Full Restoration of <em>Brucella</em>-Infected Dendritic Cell Functionality through Vγ9Vδ2 T Helper Type 1 Crosstalk

<div><p>Vγ9Vδ2 T cells play an important role in the immune response to infectious agents but the mechanisms contributing to this immune process remain to be better characterized. Following their activation, Vγ9Vδ2 T cells develop cytotoxic activity against infected cells, secrete large amounts of cytokines and influence the function of other effectors of immunity, notably cells playing a key role in the initiation of the adaptive immune response such as dendritic cells. <em>Brucella</em> infection dramatically impairs dendritic cell maturation and their capacity to present antigens to T cells. Herein, we investigated whether V T cells have the ability to restore the full functional capacities of <em>Brucella</em>-infected dendritic cells. Using an in vitro multicellular infection model, we showed that: 1/<em>Brucella</em>-infected dendritic cells activate Vγ9Vδ2 T cells through contact-dependent mechanisms, 2/activated Vγ9Vδ2 T cells induce full differentiation into IL-12 producing cells of <em>Brucella</em>-infected dendritic cells with functional antigen presentation activity. Furthermore, phosphoantigen-activated Vγ9Vδ2 T cells also play a role in triggering the maturation process of dendritic cells already infected for 24 h. This suggests that activated Vγ9Vδ2 T cells could be used to modulate the outcome of infectious diseases by promoting an adjuvant effect in dendritic cell-based cellular therapies.</p> </div

<i>Brucella</i>-infected DCs induce Vγ9Vδ2 T cells to produce IFN-γ even when they are added 24 h p.i.

<p><i>Brucella</i>-infected DCs (MOI = 20) were cultured alone or in the presence of untreated or stimulated (0.2 nM HMB-PP) Vγ9Vδ2 T cells. Vγ9Vδ2 T cells were added to DCs 1 h or 24 h p.i. At 24 h, 48 h and 72 h p.i., supernatants were collected. IFN-γ was assessed by ELISA test. Data shown are the mean +/− SD of triplicates and representative of three independent experiments performed with cells from different donor. A significant difference between infected DCs in the presence or not of Vγ9Vδ2 T cells was calculated by using Student’s t test and is indicated by (**) where p<0.01 and (***) where p<0.001.</p

Cell-cell contacts between Vγ9Vδ2 T cells and infected DCs are required for maturation process.

<p><i>Brucella</i>-infected DCs (MOI = 20) were cocultured in contact (black bars) or separated by a trans-well chambers (insert, white bars) with Vγ9Vδ2 T cells in the presence or not of HMB-PP (0.2 nM). At 48 h p.i., supernatants were collected and cells were stained. CD83 (A, left panel) and CD86 (B, left panel) expression analyses were realized on CD1a+ cells by flow cytometry. Data shown are the mean +/− SD of three independent experiments performed with cells from different donors. Representative flow cytometry histograms show the expression of CD83 (A, right panel) and CD86 (B, right panel) of infected DCs alone (filled grey), cocultured in contact (grey line) or separated (black line) with HMB- or not- stimulated Vγ9Vδ2 T cells. IL-12 was assessed by ELISA test in the collected supernatants (C). Data shown are the mean +/− SD of triplicates and are representative of the three experiments. A significant difference between infected DCs in the presence or not of Vγ9Vδ2 T cells was calculated by using Student’s t test and is indicated by (*) where p<0.05 and (**) where p<0.01. Significant differences between infected DCs in contact or not with Vγ9Vδ2 T cells and between HMB- or not- stimulated Vγ9Vδ2 T cells are indicated directly on the graph.</p

Role of IFN-γ and TNF-α on DC maturation induced by Vγ9Vδ2 T cells.

<p>DCs (DC), infected DCs (inf DC) and infected DCs in the presence of Vγ9Vδ2 T cells (infDCs+γδT) were cultured with neutralizing mAbs to IFN-γ (white bars), TNF-α (striped white bars) or with isotype control Ab (black bars). At 48 h p.i., supernatants were collected and cells were stained with FITC-conjugated mAbs to CD83 (A), CD86 (B). CD83 and CD86 expression analysis were realized on CD1a+ cells by flow cytometry. IL-12 was assessed by ELISA in the collected supernatants (C). Data shown are the mean +/− SD of triplicates and are representative of the three experiments. The Student’s <i>t</i> test was used to calculate significant differences between: - 1: infected DCs in the presence or not of Vγ9Vδ2 T cells and was indicated by (*) where <i>p</i><0. 05 and (**) where <i>p</i><0.01 and 2: infected DCs cocultured with Vγ9Vδ2 T cells in the presence of neutralizing mAb or isotype control and is directly indicated on the graph.</p

Vγ9Vδ2 T cells induce full maturation of <i>Brucella</i>-infected DCs. Non- or <i>Brucella</i>-infected DCs (MOI = 2, 5, 20, and 50) were cocultured with Vγ9Vδ2 T cells.

