Fig. 4: CD28 costimulation inhibits CD73 upregulation on TCR-activated CD8⁺ T cells.

a Naïve CD28WTCD8⁺ T cells were sorted and stimulated with anti-CD3 antibody in the presence (red) or absence (black) of anti-CD28 antibody. Histogram of CD73 expression on CD8⁺ T cells on day 5 after stimulation. The data presented are representative of one of the four independent experiments. b As a, relative MFI of CD73 expression. (n = 4). MFI of CD73 in cells stimulated with anti-CD3 antibody (black) was taken as 1.0. MFI of CD73 in cells stimulated with anti-CD3/28 antibodies (red) was normalized against that in cells stimulated with anti-CD3 antibody (relative MFI). c Naïve CD8⁺ T cells from CD28KO (black) and CD28WT (red) mice were stimulated with anti-CD3/28 antibodies for 5 days and the cells were subjected to flow cytometric analysis for CD39 and CD73 expressions. The data presented are representative of one of the three independent experiments. d Naïve CD8⁺ T cells from CD28KO and CD28WT mice were stimulated with anti-CD3/28 antibodies for 5 days and the cell lysate was subjected to Western blot analysis for CD73 expression. The blot represents three independent experiments. e Naïve CD8⁺ T cells from P14 CD28KO (black) and P14 CD28WT (red) mice were stimulated with M2 peptide-pulsed B cell blasts. On day 5 after stimulation, cells were subjected to flow cytometric analysis for CD39 and CD73 expressions. The data presented are representative of one of the three independent experiments. f CD28^hi (red) and CD28^lo (black) populations sorted from naïve CD28WTCD8⁺ T cells were stimulated with anti-CD3/28 for 5 days and subjected to flow cytometric analysis of CD39 and CD73 expressions. The data presented are representative of one of the three independent experiments. g Naïve CD28WTCD8⁺ T cells were stimulated by anti-CD3 (black) or by anti-CD3/28 (red) antibodies in the presence (blue) or absence of LY294002 (3 μM). Histogram of CD73 expression on CD8⁺ T cells on day 5 after stimulation. The data presented are representative of one of the three independent experiments. h As g, relative MFI with that stimulated with anti-CD3 antibody as 1.0 (black). Anti-CD3/28, red. Anti-CD3/28 + LY294002, blue. (n = 3). i Culture supernatants of anti-CD3 (black)- and anti-CD3/28 (red)-stimulated CD8⁺ T cells from CD73^+/+ (n = 8) and CD73^−/− (n = 5) mice were collected after 2-h incubation in serum-free IL-2-containing DMEM and were subjected to UPLC-MS/MS analysis to quantify adenosine. j The capacity of CD8⁺ T cells to degrade AMP into adenosine was analyzed after 2-h incubation with AMP_13C,15N isotope (AMP*, 37.5 μM). Culture supernatants of anti-CD3 (black)- and anti-CD3/28 (red) antibodies-stimulated CD8⁺ T cells from CD73^+/+ (n = 7) and CD73^−/− (n = 5) mice were collected after 2-h incubation in serum-free IL-2-containing DMEM and were subjected to UPLC-MS/MS analysis to quantify adenosine_13C,15N. Neither pre-incubation of cells with CD73 inhibitor iCD73 (n = 5) before the addition of AMP_13C,15N nor stimulated CD73^−/− CD8⁺ T cells produced significant amounts of adenosine_13C,15N, confirming the role of CD73 in the production of adenosine. Statistical evaluations were performed using the Student’s t-test with data expressed as the mean ± standard error of the mean (b, h, i, j). *p < 0.05, **p < 0.01, ***p < 0.001.