Fig 1.
CD69 is expressed on CD8+ T cells in the absence of recent T cell activation.
The phenotype of CD8+ T cells isolated from peripheral blood, spleen and tonsils were examined by flow cytometry. (A) The mean (± SD; n = 13 for blood and spleen and n = 7 for tonsils) percent of CD69+CD8+ T cells in these compartments. (B) CD69 expression on naïve (CCR7+CD45RA+), central memory (TCM, CCR7+CD45RA—), effector memory (TEM, CCR7—CD45RA—) and TEMRA (CCR7—CD45RA+) CD8+ T cells from blood (blue), spleen (red) and tonsils (green). Individual dots represent different donor samples. (C and D) Representative flow cytometry histogram plots show the expression levels of activation markers CD25, CD137, HLA-DR and KLRG1 between the CD69+CD8+ T cells (red) and CD69—CD8+ T cells (blue) in the spleen (C) and tonsils (D). Data is representative of 5 independent experiments. (E) Graph shows the relative expression levels of BCL2 between CD69+ TEM CD8+ T cells and CD69—TEM CD8+ T cells from human spleen (n = 5). P = 0.0625 by one-way ANOVA.
Fig 2.
Two subsets of CD8+ T cells are retained within human lymphoid tissues.
The co-expression of CD69 and CD103 by CD8+ T cells isolated from spleen and tonsils were determined by flow cytometry. (A and B) Representative flow cytometry plots (left) and graphs (right; mean ± SEM; n = 10) show the proportion of CD69+CD8+ T cells expressing CD103 in human spleen (A) and tonsils (B). (C—E) Representative flow cytometry plots showing the expression levels of CCR7, CD45RA and CD11a between CD69—CD103—(blue), CD69+CD103—(red) and CD69+CD103+ (green) CD8+ T cell subsets from the spleen (C) and tonsils (D) and the expression of PD-1, TIM3 and BTLA in tonsils (E). Data is representative of 3–5 independent experiments. (F) Relative expression of KLF2 and S1PR1 in purified CD8+ T subsets from the spleen (left panels) and tonsils (right panels). Individual dots represent different donor samples (n = 8 for spleen and n = 3 for tonsils). Statistical analysis was performed using one-way ANOVA and Tukey test. P<0.05 is noted with * and P<0.0005 is noted with ***.
Fig 3.
CD69+CD103+CD8+ T cells localize near the epithelial barrier in tonsils.
The locations of CD69+CD8+ T cell subsets were determined by immunohistochemistry. (A) Immunofluorescence microscopy images of human tonsils show the localization of CD8 (green), CD69 (blue) and CD103 (red). Scale bar represents 100 μm. (B) Higher magnification of areas 1 & 2 show the localization of CD103+CD69+CD8+ and CD103CD69+CD8+ T cells within the tonsils. (C) White arrowheads point to CD103+CD69+CD8+ T cells in areas 1 and CD103—CD69+CD8+ T cells in area 2. (C) Quantitative analysis of the distance of CD103+CD3+CD8+ T cells from the epithelium shows majority localizing near the epithelial surface (P = 0.0022 by two-tailed Mann Whitney U-test). (D) Immunofluorescence microscopy of human spleen sections shows the localization of CD8 (green), CD69 (blue) and CD103 (red). Scale bar represents 100 μm. Higher magnification of areas 1 and 2 show the distribution of CD103+CD69+CD8+ and CD103—CD69+CD8+ T cells. White arrowheads in area 1 show the CD103+CD69+CD8+ T cells and in area 2 show the CD103—CD69+CD8+ T cells.
Fig 4.
IL-15 and TGF-β co-operate to extinguish expression of KLF2 and S1PR1.
(A) Representative flow cytometry plots show the expression of CD69 and CD103 by CD8+ T cells following 7 day culture under different conditions: unstimulated (US), IL-15 or IL-15 + TGF-β and polyclonal stimulation (TAE). (B) Plot shows the proportion of CD69+CD103- (left panel) and CD69+CD103+ (right panel) CD8+ T cells following 7 day culture with different cytokines. Data are represented as the mean and SEM of 5–11 donors. (C) Representative flow cytometry plots show the expression of CD69, CD137 and dilution of cell trace violet (CTV) dye following stimulation of circulating CD8+ T cells for 7 days with TAE beads (upper panels) or IL-15 (lower panels). (D) Representative flow cytometry plot and graph show the expression of CD69 and the dilution of cell trace violet dye following stimulation of purified circulating naïve (n = 4), TCM (n = 2), TEM (n = 8) and TEMRA (n = 5) CD8+ T cells for 7 days with IL-15. (E-F) Plots show the relative expression of S1PR1 (E) and KLF2 (F) in CD69+ or CD69—CD8+ T cells following culture for 7 days with no stimulation or stimulation with IL-15 with and without TGF-β. Purified circulating CD8+ T cells were cultured for 7 days and the resulting CD69+ and CD69—populations were purified by cell sorting. The expression levels of KLF2 and S1pr1 were quantified by RT-PCR. Individual dots represent different samples and the data is represented as the mean ± SEM. Statistical analysis was performed using one-way ANOVA and Tukey test. P<0.05 is noted with * and P<0.005 is noted with **. (G-H) The ability of IL-15 induced CD69+CD8+ T cells to migrate to S1P and CCL5 (20 nM) was tested in trans-well migration assays. Cultured cells were sorted as stated above (for F) and their ability to migrate towards different concentrations of S1P (G) or CCL5 (20 nM) (H) was determined. Data represent the mean and SEM of three independent experiments using three different donors. Statistical analysis was performed using two-way ANOVA and the p value was < 0.05.
Fig 5.
Constitutive expression of IL-15 within tonsils.
Immunofluorescence microscopy of frozen section of the tonsils show the presence of IL-15 expressing cells in the T cell areas and the epithelial lining of the tissue. Sections were stained with anti-IL-15 (red), anti-CD8 (green) and anti-IgM (blue). Yellow dashed line marks the epithelial barrier surface and the white dashed-lines show B cell follicles (B). ‘T” indicates the extra-follicular regions where T cells localize.
Fig 6.
Selective retention of EBV-specific CD8+ T cells in tissues.
Virus-specific cells were determined by staining single cell suspensions with soluble peptide-MHC complexes (dextramers). (A) The graphs show the normal range and the line at mean value for each virus-specific population in blood (blue), spleen (red) and tonsils (orange). (B) Representative flow cytometry plots show the dual expression of CD69 and CD103 on EBV and CMV-specific CD8+ T cells in blood, spleen and tonsils. (C) Graphs show the proportions of CD69—CD103—, CD69+CD103—and CD69+CD103+ EBV-specific and CMV-specific CD8+ T cells from spleen and tonsils. Data is presented as the mean ± SEM of n = 5 (for EBV specific cells in tonsils and CMV-specific cells in spleen), n = 6 (EBV-specific cells in spleen) and n = 3 (CMV-specific cells in tonsils) different samples. (D) Representative flow cytometry overlayed histogram plots show no difference in the expression of HLA-DR between CD69—(blue) and CD69+ (red) EBV-specific CD8+ T cells in the tonsils (left) and spleen (right).