Various deletions and mutations of MMP-2 cDNA allow MMP-2_560–568 epitope generation by the endogenous pathway in HLA-A*0201 transfected COS-7 cells.

<p>COS-7 cells were cotransfected with HLA-A*0201 plasmid and with (A) plasmids coding for deleted MMP-2 or (B) plasmids coding for mutated MMP-2. 48 h later, M134.12 CTL clones were added to transfected COS-7 cells (E/T ratio 1∶3) and the TNF response was tested after 6 h on Wehi-13 cells. Standard deviations were obtained from duplicates. cDNA NA134-A corresponding to the C-terminal part of MMP-2 contains MMP-2_560-568 epitope and was used as positive control. Transfection efficiency was controlled with GFP transfected COS-7 cells. PS corresponding to the signal sequence (pre), PD corresponding to the prodomaine (pro) and PEX corresponding to the hemopexine domaine of the MMP-2. Data are representative of at least two independent experiments. Error bars indicate standard deviations of duplicates.</p

Mutated MMP-2 proteins are more rapidly degraded than the wild-type enzyme.

<p>COS-7 cells transfected with indicated plasmids were pulse-labeled with [³⁵S] methionine/cystein for 15 min and chased for 0–24 h. MMP-2 immunoprecipitates were separated by SDS-PAGE and analyzed by autoradiography (A). Data were plotted to indicate the residual protein remaining where the amount of this protein at 0 h time point was calculated to represent 100% of total MMP-2 in each case (B). Data are representative of at least two independent experiments.</p

Hypothetical models for MMP-2_560–568 epitope generation by cross-presentation and by endogenous pathway.

<p>Cross-presentation (1): Newly synthesized wild-type MMP-2 acquire disulfide bonds in the endoplasmic reticulum (ER) before joining the secretory pathway. In the extracellular environment, physiologic activation of the pro-MMP-2 induce the cleavage of the propeptide domain which contains a disulfide bridge (C60-C65: unique to the MMP-2). MMP-2 active form then interact with the integrine αvβ3 and is internalized in clathrin-coated vesicle. Finally MMP-2 is transported to the cytosol, in an unknown mechanism, and degraded by the proteasome. Peptides generated can reach the endogenous pathway (peptides are transported in the ER through TAP, bind to HLA-A*0201 and transported to the cell surface). Endogenous presentation (2): Mutated MMP-2 lacking a disulfide bond can't join the secretory pathway and is retrotranslocated via Sec61. In the cytosol, mutated MMP-2 is degraded by the proteasome and resulting peptides are loaded on MHC class I molecules.</p

Disulfide bond deletion permit MMP-2_560–568 epitope generation by the endogenous pathway in HLA-A*0201+/αvβ3- human tumor cells.

<p>Melanoma cell line M117 and non small cell lung carcinoma line 1355 were transfected with plasmids coding for cystein deleted MMP-2. 48 h later, M134.12 CTL clones were added to tumor cells (E/T ratio 1∶3) and the TNF response was tested after 6 h on wehi-13 cells. Standard deviations were obtained from duplicates. cDNA NA134-A corresponding to the C-terminal part of MMP-2, contains MMP-2_560–568 epitope and was used as positive control. Transfection efficiency was controlled with GFP transfected tumor cells. Data are representative of at least two independent experiments. Error bars indicate standard deviations of duplicates. p<0.005 was considered significant.</p

Reactivity of HLA-E-restricted CD8 T cells against allogeneic endothelial cells.

<p>A/Surface expression of HLA-E (thick lines) and total HLA-I (dotted lines) molecules by two representative endothelial cultures (HAEC). RFI are indicated. B/Cytokine production by HLA-E-restricted CD8 T cells upon stimulation with endothelials cultures. MART.22 T cells were fixed, permeabilized and stained for intracellular TNF-α following 6 h of incubation with HAECs (thick line) or not (thin line). Data are expressed as percentage of intracellular cytokine secreting T cells upon stimulation with HAECs. C/Degranulation of HLA-E-restricted CD8 T cells upon stimulation with endothelial cultures. MART.22 T cells were incubated for 4 h with HAECs (thick line) or not (thin line) in the presence of anti-CD107a antibody. Results are expressed as percentages of surface CD107a positive T cells upon stimulation with endothelial cells.</p

Leader sequence peptides derived from HCMV-UL40/HLA-I molecules and recognition by HLA-E-restricted T cell clone.