<p>At 48 h p.i., supernatants were collected and cells were stained with FITC-conjugated mAbs to CD83 (A), CD86 (B). CD83 and CD86 expression analyses were realized on CD1a+ cells by flow cytometry and the results were expressed as percentage of positive cells for CD83 and mean fluorescence intensity (MFI) for CD86. Data shown are the mean +/− SD of three independent experiments performed with cells from different donors. IL-12 was assessed by ELISA test in the collected supernatants (C). Data shown are the mean +/− SD of triplicates and are representative of three experiments. A significant difference between infected DCs in the presence or not of Vγ9Vδ2 T cells was calculated by using Student’s t test and is indicated by (*) where p<0.05 and (**) where p<0.01.</p

Activated Vγ9Vδ2 T cells are still able to induce maturation of <i>Brucella</i>-infected DCs even when they are added 24 h p.i.

<p>A/<i>Brucella</i>-infected DCs (MOI = 20) were cultured alone or in the presence of stimulated (0.2 nM HMB-PP) Vγ9Vδ2 T cells. Vγ9Vδ2 T cells were added to DCs 1 h or 24 h p.i. At 24 h, 48 h and 72 h p.i., supernatants were collected and cells were stained. CD83 and CD86 expression analysis were realized on CD1a+ cells. IL-12 was assessed by ELISA test in the collected supernatants. Data shown are the mean +/− SD of triplicates and representative of three independent experiments performed with cells from different donor. A significant difference between infected DCs in the presence or not of Vγ9Vδ2 T cells was calculated by using Student’s t test and is indicated by (*) where p<0. 05 and (**) where p<0.01. (B) <i>Brucella</i>-infected DCs (MOI = 20) were cultured alone or in the presence of stimulated (0.2 nM HMB-PP) Vγ9Vδ2 T cells. Vγ9Vδ2 T cells were added either 1 h or 24 h p.i. In all cases, CFSE-stained naive CD4+ T cells were added 24 h p.i. with DC/T cell ratio 0.1. After 5 days, CD4+ T cell proliferation was analyzed by flow cytometry and the proliferation index was calculated by comparison with non-infected cells. Data shown are the mean of duplicates +/− SD of three independent experiments performed with cells from different donors. Statistical difference is calculated by comparison with <i>Brucella</i>-infected DCs by using wilcoxon rank test and is indicated by (*) where p<0. 05.</p

Analysis of the TCR Vb repertoire of pp65_495–502/A*0201- or BZLF1/B*3501-sorted T cell lines, using TCR Vβ-specific mAb.

<p>The percentage of pp65/A*0201- or BZLF1/B*3501-specific T cells stained by the various anti-TCR Vβ mAb is mentioned according to the IMGT nomenclature (Beckman Coulter anti-TCR Vβ name is indicated in bracket). All T cell lines were derived from PBL, either from healthy donors or from RA patients.</p>a<p>Percentage determined by TCR sequencing (ref. 28). T cell lines exhibiting alloreactivity are marked in bold.</p

Allorecognition of human endothelial cell cultures by CD8 T cell lines enriched in herpesvirus-specific T cells.

<p>T cell lines from D01 and D03, sorted with pp65_495–503/A*0201 multimers, were tested against HAEC #4373, #3315, and #3376, while T cell line from D15, sorted with BZLF1_54–64/B*3501 multimers, was tested against HAEC #3643 (Cw*0602+) and #1415 (Cw*0602-) for TNF-α production (<b>A</b>) and cytotoxicity (Effector-Target ratio 15∶1) (<b>B</b>). TNF-α production was measured as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012120#pone-0012120-g002" target="_blank">Figure 2</a> legend. Only the endothelial cell lines expressing the relevant allogeneic HLA allele induced cytotoxicity and TNF production by the CD8 T cell lines tested. The data are representative of 3 different experiments.</p

Enrichment in pp65_495–503/A*0201- or BZLF1_54–64/B*3501-specific T cells after sorting of CD8 T cells with pMHC magnetic multimers.

<p>(<b>A</b>) CD8 T cells, derived from the PBL from D01 and D03 were sorted with 245V mutated pp65_495–503/A*0201 multimers, then expanded in culture, and stained with PE-conjugated pp65_495–503/A*0201 tetramers and FITC-conjugated anti-CD3. (<b>B</b>) CD8 T cells derived from the PBL from D15 were sorted with 245V mutated BZLF1_54–64/B*3501 multimers, and then expanded in culture. Unsorted and sorted T cells were stained with PE-conjugated BZLF1/B35 tetramers and FITC-conjugated anti-CD3. The percentage of positive cells is indicated in the upper right quadrant.</p

HLA class I typing of LCL.

<p>ND : Not determined. LCL and the HLA triggering allorecognition are indicated in bold.</p