Functional properties of the M314.132 DP T cell clone.

<p>A/ Cytokine production analysis. DP T cell clone was fixed, permeabilized and stained for cytokines following autologous melanoma stimulation. Data are expressed as mean % of intracellular cytokine secreting cells. Open histograms correspond to the analysis of cytokine production by unstimulated M314.132 DP T cell clone (negative control). B/Lysis of the M314 autologous melanoma cell line (closed circles) by M314.132 DP T cell clone. The M132 cell line was used as negative control target (open circles). 51Cr-labeled tumor cells were co-cultured with T cells at various E/T ratios. Chromium release in the supernantants was measured after a 4-h incubation period. C/ Phenotypic characterization of M314 DP T cell clone. D/ Proliferation capacity. CFSE-labeled T cell clones were stimulated with anti-CD3 (OKT3). The CD8 T cell clone used as positive control was obtained by limiting dilution of melanoma specific CD8 T cells. As negative control, T cell clones were maintained in the absence of any stimulation (Not Stimulated: NS).</p

Reactivity of M314.132 DP T cell clone against normal cell lines.

<p>A/ TNF secretion by the M314.132 DP T cell clone in response to melanocytes. 10⁴ DP T cells were added to 3×10⁴ HLA-A*2402 positive or negative melanocytes, and the clone reactivity was assessed by a TNF release assy. B/ TNF secretion by the M314.132 DP T cell clone to HLA-A*2402 transfected (+) or non-transfected (−) normal cells of different origins and/or species. Results are expressed as relative reactivity to the indicated cells in comparison with TNF secretion (100%) induced by M314 autologous melanoma cells. C/ Lack of recognition of HLA-A*2402 EBV-B lymphocytes. DP T cell clone was fixed, permeabilized and stained for cytokines following stimulation with EBV-B cell lines expressing or not HLA-A*2402 molecules. Data are expressed as mean % of intracellular TNF-α secreting cells. D/ TNF response of DP T cell clone toward melanoma cells treated with or without IFN-γ. Melanoma cells were cultured in the presence or absence of 100U/ml rIFN-γ for 15 days. DP T cell clone and two CD8 T cell clones used as controls were fixed, permeabilized and stained for TNF following stimulation with untreated (white bars) or IFN-γ-treated (hatched bars) melanoma cells. Data are expressed as mean % of intracellular TNF-α secreting cells.</p

Comparison of cytokine production capacities of DP T cells with that of SP subpopulations by FACS analysis.

<p>Data are expressed as mean % of intracellular cytokine secreting cells in response to anti-CD3 stimulation (n = 5). Significance increase of cytokines production by DP T cells was evaluated by Tukey-Kramer's test. *P<0.05,***P<0.001. No cytokine production was observed by unstimulated subpopulations.</p

Distribution of T cells subsets based on CD3, CD4, CD8 amongst melanoma patients PBMC and tumor associated lymphocytes and healthy donor PBMC.

<p>Results are expressed as median fraction of cells expressing the marker(s) +/− SD among total cells. Significant differences were evaluated by comparison with similar cell fractions among melanoma cancer patient PBMC using the Tukey-Kramer's test. *P<0.05, **P<0.01, ***P<0.001.</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

Reactivity of M314.132 DP T cell clone against tumor cell lines.

<p>A and B/ TNF secretion by the M314.132 DP T cell clone in response to tumor cell lines. 10⁴ DP T cells were added to 3×10⁴ M314 melanoma cells (A) or other tumor cells (B). All tumor cell lines which are not recognized by the DP clone do not express the HLA-A*2402 and/or 2301 molecules. C/ TNF secretion by the M314.132 DP T cell clone to HLA-A*2402 transfected tumor cells. Melanoma (n = 10), breast carcinoma (n = 2), renal carcinoma (n = 1), ovarian carcinoma (n = 1), myeloma (n = 1), and glioblastoma (n = 3) cell lines were transiently transfected with 100ng of HLA-A*2402 plasmid with a lipofectamine reagent kit. 10⁴ DP T cells were added to 3×10⁴ target cells, and the DP T cell clone reactivity was assessed by a TNF release assay. Results are expressed as relative reactivity to the indicated cells in comparison with TNF secretion (100%) induced by M314 autologous melanoma cells.</p

Analysis of polyclonal DP T cells in melanoma: repertoire diversity and autologous-tumor reactivity.

<p>A/ Repertoire diversity of DP T cells populations in tumors from four melanoma patients was assessed by labeling with 24 anti-Vβ mAbs. Insets indicate the percentage of the specific population characterized with this panel. B/ Percentage of TNF-producing cells in the SP CD8 and DP sub-populations in response to the autologous melanoma cell line. The autologous tumor cells lines were established from metastatic lymph nodes of 21 melanoma patients and were tested after two at four weeks of culture initiation. 10⁶ ILNL and 2×10⁶ melanoma cells were incubated for 6 h in the presence of Brefeldin A, stained with CD4 and CD8 mAbs, fixed, and stained with anti-TNF Ab in a permeabilization buffer. 10⁵ cells were then analyzed by flow cytometry.</p

DP T cell clone selection and characterization.

<p>A/. Distribution of CD3⁺ T cells subsets based on CD4, CD8 in the M314 ILNL population. Lymphocytes were analyzed after in vitro expansion by three-color flow cytometry using antibodies specific for CD3, CD4 and CD8α or CD8β. Percentage of Vβ13.2 TCR expressing cells was examined on gated the 14% of DP T cells. The Vβ13.2 TCR expressing cells DP T cells were then sorted by FACS. B and C/ TNF secretion by the M314.132 DP T cell clone in response to the autologous melanoma cell line. 10⁴ DP T cells were added to 3×10⁴ M314 melanoma cells in the presence or not of blocking antibodies directed against class I (W6/32), B/C/A24 (B1.23.2), class II (206) HLA and against CD4 and CD8 molecules at the indicated dilutions or concentrations. DP T cell clone reactivity was assessed by a TNF release assay. D/ Time course of CD3 (white) and TCR (black) down-regulation in M314.132 DP T cell clone stimulated with autologous melanoma cell line.</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