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.

Following many viral infections, there are large expansions of Ag-specific CD8+ T cells. After viral clearance, mechanisms exist to ensure that the vast majority of effector cells undergo apoptosis. In studies of thymocyte apoptosis, loss of mitochondrial potential (ΔΨm) and excess production of reactive oxygen intermediates have been implicated as key events in cellular apoptosis. The purpose of the experiments presented in this work was to determine these parameters in Ag-specific CD8+ T cells during a physiological response such as viral infection. Using lymphocytic choriomeningitis virus infection of mice, we found that Ag-specific CD8+ effector T cells that had undergone recent TCR stimulation had an increased ΔΨm. These cells also had increased levels of superoxide. As these cells progressed through the contraction of the immune response, their potential decreased, but superoxide levels remained similar to naive cells. One of the consequences of reduced mitochondrial potential is membrane permeability and subsequent caspase activation. We examined both the enzymatic activity and levels of cleaved caspase 3, an effector caspase, and could only detect increased levels in Ag-specific CD8+ T cells on day 5 postinfection, a time point in which virus was still present. This contrasts with Ag-specific effector cells examined during the contraction phase that had no detectable caspase activity directly ex vivo. These data suggest that the apoptotic program begins earlier than previously expected on day 5, during the expansion phase.

During many viral infections, antigen-specific CD8؉ T cells undergo large-scale expansion. After viral clearance, the vast majority of effector CD8 ؉ T cells undergo apoptosis. Previous studies have implicated reactive oxygen intermediates (ROI) in lymphocyte apoptosis. The purpose of the experiments presented here was to determine the role of ROI in the expansion and contraction of CD8 ؉ T cells in vivo during a physiological response such as viral infection. Mice were infected with lymphocytic choriomeningitis virus (LCMV) and treated with Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), a metalloporphyrinmimetic compound with superoxide dismutase activity, from days 0 to 8 postinfection. At the peak of CD8؉ -T-cell response, on day 8 postinfection, the numbers of antigen-specific cells were 10-fold lower in MnTBAPtreated mice than in control mice. From days 8 to 30, a contraction phase ensued where the numbers of antigen-specific CD8 ؉ T cells declined 25-fold in vehicle-treated mice compared to a 3.5-fold decrease in MnTBAP-treated mice. Differences in contraction appeared to be due to greater proliferation in drug-treated mice. By day 38, the numbers of antigen-specific CD8؉ memory T cells were equivalent for the two groups. The administration of MnTBAP during secondary viral infection had no effect on the expansion of antigen-specific CD8 ؉ secondary effector T cells. These data suggest that ROI production is critical for the massive expansion and contraction of antigen-specific CD8 ؉ T cells during primary, but not secondary, viral infection.

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The inverse relationship between gestational age at birth and postviral respiratory morbidity suggests that infants born preterm (PT) may miss a critical developmental window of T cell maturation. Despite a continued increase in younger PT survivors with respiratory complications, we have limited understanding of normal human fetal T cell maturation, how ex utero development in premature infants may interrupt normal T cell development, and whether T cell development has an effect on infant outcomes. In our longitudinal cohort of 157 infants born between 23 and 42 weeks of gestation, we identified differences in T cells present at birth that were dependent on gestational age and differences in postnatal T cell development that predicted respiratory outcome at 1 year of age. We show that naive CD4+ T cells shift from a CD31-TNF-α+ bias in mid gestation to a CD31+IL-8+ predominance by term gestation. Former PT infants discharged with CD31+IL8+CD4+ T cells below a range similar to that of full-term born infants were at an over 3.5-fold higher risk for respiratory complications after NICU discharge. This study is the first to our knowledge to identify a pattern of normal functional T cell development in later gestation and to associate abnormal T cell development with health outcomes in infants.