Fig 1.
CD39 and CD73 expression on human T cells following activation.
PBMCs were stimulated with anti-CD3 mAb. Flow cytometric analysis of CD39, CD73 and CD25 on CD4+ and CD8+ T cells was performed on days 0–6. Representative concatenated dot plots for surface expression of CD39, CD73 and CD25 on CD4+ T cells (A) and CD8+ T cells (B) are shown. (C) Changes in percentages (upper panel) and MFI (lower panel) of CD39 and CD73 expression following stimulation. Mean ± SEM of three independent experiments are shown. Unpaired t test, comparison with day 0, * p<0.05, ** p<0.01, *** p<0.001.
Fig 2.
CD39 and CD73 expression profiles of human peripheral blood T-cell subsets.
(A) Gating strategy for conventional CD4+ and CD8+ T cells from human peripheral blood. CD25+CD127— Treg cells were excluded. (B) CD39 and CD73 as well as CCR7 and CD45RA expression profile of conventional CD4+ and CD8+ T cells. Naive: CCR7+CD45RA+, effector/effector memory (EM): CCR7—CD45RA—, central memory (CM): CCR7+CD45RA— and TEMRA: CCR7—CD45RA+. (C) Percentages of CD39 and CD73 expression on CD4+ and CD8+ T-cell subsets from the blood of 10 healthy donors and the means are shown. ANOVA multiple comparison, * p<0.05, ** p<0.01, *** p<0.001.
Fig 3.
CD39 and CD73 expression on CD4+ and CD8+ T cells from the synovial fluid of inflamed joints.
T cells from the peripheral blood and synovial fluid of JIA patients were analyzed for the expression of HLA-DR, CD39 and CD73. Treg cells were excluded from analysis (see Fig 2A for gating strategy). (A, B) Representative expression profiles for CD39, CD73 and HLA-DR on conventional CD4+ T cells (A) and CD8+ T cells (B) from peripheral blood (PB) and synovial fluid (SF). (C) Percentages of HLA-DR+ cells among CD4+ and CD8+ T cells in PB and SF. (D) Percentages of CD39+ cells (top) and of CD73+ cells (bottom) among HLA-DR+ and HLA-DR—subsets of CD4+ and CD8+ T cells in peripheral PB and SF. Results for blood and SF of 6 JIA patients are shown. Paired t test, * p<0.05, ** p<0.01, *** p<0.001, ns p>0.05.
Fig 4.
CD39 and CD73 expression profiles of T cells during the course of L. monocytogenes infection.
Wildtype mice were i.v. infected with 1×105 LmOVA. At indicated time points, surface expression of CD39 and CD73 was analyzed on CD4+ and CD8+ T cells from spleens. (A) Representative concatenated dot plots for CD39 and CD73 expression on CD4 and CD8-gated cells. CD39 staining was controlled with cells from CD39-/- mice, CD73 staining with an isotype control. (B) MFI of CD39 and CD73 staining on CD4 and CD8-gated cells. (C, D) Percentages of CD39+ and CD73+ cells among CD4+ (C) and CD8+ T cells (D). Shown are %-values for total, for CD62L+CD44— and for CD62L—CD44+ T-cell populations (see S1 Fig for gating strategy and cell numbers). Values in B-D give the mean ± SEM for three independently analyzed mice per time point and are representative for three independent experiments. Unpaired t test, comparison with day 0 (in C and D only shown for total T-cell populations), * p<0.05, ** p<0.01, *** p<0.001.
Fig 5.
CD39 and CD73 expression profiles of listeria-specific T cells.
Wildtype mice were i.v. infected with 1×105 LmOVA. (A-E) Spleens cells were isolated at the indicated time points and stimulated with LLO189-201 and OVA257-264 peptides for 4h, and then analyzed for expression IFN-γ and CD39 or CD73. Responding T cells were identified by intracellular IFN-γ staining. (A) Numbers of CD4+ and CD8+ T cells per spleen responding to peptide stimulation with IFN-γ production. Representative FACS plots of CD4 (B) and CD8-gated cells (C). Numbers give the %-values for quadrants. Numbers of total, CD39+, CD39—, CD73+, and CD73—cells for IFN-γ+ CD4+ (D) and for IFN-γ+ CD8+ T cells (E). Bars give the mean ± SEM. Representative results of CD39 (F) and CD73 expression (G) on OVA257-264-dextramer+ CD8+ T cells at d9 post infection. Open histograms show dextramer+ and filled histograms dextramer—CD8+ T cells. Numbers give the MFI. Bars give the mean ± SEM. Results are representative for at least two independent experiments with three or five individually analyzed mice per group and time point. Paired t test, * p<0.05, ** p<0.01.
Fig 6.
Innate response of CD39-/- mice against L. monocytogenes.
Wildtype and CD39-/- mice were i.v. infected with 5×103 Lm. (A) Bacterial burden in spleens was determined at d2 post infection. Combined results for 10 individually analyzed mice from two independent experiments and the median are shown. cfu, colony forming units. Mann Whitney test, * p<0.05. (B) Wildtype mice were infected with 1×105 LmOVA. At the indicated time points, neutrophils and inflammatory monocytes from the spleen were analyzed for the expression of CD39 and CD73 by flow cytometry. Representative concatenated dot plots for surface expression of CD39 and CD73 on neutrophils and inflammatory monocytes are shown. (See S2 Fig for mean values for groups of mice.) (C, D) Spleen cells from wildtype and CD39-/- mice were cultured with Lm overnight. Supernatants were collected and the concentration of IL-1β (C) and TNF-α (D) was determined by ELISA. Bars present the mean ± SEM of eight values. (E) Wildtype and CD39-/- mice were i.v. infected with 5×103 Lm. On day 2 post infection, spleen cells were isolated and expression of TNF-α was directly analyzed in inflammatory monocytes by intracellular cytokine staining and flow cytometry. Representative histograms and frequency of TNF-α+ inflammatory monocytes are given. Bars present the mean ± SEM of five individually analyzed mice. Results are representative for two independent experiments. Unpaired t test, ** p<0.01.
Fig 7.
Acquired immune response of CD39-/- mice against L. monocytogenes.
Wildtype and CD39-/- mice were i.v. infected with 5×103 Lm. (A) Bacterial burden in spleens was determined at d7 post infection. Combined results for five individually analyzed mice and the median are shown. cfu, colony forming units. Mann Whitney test, ns p>0.05. (B) Wildtype and CD39-/- mice were i.v. infected with 1×105 LmOVA. On day 9 post infection, CD8+ T cells were analyzed with OVA257-264-dextramers. Representative dot plots and frequencies of dextramer+ cells among CD8+ T cells. Bars give the mean ± SEM of seven individually analyzed mice. Combined data of two experiments. Unpaired t test, *** p<0.001.