Regulation of CD8+ T Cell Responses to Retinal Antigen by Local FoxP3+ Regulatory T Cells

While pathogenic CD4 T cells are well known mediators of autoimmune uveoretinitis, CD8 T cells can also be uveitogenic. Since preliminary studies indicated that C57BL/6 mice were minimally susceptible to autoimmune uveoretinitis induction by CD8 T cells, the basis of the retinal disease resistance was sought. Mice that express β-galactosidase (βgal) on a retina-specific promoter (arrβgal mice) were backcrossed to mice expressing green fluorescent protein (GFP) and diphtheria toxin (DTx) receptor (DTR) under control of the Foxp3 promoter (Foxp3-DTR/GFP mice), and to T cell receptor transgenic mice that produce βgal-specific CD8 T cells (BG1 mice). These mice were used to explore the role of regulatory T cells in the resistance to retinal autoimmune disease. Experiments with T cells from double transgenic BG1 × Foxp3-DTR/GFP mice transferred into Foxp3-DTR/GFP × arrβgal mice confirmed that the retina was well protected from attempts to induce disease by adoptive transfer of activated BG1 T cells. The successful induction of retinal disease following unilateral intraocular administration of DTx to deplete regulatory T cells showed that the protective activity was dependent on local, toxin-sensitive regulatory T cells; the opposite, untreated eye remained disease-free. Although there were very few Foxp3+ regulatory T cells in the parenchyma of quiescent retina, and they did not accumulate in retina, their depletion by local toxin administration led to disease susceptibility. We propose that these regulatory T cells modulate the pathogenic activity of βgal-specific CD8 T cells in the retinas of arrβgal mice on a local basis, allowing immuno regulation to be responsive to local conditions

Viral Sequestration of Antigen Subverts Cross Presentation to CD8+ T Cells

Virus-specific CD8+ T cells (TCD8+) are initially triggered by peptide-MHC Class I complexes on the surface of professional antigen presenting cells (pAPC). Peptide-MHC complexes are produced by two spatially distinct pathways during virus infection. Endogenous antigens synthesized within virus-infected pAPC are presented via the direct-presentation pathway. Many viruses have developed strategies to subvert direct presentation. When direct presentation is blocked, the cross-presentation pathway, in which antigen is transferred from virus-infected cells to uninfected pAPC, is thought to compensate and allow the generation of effector TCD8+. Direct presentation of vaccinia virus (VACV) antigens driven by late promoters does not occur, as an abortive infection of pAPC prevents production of these late antigens. This lack of direct presentation results in a greatly diminished or ablated TCD8+ response to late antigens. We demonstrate that late poxvirus antigens do not enter the cross-presentation pathway, even when identical antigens driven by early promoters access this pathway efficiently. The mechanism mediating this novel means of viral modulation of antigen presentation involves the sequestration of late antigens within virus factories. Early antigens and cellular antigens are cross-presented from virus-infected cells, as are late antigens that are targeted to compartments outside of the virus factories. This virus-mediated blockade specifically targets the cross-presentation pathway, since late antigen that is not cross-presented efficiently enters the MHC Class II presentation pathway. These data are the first to describe an evasion mechanism employed by pathogens to prevent entry into the cross-presentation pathway. In the absence of direct presentation, this evasion mechanism leads to a complete ablation of the TCD8+ response and a potential replicative advantage for the virus. Such mechanisms of viral modulation of antigen presentation must also be taken into account during the rational design of antiviral vaccines

Presented at the 1996 annual meeting of the Association for Research and Vision in Ophthalmology

Purpose. This study was designed to determine whether Alamar blue could be used to evaluate corneal endothelial cell viability in vitro. Methods. Alamar blue incorporates a proprietary redox indicator that changes color in response to metabolic activity. Primary rabbit endothelial cells were subcultured on 96-well plates at densities ranging from 1,250 to 40,000 cells per well. After 12 hours' incubation, Alamar blue was added to each well and absorbance measured hourly from 1 to 9 hours. Sodium azide-killed cells were used as a control. Alamar blue conversion was also compared with Conclusions. Alamar blue reduction measures endothelial cell viability and can readily differentiate cell concentrations. It demonstrates several advantages over [ 3 H]-thymidine: It can assay nonproliferating endothelial cell metabolism, it allows rapid assessment of large numbers of samples, it can differentiate endothelial cell concentrations, it is nontoxic, it is nonradioactive and allows for simple disposal, it is less cosdy, and it allows for continuous monitoring of endothelial cell metabolism and viability. Invest Ophthalmol Vis Sci. 1997; 38:1929-1933 L he ability to measure corneal endothelial cell viability in vitro accurately is important for applications that include measuring the toxicologic effects of intraocular drugs and irrigating solutions and basic physiologic studies of endothelial cell metabolism. Corneal endothelial cell viability has special importance in eye banks, for which assessment of cell viability is crucial to the development of storage media for donor corneas. The ideal method of measuring corneal endothelial cell viability would not be toxic to the endothelial cell, allowing continuous or repeated measurements; would have a range of sensitivity that allows measurement of degrees of viability; would not be de

Immunoproteasome deficiency modifies the alternative pathway of NFκB signaling.

Immunoproteasome is a protease abundant in immune cells and also present, albeit at lower concentrations, in cells outside the immune system. Recent evidence supports a novel role for the immunoproteasome in the cellular stress response potentially through regulation of NFκB signaling, which is the primary response to multiple stressors. The current study tests whether the Classical or Alternative Pathways are regulated by immunoproteasome following chronic TNFα exposure in cultured retinal pigment epithelial cells isolated from wild-type mice and mice deficient in one (LMP2, L2) or two (LMP7 and MECL-1, L7M1) immunoproteasome subunits. Assays were performed to assess the expression of NFκB responsive genes, the content and activity of NFκB transcription factors (p65, p50, p52, cRel, RelB), and expression and content of regulatory proteins (IκBα, A20, RPS3). Major findings include distinct differences in expression of NFκB responsive genes in both KO cells. The mechanism responsible for the altered gene expression could not be established for L7M1 since no major differences in NFκB transcription factor content or activation were observed. However, L2 cells exhibited substantially higher content and diminished activation of NFκB transcription factors associated with the Alternative Pathway and delayed termination of the Classical Pathway. These results provide strong experimental evidence supporting a role for immunoproteasome in modulating NFκB signaling