On the lifespan of virgin T lymphocytes.

To study the lifespan of virgin T lymphocytes, we removed the thymus from adult female mice and then, at various times afterward, tested their ability to mount an immune response to a newly encountered Ag, the male Ag H-Y. We found that unprimed thymectomized mice were able to generate a primary response to H-Y for some time after thymectomy but lost this ability at approximately 6 mo. In contrast, mice that were primed to H-Y just after thymectomy continued to display immunological memory to H-Y for >1 year. These experiments show that primary immune responses disappear in the absence of a thymus.     

The JAM Test and its daughter P-JAM: simple tests of DNA fragmentation to measure cell death and stasis

Cytotoxic T lymphocytes, and other death-inducing agents, have at least two different ways of killing their targets: drilling holes in the target cell membrane, or triggering the targets to commit suicide. The JAM Test is a method that measures the DNA fragmentation that accompanies cell suicide. We label target cells with radioactive DNA-precursor nucleotides and harvest them onto fiberglass filters, which trap large pieces of DNA but pass smaller fragments of apoptotic cells. As a general measure of apoptosis, the JAM Test described here is faster (can be completed in 4 h [or less if labeling is done the night before]), more quantitative, easier, more sensitive, more flexible and cheaper than most other current assays of apoptosis. The P-JAM, also discussed, additionally allows for assessment of death in cells that don't fragment their DNA, and allows for assays of agents that induce cell stasis rather than death.     

Interferon-induced expression of MxA in the respiratory tract of rhesus macaques is suppressed by influenza virus replication

To determine the relationship between influenza A virus replication and innate antiviral immune responses, rhesus monkeys were given oseltamivir before influenza A/Memphis/7/01 (H1N1) challenge. We found that oseltamivir treatment significantly reduced viral replication in the trachea (p < 0.029). Further, in the trachea of both treated and untreated monkeys the mRNA levels of most innate antiviral molecules in the IFN-alphabeta pathway were dramatically increased by 24 h postinfection. However, the mRNA level of a single IFN-stimulated gene, MxA (myxovirus resistance A), the IFN-stimulated gene known to be critical in blocking influenza virus replication, was significantly lower in the tracheal lavages of untreated monkeys than in the oseltamivir-treated monkeys (p = 0.05). These results demonstrate for the first time that uncontrolled influenza A virus replication actively suppresses MxA gene expression and emphasize the critical role of innate immunity in controlling influenza virus replication in vivo.     

Exogenous IFN-alpha administration reduces influenza A virus replication in the lower respiratory tract of rhesus macaques

To determine the role of innate immune responses in controlling influenza A virus replication, rhesus macaques (RM) were administered pegylated IFN-alpha prior to virus challenge. Systemic and mucosal pegylated IFN-alpha administration induced expression of the interferon-stimulated genes (ISG) MxA and OAS in the airways. RM treated with IFN-alpha 24 hours prior to influenza virus challenge had significantly lower peak vRNA levels in the trachea compared to untreated animals. In addition to blunting viral replication, IFN-alpha treatment minimized the weight loss and spike in body temperature after influenza infection of RM. These results confirm the importance of IFN-alpha induced innate immune responses in the rapid control of influenza A virus replication in primates.     

Testing time-, ignorance-, and danger-based models of tolerance

In this study, we present data showing that tolerance to Ags in the periphery is not determined by the time at which the Ag appears, or by special properties of tissues in newborn mice or newly developing immune systems. We placed male grafts onto immunoincompetent female mice, allowed the grafts to heal for up to 5 mo, and then repopulated the recipients with fetal liver stem cells. We found that the newly arising T cells were neither tolerant nor ignorant of the grafts, but promptly rejected them, though they did not reject female grafts, nor show any signs of autoimmunity. We also found that the H-Y Ag was continuously cross-presented on host APCs, that this presentation was immunogenic, not tolerogenic, and that it depended on the continuous presence of the graft. In searching for the stimulus that might activate the host APCs, we analyzed mRNA expression with a highly sensitive real-time quantitative PCR assay. By using two different "housekeeping" molecules for comparison, we analyzed the message levels for several stress and/or inflammatory molecules in the healed grafts. We found that the long-healed grafts were not equivalent to "normal" skin because the healed grafts expressed lower levels of GAPDH. Altogether, these data suggest that acceptance vs rejection of peripheral tissues is not attributable to ignorance, timing-based tolerance, or special circulation properties of naive T cells in neonatal tissues. It is more likely attributable to an aspect of the context of Ag presentation that remains to be identified.