<p>Autologous HLA class I alleles of the transplant recipient are indicated in bold.</p>a<p>MART.22 HLA-E-restricted T cell clone activity in response to.221 cells pulsed with different peptides (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050951#pone-0050951-g002" target="_blank">Figure 2</a>).</p>b<p>These peptides are identical to peptides contained in the UL40 ORF from various CMV strains.</p>c<p>These pepides have previously been described for their ability to trigger HLA-E restricted CD8 T cell responses.</p

Characterization of CMV/HLA-I-derived peptides recognized by HLA-E-restricted CD8 T cells.

<p>A/TNF production in response to stimulation with.221 cells pulsed with synthetic peptides.221 cells were incubated for 1 h with range concentrations of the indicated peptides before addition of MART.22 T cells. After 6 h, T cells were fixed, permeabilized and stained for intracellular TNF-α. Results are expressed as percentage of TNF-producing T cells. B/Peptide-MHC tetramer staining of HLA-E-restricted CD8 T cells. MART.22 T cells were incubated for 1 h with biotyniled HLA-E monomers refolded with the indicated peptides and tetramerized with PE-coupled streptavidin. Peptide-HLA-E tetramers staining was assessed by flow cytometry and RFI are indicated.</p

Regulation of HLA-E-restricted CD8 T cells reactivity against allogeneic endothelial cells by NK receptor.

<p>A/Reactivity of HLA-E-restricted T cells against poorly recognized (HAEC#116) or unrecognized (HAEC#402) endothelial cultures pulsed with synthetic peptides. HAECs were incubated for 1 h with range concentrations of the indicated peptides before MART.22 T cells were added. After 6 h, T cells were fixed, permeabilized and stained for intracellular TNF-α. Results are expressed as percentage of TNF-producing T cells. B/Impact of KIR2DL2-ligands expression by HAECs on HLA-E-restricted T cells alloreactivity. Percentages of TNF-producing MART.22 T cells are shown for HAECs with none, one or two protective HLA-C alleles. C/Reactivity of HLA-E-restricted T cells against unrecognized endothelial cultures (HAEC#402) in the presence of blocking antibodies. HAECs were incubated with MART.22 T cells in the presence or not of indicated concentrations of blocking antobodies. After 6 h, T cells were fixed, permeabilized and stained for intracellular TNF-α. Results are expressed as percentage of TNF-producing T cells.</p

Expression of NK receptors by HLA-E-restricted CD8 T cells and functional characterization.

<p>A/Surface expression of NK receptors by HLA-E-restricted CD8 T cells. RFI of stained T cells (thick line) are indicated. B/Modulation of HLA-E restricted CD8 T cells reactivity through NKR engagement. ⁵¹Cr-labeled P815 cells were preincubated with the indicated concentration of anti-CD3 antibody in the presence or not of the indicated anti-NKR antibody for 1 h. Then, MART.22 T cells were added for 4 h. Redirected cytotoxic activity was assessed through measure of Chromium release in the supernatants. Percentages of specific lysis are indicated. Means and standard deviations of triplicate wells are shown for one representative experiments out of three performed.</p

Functional characterization of HLA-E-restricted CD8 T cells.

<p>A/Induction of strong and rapid Ca²⁺ responses within activated HLA-E-restricted CD8 T cells. B-EBV 721.221 cells transfected (.221-E) or not (.221) with HLA-E and the leader sequence of HLA-B*08, were incubated with MART.22 T cells loaded with Fura-2 (1∶1 ratio). T cell intracellular Ca²⁺ levels were monitored by videomicroscopy for the indicated acquisition time. Graphs represent the kinetics of intracellular Ca2⁺ levels (340/380 nm ratio). Values correspond to the mean of emission measured among all T cells present in the field (approximatively 20 cells per experiment). Results are representative of two independent experiments. B/Degranulation of HLA-E-restricted CD8 T cells upon stimulation.221-E cells (thick line) or.221 cells (thin line) were incubated for 4 h with MART.22 T cells in the presence of anti-CD107a antibody. Results are expressed as pourcentages of surface CD107a positives T cells upon stimulation with.221-E cells. C/Cytotoxic activity of HLA-E restricted CD8 T cells. 10³⁵¹Cr-labeled.221-E cells (squares) or.221 cells (circles) were co-cultured for 4 h with MART.22 T cells at various E/T ratios. Cytotoxic activity was assessed through measure of Chromium release in the supernatants. Percentages of specific lysis are indicated. Means and standard deviations of triplicate wells are shown for one out of three comparable experiments. D/Cytokine production analysis of HLA-E restricted CD8 T cells. MART.22 T cells were fixed, permeabilized and stained for intracellular cytokines following 6 h of incubation with.221-E cells (thick line) or.221 cells (thin line). Data are expressed as mean % of intracellular cytokine secreting cells upon stimulation with.221-E cells.</p