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

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

Expression of NFκB Responsive Genes Following TNFα Stimulation.

<p>Measures of gene expression were performed by quantitative RT-PCR for transcription factors <i>nfκb2</i> (p105) (<b>A</b>), <i>nfκb1</i> (p100) (<b>B</b>), <i>relB</i>(<b>C</b>), inhibitors of the NFκB pathway <i>iκbα</i> (<b>D</b>) and <i>a2 0</i>(<b>E</b>), and three prototypic responsive genes, inducible nitric oxide synthase (<i>inos</i>) (<b>F</b>), cyclooxygenase 2 (<i>cox2</i>) (<b>G</b>), and interleukin-6 (<i>il-6</i>) (<b>H</b>). Graph shows changes in expression relative to WT 0 (no TNFα) following stimulation of RPE cells from WT or KO (L7M1, L2) mice with TNFα (10 ng/ml). The response was normalized to ARBP for each sample. Data shown in A, D, G, and H are the mean (± SEM) of three independent experiments performed in triplicate. Data shown in B, C, E and F are the mean (± SEM) of two independent experiments performed in triplicate. Two-way ANOVA results are shown in each panel for (S) strain, (T) time post-TNFα, and (SxT) interaction. One-way ANOVA was performed for each strain over time to determine if there was a significant treatment effect. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056187#s3" target="_blank">Results</a> of post-hoc comparisons showing significant difference to no TNFα are indicated by * (p<0.05). (<b>I</b>) Secreted IL-6 was measured in culture media by ELISA. (a) IL-6 content was measured 48 hours after either a media change (no TNF) or a single dose of TNFα (1X TNF). (b) IL-6 content was measured after either a media change (no TNF), a single dose of TNFα followed by a media change (wash out) 48 hrs later (1X WO), or a single dose of TNFα followed by a second dose of TNFα (2X TNF) 48 hrs later. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056187#s3" target="_blank">Results</a> of one-way ANOVA comparing each cell line per treatment indicates significant differences by # (p<0.05). Data shown are the mean (± SEM) of three independent experiments run in triplicate.</p

Characterization of Murine RPE Cell Lines.

<p>(<b>A</b>) Gels show RT-PCR products for pigment epithelium-derived factor (PEDF), bestrophin1 (Best1), and βactin in cultured RPE isolated from WT, L7M1, and L2 mice. (R) mouse RPE (positive control), (M) Muscle, and (L) lens, negative controls, (N) no template control. (<b>B</b>) Western blots showing reaction for RPE-65 and βactin in cultured RPE cells (50 µg per lane). (C) Reaction from RPE harvested from mice (positive control). (<b>C</b>) Western blots showing reactions for proteasome subunits from cultured cells (RPE culture) and RPE harvested from mice, (RPE in vivo). Protein loads were 5 µg per lane for α7, β1, and β5. Protein loads for LMP2 and LMP7 were 15 µg for cultured RPE and 25 µg for RPE tissue harvested from mice. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. The 20 s reaction was used as a positive control. (<b>D</b>)Summary of proteasome subunit content measured from Western blots at baseline (no TNFα) in RPE culture (cc) (n = 2 cell lines/group) and in vivo RPE harvested from mice (iv) (n = 3 mice/group). *p = 0.01, **p = 0.004. (<b>E</b>) Flow cytometry of phagocytosed 1 µm YG-labeled latex beads. Bars represent the number of beads internalized by individual cells. Graph summarizes the percent cells containing 1 or more beads. Data shown are representative of two independent experiments. (<b>F</b>) Proteasomal catalytic activity in WT and i-proteasome-deficient mice (L7M1 and L2). Hydrolysis of the fluorogenic peptides LLVY-AMC, VGR-AMC, and LLE-AMC measured the chymotrypsin-like (Chymo), trypsin-like (Trypsin), and caspase-like (Caspase) activities, respectively. (<b>G</b>) Summary of proteasome subunit content measured from Western blots at baseline (no TNFα) and after TNFα (10 ng/mL) stimulation. Subunit immune reactions were normalized to a standard sample run on each blot and to the total proteasome content for each preparation. The content of each subunit is shown relative to the reaction at baseline. Data shown are the mean (±SEM) of three independent experiments.</p

and activation of p105/p50 and p100/p52.

<p>(<b>A,C</b>) Protein content of transcription factor precursor p105 and its active form, p50 (A) and precursor p100 and its active form, p52, were measured from Western blots using cytoplasmic fractions from cells harvested without TNFα or after 30 min and 3 hrs of TNFα stimulation. TNFα treated HeLa cells (+) were used as a positive control. Immune reactions were normalized to a standard sample (std) run on each blot. Protein loads were 10 µg per lane. Graphs show the mean (± SEM) of six independent experiments from n = 2 cell lines/group. (<b>B,D</b>) Transcription factors p50 (B) and p52 (D) activation were monitored using the Trans-Am NFκB Transcription Factor assay kit and the nuclear fraction from cells harvested before and after TNFα stimulation. Graphs show the mean (± SEM) of five independent experiments done in duplicate. (Data are from n = 2 cell lines/group) Two-way ANOVA results are shown in each panel for (S) strain, (T) time post TNFα and (SxT) interaction. One-way ANOVA was performed for each strain over time to determine if there was a significant treatment effect. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056187#s3" target="_blank">Results</a> of post-hoc comparisons showing significant difference with no TNFα are indicated by * (p<0.05).</p