. This contrasts with CD8⁺D^bGP33-41⁺and CD8⁺D^bGP276-286⁺T cells that underwent similar decreases in numbers in both Bim mutant and wild-type mice. Increased numbers of CD8⁺D^bNP396-404⁺T cells in Bim mutant mice were due to lack of apoptosis and could not be explained by altered proliferation, differential homing to tissues, or increased help from CD4⁺T cells. When viral titers were examined, high levels were initially observed in both groups, but in Bim mutant mice, clearance from the spleen and sera was slightly accelerated. These experiments demonstrate the critical role of Bim during chronic viral infection to down-regulate CD8⁺T-cell responses and have implications for designing strategies for optimizing immunotherapies during situations where antigen persists, such as chronic infection, autoimmune syndromes, and cancer.

Infection with influenza A virus can lead to increased susceptibility to subsequent bacterial infection, often with Streptococcus pneumoniae. Given the substantial modification of the lung environment that occurs following pathogen infection, there is significant potential for modulation of immune responses. In this study, we show that infection of mice with influenza virus, followed by the noninvasive EF3030 strain of Streptococcus pneumoniae, leads to a significant decrease in the virus-specific CD8⁺ T cell response in the lung. Adoptive-transfer studies suggest that this reduction contributes to disease in coinfected animals. The reduced number of lung effector cells in coinfected animals was associated with increased death, as well as a reduction in cytokine production in surviving cells. Further, cells that retained the ability to produce IFN-γ exhibited a decreased potential for coproduction of TNF-α. Reduced cytokine production was directly correlated with a decrease in the level of mRNA. Negative regulation of cells in the mediastinal lymph node was minimal compared with that present in the lung, supporting a model of selective regulation in the tissue harboring high pathogen burden. These results show that entry of a coinfecting pathogen can have profound immunoregulatory effects on an ongoing immune response. Together, these findings reveal a novel dynamic interplay between concurrently infecting pathogens and the adaptive immune system.