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

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

DataSheet_1_Human gut microbiota-reactive DP8α regulatory T cells, signature and related emerging functions.pdf

In mice, microbiota-induced Tregs both maintain intestinal homeostasis and provide resistance to immuno-pathologies in the adult. Identifying their human functional counterpart therefore represents an important goal. We discovered, in the human colonic lamina propria and blood, a FoxP3-negative IL-10-secreting Treg subset, which co-expresses CD4 and CD8α (hence named DP8α) and displays a TCR-reactivity against Faecalibacterium prausnitzii, indicating a role for this symbiotic bacterium in their induction. Moreover, supporting their role in intestinal homeostasis, we previously reported both their drastic decrease in IBD patients and their protective role in vivo against intestinal inflammation, in mice. Here, we aimed at identifying the genomic, phenotypic and functional signatures of these microbiota-induced Tregs, towards delineating their physiological role(s) and clinical potential. Human F. prausnitzii-reactive DP8α Treg clones were derived from both the colonic lamina propria and blood. RNA-sequencing, flow cytometry and functional assays were performed to characterize their response upon activation and compare them to donor- and tissue-matched FoxP3+ Treg clones. DP8α Tregs exhibited a unique mixed Tr1-like/cytotoxic CD4+ T cell-profile and shared the RORγt and MAF master genes with mouse gut microbiota-induced FoxP3+ Tregs. We revealed their potent cytotoxic, chemotactic and IgA-promoting abilities, which were confirmed using in vitro assays. Therefore, besides their induction by a Clostridium bacterium, DP8α Tregs also partake master genes with mouse microbiota-induced Tregs. The present identification of their complete signature and novel functional properties, should be key in delineating the in vivo roles and therapeutic applications of these unique human microbiota-induced Tregs through their study in pathological contexts, particularly in inflammatory bowel diseases.</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

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

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

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