Comparison of caudal vein and retro orbital injections.

<p> After classical busulfan pre-conditioning (2x25 mg/Kg), female mice received 1000 non manipulated CD34+ cells by i.v. injection either in the caudal vein or retro orbital sinus. Their bone marrow were analyzed 8 weeks later for <b>A.</b> total huCD45 chimerism, <b>B.</b> huCFU content. Each mouse represented by (●), Median represented by (─).</p

Impact of gender on human cells engraftment.

<p>After classical busulfan pre-conditioning (2x25 mg/Kg), female and male mice received 100, 500 or 1000 non manipulated CD34+ cells by i.v. injection in the retro orbital sinus. Their bone marrow were analyzed 8 weeks later for <b>A.</b> total huCD45 chimerism, <b>B.</b> huCFU content. Each mouse represented by ● (female) or ○ (male), Median represented by (─).</p

Impact on engraftment level of time delay between mice pre-conditioning and cell injections.

<p>Female mice received 2 doses of busulfan (25mg/Kg each) at 24 hours, 4 days or 7 days intervals. Mice received 1000 non manipulated CD34+ cells by caudal injection and their bone marrow were analyzed 8 weeks later for <b>A.</b> total huCD45 chimerism, <b>B.</b> B lymphoid (▪) versus myeloid (□) cells ration in CD45+ cells, and <b>C.</b> huCFUs content per femur. Each mouse represented by (●), Median represented by (─).</p

γδ T cell recovery rescues CD3ε^−/− mice from MCMV-induced death.

<p><b>A.</b> Bone marrows (BM) from TCRδ^−/−, TCRα^−/−, CD3ε^+/− and CD3ε^−/− mice (10 of each) were transferred into CD3ε^−/− recipient mice at day 0 (1 donor BM/recipient). At days 15, 30, 60 and 90, blood samples were collected (5 for each grafted mouse line) in order to follow αβ/γδ T cell reconstitution in peripheral blood. The evolution of the proportions of αβ T cells in CD3ε^+/− > CD3ε^−/− and TCRδ^−/− > CD3ε^−/− mice are shown (top) as well as the evolution of γδ T cells in CD3ε^+/− > CD3ε^−/− and TCRα^−/− > CD3ε^−/− mice (bottom). Results are expressed as percentages among peripheral blood lymphocytes ± SD. <b>B.</b> Three months post-graft, CD3ε^+/− > CD3ε^−/−, TCRα^−/− > CD3ε^−/−, TCRδ^−/− > CD3ε^−/− and CD3ε^−/− > CD3ε^−/− mice (10 of each) were infected i.p. with 2.10³ PFU of MCMV and monitored daily for mortality. This experiment was repeated twice with concordant results. <b>C.</b> γδ T cells from uninfected or 14-days infected TCRα^−/− mice were purified and i.v. transferred (8–9.10⁵ cells, 92–93% purity) into CD3ε^−/− mice (8–9 recipients). 24h after transfer, reconstituted CD3ε^−/− mice were challenged with 2.10³ PFU of MCMV and monitored daily for mortality. 7 untransferred CD3ε^−/− were used as controls. This experiment was repeated twice.</p

MCMV dissemination in lungs, spleen, liver, intestine and salivary glands from T cell competent and T cell deficient mice.

<p>TCRδ^−/−, TCRα^−/−, CD3ε^+/−, and CD3ε^−/− mice were infected i.p. with 2.10³ PFU of MCMV. At indicated days post-infection, 4 mice of each mouse line were dissected and MCMV gB was quantified in organs by real time PCR. The experiment was repeated 3 times under similar conditions. Histograms represent means of MCMV DNA copy number (per 100 ng genomic DNA) ± SD of all mice from the three experiments (n = 4x3 mice). Statistical differences between viral loads in TCRα^−/− versus CD3ε^+/− mice, and in TCRα^−/− versus CD3ε^−/− mice are shown.</p

Mobilization of γδ T cells in MCMV-infected organs from TCRα^−/− mice.

