Given the global impact of persistent infections on the human population, it is of the utmost importance to devise strategies to noncytopathically purge tissues of infectious agents. The central nervous system (CNS) poses a unique challenge when considering such strategies, as it is an immunologically specialized compartment that contains a nonreplicative cell population. Administration of exogenously derived pathogen-specific memory T cells (referred to as adoptive immunotherapy) to mice burdened with a persistent lymphocytic choriomeningitis virus (LCMV) infection from birth results in eradication of the pathogen from all tissues, including the CNS. In this study, we sought mechanistic insights into this highly successful therapeutic approach. By monitoring the migration of traceable LCMV-specific memory CD8+ T cells after immunotherapy, it was revealed that cytotoxic T lymphocytes (CTLs) distributed widely throughout the CNS compartment early after immunotherapy, which resulted in a dramatic elevation in the activity of CNS antigen-presenting cells (APCs). Immunotherapy induced microglia activation as well as the recruitment of macrophages and dendritic cells (DCs) into the brain parenchyma. However, DCs emerged as the only CNS APC population capable of inducing memory CTLs to preferentially produce the antiviral cytokine tumor necrosis factor-α, a cytokine demonstrated to be required for successful immunotherapeutic clearance. DCs were also found to be an essential element of the immunotherapeutic process because in their absence, memory T cells failed to undergo secondary expansion, and viral clearance was not attained in the CNS. These experiments underscore the importance of DCs in the immunotherapeutic clearance of a persistent viral infection and suggest that strategies to elevate the activation/migration of DCs (especially within the CNS) may facilitate pathogen clearance.

Although both self- and pathogen-specific T cells can participate in tissue destruction, recent studies have proposed that after viral infection, bystander T cells of an irrelevant specificity can bypass peptide-MHC restriction and contribute to undesired immunopathological consequences. To evaluate the importance of this mechanism of immunopathogenesis, we determined the relative contributions of Ag-specific and bystander CD8+ T cells to the development of CNS disease. Using lymphocytic choriomeningitis virus (LCMV) as a stimulus for T cell recruitment into the CNS, we demonstrate that bystander CD8+ T cells with an activated surface phenotype can indeed be recruited into the CNS over a chronic time window. These cells become anatomically positioned in the CNS parenchyma, and a fraction aberrantly acquires the capacity to produce the effector cytokine, IFN-γ. However, when directly compared with their virus-specific counterparts, the contribution of bystander T cells to CNS damage was insignificant in nature (even when specifically activated). Although bystander T cells alone failed to cause tissue injury, transferring as few as 1000 naive LCMV-specific CD8+ T cells into a restricted repertoire containing only bystander T cells was sufficient to induce immune-mediated pathology and reconstitute a fatal CNS disease. These studies underscore the importance of specific T cells in the development of immunopathology and subsequent disease. Because of highly restrictive constraints imposed by the host, it is more likely that specific, rather than nonspecific, bystander T cells are the active participants in T cell-mediated diseases that afflict humans.

Summary DM catalyses class II‐associated invariant chain peptide (CLIP) release, edits the repertoire of peptides bound to major histocompatibility complex (MHC) class II molecules, affects class II structure, and thereby modulates binding of conformation‐sensitive anti‐class II antibodies. Here, we investigate the ability of DM to enhance the cell surface binding of monomorphic antibodies. We show that this enhancement reflects increases in cell surface class II expression and total cellular abundance, but notably these effects are selective for particular alleles. Evidence from analysis of cellular class II levels after cycloheximide treatment and from pulse‐chase experiments indicates that DM increases the half‐life of affected alleles. Unexpectedly, the pulse‐chase experiments also revealed an early effect of DM on assembly of these alleles. The allelically variant feature that correlates with susceptibility to these DM effects is low affinity for CLIP; DM‐dependent changes in abundance are reduced by invariant chain (CLIP) mutants that enhance CLIP binding to class II. We found evidence that DM mediates rescue of peptide‐receptive molecules from inactive forms in vitro and evidence suggesting that a similar process occurs in cells. Thus, multiple mechanisms, operating along the biosynthetic pathway of class II molecules, contribute to DM‐mediated increases in the abundance of low‐CLIP‐affinity alleles.

