Suppression of autoimmune disease after vaccination with autoreactive T cells that express Qa-1 peptide complexes

The ability of autoreactive T cells to provoke autoimmune disease is well documented. The finding that immunization with attenuated autoreactive T cells (T cell vaccination, or TCV) can induce T cell–dependent inhibition of autoimmune responses has opened the possibility that regulatory T cells may be harnessed to inhibit autoimmune disease. Progress in the clinical application of TCV, however, has been slow, in part because the underlying mechanism has remained clouded in uncertainty. We have investigated the molecular basis of TCV-induced disease resistance in two murine models of autoimmunity: herpes simplex virus-1 (KOS strain)–induced herpes stromal keratitis and murine autoimmune diabetes in non-obese diabetic (NOD) mice. We find that the therapeutic effects of TCV depend on activation of suppressive CD8 cells that specifically recognize Qa-1–bound peptides expressed by autoreactive CD4 cells. We clarify the molecular interaction between Qa-1 and self peptides that generates biologically active ligands capable of both inducing suppressive CD8 cells and targeting them to autoreactive CD4 cells. These studies suggest that vaccination with peptide-pulsed cells bearing the human equivalent of murine Qa-1 (HLA-E) may represent a convenient and effective clinical approach to cellular therapy of autoimmune disease

Bortezomib-induced anti-MCL activity involves oxidative stress-mediated apoptosis.

<p>(<b>A</b>) Bortezomib-induced apoptosis and its inhibition by pretreatment of cells with a ROS scavenger, N-acetyl-L-cysteine (NAC). Apoptosis was determined by Annexin-V/7-AAD dual staining method using FACS. Cells that showed positive staining for both Annexin-V/7AAD were considered apoptotic/late apoptotic. Error bars indicate the standard error of the means (SEM) from two independent experiments. Statistical significance is indicated as (*) for <i>p</i>>0.05 and (**) for <i>p</i>>0.01 as compared to the respective untreated control. (<b>B</b>) Bortezomib-induced ROS generation in MCL cells. Cells were treated for 24 h with 20 nM of bortezomib for fluorimetric assessment of a ROS-specific cell permeable probe, DCH-FDA by FACS as described in materials and methods. After DCH-FDA labeling (10 µM for 20 min/37°C), cells were washed and resuspended in PBS. Mean fluorescence intensities of control and bortezomib-treated samples were determined by FACS. Results are expressed as relative fold of increase in MFI compared to control.</p

Bortezomib treatment increases nuclear localization of BACH2 in bortezomib susceptible cells.

<p>Using ‘AMIRA’ software, confocal images of Jeko (A) and SP53 (B) cells were analyzed to quantitate the BACH2 fluorescence in the cytoplasm and nucleus of control and treated cells. Three random images were analyzed for each cell line. Cytoplasmic and nuclear gating of fluorescence was done using de-segmentation analysis of software using confocal images of both the channels (BACH2, red and Draq5, blue). Representative gated images for cytoplasmic and nuclear marking of control and bortezomib samples for each cell line were shown. Data were collected from analysis of three images and expressed as mean ± SD.</p

Bortezomib treatment did not increase nuclear localization of BACH2 in bortezomib resistant cells.

<p>Using ‘AMIRA’ software, confocal images of Mino (A) and Rec-1 (B) cells were analyzed to quantitate the BACH2 fluorescence in the cytoplasm and nucleus of control and treated cells. Three random images were analyzed for each cell line. Cytoplasmic and nuclear gating of fluorescence was done using de-segmentation analysis of software using confocal images of both the channels (BACH2, red and Draq5, blue). Representative gated images for cytoplasmic and nuclear marking of control and bortezomib samples for each cell line were shown. Data were collected from analysis of three images and expressed as mean ± SD.</p

BACH2 nuclear translocation suppresses expression of pro-survival and anti-oxidative genes in bortezomib sensitive cell lines (Jeko and SP53) but not in bortezomib resistant cell lines (Mino and Rec-1).

<p>Jeko, SP53, Mino, Rec-1 cells were treated with bortezomib (20 nM) for 24 h and total RNA was isolated by an RNA extraction kit. Purified RNA (25 ng) was used for cDNA synthesis using oligo dT primers. cDNAs of control and treated samples were used to determine the level of gene expression changes after bortezomib treatment. Expression level for each gene (<i>HO-1, MCL-1, Nrf2, CAT</i> and <i>Gss</i>) was normalized with β-actin expression. Data were expressed as mean ± SD of four experiments done in duplicates or triplicates for each gene and plotted as a relative fold of decrease or increase in bortezomib-treated samples compared to the respective gene expression in untreated controls.</p