<p>TCRα^−/− mice were infected i.p. with 2.10³ PFU of MCMV. At indicated post-infection days, 5–9 mice were sacrificed, immune cells prepared from each organ and γδ T cells stained as shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004702#ppat.1004702.s002" target="_blank">S2 Fig.</a><b>A.</b> Kinetics of absolute γδ T cell numbers determined as described in methods. Presented data are mean ± SEM of 8–9 mice from one representative of 2 experiments. <b>B</b>. CD62L and CD44 expression by lymphocytes was evaluated by flow cytometry, with the presented gating strategy (lungs shown as example). <b>C.</b> Longitudinal analysis of γδ T cell phenotype in all organs. Results are pooled from 2 independent experiments representing a total of 13–14 mice (means ± SEM). Statistical differences of cell numbers and percentages between day 3 and other time points are shown, as well as statistical differences between days 0 and 56 (solid line).</p

NK-independent antiviral protective effect of γδ T cells.

<p><b>A.</b> TCRα^−/− and CD3ε^−/− mice were uninfected (Day 0) or infected i.p. with 2.10³ PFU of MCMV. At indicated days post-infection, 5–6 mice were sacrificed and immune cells were isolated from each organ for flow cytometry analysis. Absolute numbers of NK cells were calculated as described in methods. Black and white symbols represent individual TCRα^−/− and CD3ε^−/− mice respectively, and horizontal lines represent the mean of 10–12 mice pooled from 2 independent experiments. Differences were evaluated using the Mann-Whitney test: * = p<0,05, ** = p<0,01, *** = p<0,001. <b>B.</b> γδ T cells from 14-days infected TCRα^−/− mice were purified and i.v. transferred (1.10⁶ cells, 97% purity) into Rag^−/−γc^−/− mice (10 recipients). 24h after transfer, reconstituted Rag^−/−γc^−/− mice were challenged with 2.10³ PFU of MCMV and monitored daily for mortality. 5 untransferred Rag^−/−γc^−/− mice were used as controls. Results are from one representative of 2 independent experiments. <b>C.</b> Left: flow cytometry analysis of live (7AAD⁻) CD3ε⁺panγδ⁺ T cells (upper panels) and NKp46⁺NK1.1⁺ cells (lower panels) in splenocytes from TCRα^−/−donors, before (-) and after (+) purification of γδ T cells as described in methods. Right: At day 56 post-infection of Rag^−/−γc^−/− recipients, 3 mice were sacrificed; organs removed and immune cells isolated for flow cytometry analysis of γδ and NK cells. Results are from one representative mouse.</p

Both Vγ1 and Vγ4 subset are involved in γδ T cell response to MCMV.

<p>TCRα^−/− mice were infected i.p. with 2.10³ PFU of MCMV. At indicated days post-infection, 5–9 mice were sacrificed and immune cells were prepared from each organ. Expression of Vγ1, Vγ4 and Vγ7 chains by lymphocytes was evaluated by flow cytometry (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004702#ppat.1004702.s002" target="_blank">S2 Fig.</a>). <b>A.</b> Kinetics of absolute cell numbers for each subset. Presented data are mean ± SEM of 8–9 mice from one representative of 2 experiments. <b>B</b>. CD62L and CD44 expression by γδ T cell subsets was evaluated by flow cytometry in all organs. Results are pooled from 2 independent experiments representing a total of 13–14 mice (means ± SEM). Statistical differences of cell numbers and percentages between day 3 and other time points are shown, as well as statistical differences between days 0 and 56 (solid line).</p

γδ T cell control of MCMV infection is associated with reduced organ damage.

<p><b>A.</b> TCRδ^−/−, TCRα^−/−, CD3ε^+/− and CD3ε^−/− mice were infected i.p. as indicated in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004702#ppat.1004702.g001" target="_blank">Fig. 1A</a>. 3 mice/group were bled at days 0, 3, 7, 15, 21 and just before death for biochemical analyses of AST and ALT in serums. The experiment was repeated twice and data obtained for one representative mouse/group are shown. † death of CD3ε^−/−. <b>B.</b> TCRα^−/− and CD3ε^−/− mice were uninfected, or i.p. infected with 2.10³ pfu of MCMV. Uninfected and Day 22-infected mice were sacrificed and the liver and lungs were embedded in paraffin for HES staining. Apoptotic hepatocytes are shown (arrowheads). Scale bar = 200 mm. Magnifications are indicated in the right-hand side of the figure. The data are from one representative of 3 mice for each condition.</p