IntroductionInflammation is a highly regulated physiologic response that has evolved as a mechanism to respond to infection as well as to promote healing in settings such as tissue injury. CD4 ϩ regulatory T cells (T reg ) are observed at sites of acute and chronic inflammation, 1,2 raising questions as to the molecular basis of their accumulation at these sites. Initial evidence suggested that immunosuppressive activities of T reg are diminished in the presence of inflammatory signals. 3,4 However, in several studies, the apparently opposite picture emerged, wherein T reg exposed to inflammatory signals retain potent suppressive activity. For instance, murine T reg at sites of viral infection or isolated from inflamed tissues still mediate regulatory function, 5-7 as do human T reg isolated from rheumatoid joints or inflamed colonic mucosa. 8,9 Collectively, these results point to the possibility that certain signals associated with inflammation might promote T reg activity. Here we unexpectedly identified serum amyloid A (SAA), an acute indicator of inflammation, as a novel factor that induces cellular and cytokine conditions to support the expansion of T reg while maintaining their suppressive capacity. MethodsIn vivo effects of SAA on T reg proliferation C57BL6/J mice (male, 8-10 weeks old) were purchased from Jackson Laboratory. Mice were injected intraperitoneally with recombinant human SAA (Peprotech, 30 g in 100 l PBS), purified human serum albumin (Sigma-Aldrich, 30 g in 100 l PBS), or endotoxin (Sigma-Aldrich, 0.25 ng in 100 l PBS). Animals were killed 16 hours later; peritoneal cells were harvested and stained for surface and intracellular markers to detect T reg frequency and proliferation. In vivo depletion of monocytes was performed with clodronate liposomes (Encapsula). In these experiments, 400 L of clodronate or empty liposomes were injected intraperitoneally 24 hours before SAA injection. Flow cytometry and ELISADetection of surface markers and intracellular molecules was performed. Antibodies to mouse and human proteins used in these experiments are purchased from Biolegend except for anti-human formyl peptide receptor like-1 (FPRL-1; R&D Systems), anti-human RAGE (receptor of advanced glycation end products), supressor of cytokine signaling 3 (SOCS3; Abcam), and anti-Ki-67, anti-human-pAKT (phosphonylated protein kinase B), pERK1/2 (phosphorylated extracellular signal regulated kinases 1 and 2; BD Biosciences). For in vitro experiments, the relevant subset was labeled with CFSE before suppression assays. Cells were pelleted at various time points and underwent standard staining protocols of the manufacturers. For in vivo experiments, cells were harvested from peritoneal cavity and underwent flow cytometric analysis.To detect cytokines in the plasma, cytometric bead arrays (BD Biosciences) and ELISA (R&D Systems) were used according to manufacturers' protocols. Human plasma preparationThe study was approved by the Institutional Review Board at Stanford University. All subjects provided inform...

Once a virus infection establishes persistence in the central nervous system (CNS), it is especially difficult to eliminate from this specialized compartment. Therefore, it is of the utmost importance to fully understand scenarios during which a persisting virus is ultimately purged from the CNS by the adaptive immune system. Such a scenario can be found following infection of adult mice with an immunosuppressive variant of lymphocytic choriomeningitis virus (LCMV) referred to as clone 13. In this study we demonstrate that following intravenous inoculation, clone 13 rapidly infected peripheral tissues within one week, but more slowly inundated the entire brain parenchyma over the course of a month. During the establishment of persistence, we observed that genetically tagged LCMV-specific cytotoxic T lymphocytes (CTL) progressively lost function; however, the severity of this loss in the CNS was never as substantial as that observed in the periphery. One of the most impressive features of this model system is that the peripheral T cell response eventually regains functionality at ~60-80 days post-infection, and this was associated with a rapid decline in virus from the periphery. Coincident with this "reanimation phase" was a massive influx of CD4 T and B cells into the CNS and a dramatic reduction in viral distribution. In fact, olfactory bulb neurons served as the last refuge for the persisting virus, which was ultimately purged from the CNS within 200 days post-infection. These data indicate that a functionally revived immune response can prevail over a virus that establishes widespread presence both in the periphery and brain parenchyma, and that therapeutic enhancement of an existing response could serve as an effective means to thwart long term CNS persistence.