Figure 6
Figure 6. Homeostatic cytokines regulate the differentiated expression of gut-homing markers on CD4 and CD8 TMs in vitro and in vivo. (A) IL-7 and IL-15 regulation of homing marker expression on MP CD4 and CD8 TMs in vitro. MP CD4 and CD8 TMs (gated as CD4+CD25−CD44hi or CD8+CD44hi, respectively) purified from the spleen of 1-year-old B6 mice (pool of 8 mice) were cultured in triplicates in vitro for 3 days in the presence or absence of 100 ng/mL of IL-7 or IL-15. Their expression of homing markers, as well as IL-2/15Rβ (CD122), was measured by flow cytometry. Data are shown as histogram plots and measurements of mean fluorescence intensity (MFI, presented as mean of triplicates ± SEM), and are representative of at least 3 independent experiments. Red or blue *P < .05 (CD4 or CD8 TMs cultured with cytokine compared with CD4 or CD8 TMs cultured without cytokine). (B-C) Quantification of MP CD4 and CD8 TMs (gated as CD4+CD25−CD44hi and CD8+TCRβ+CD44hi, respectively) in 9-month-old IL-15KO mice with or without supplementation of IL-15 (IP 10μg daily for 5 sequential days). (B) Tissue distribution of CD4 and CD8 TMs. (C) The fold changes of CD4 and CD8 TM numbers in response to IL-15 treatment in indicated tissues. Data are shown as mean ± SEM (n = 3). (D) Comparison of CD4 and CD8 TM formation in WT or IL-15KO mice using BLI. MFG-labeled B6 CD4 or CD8 TEs (1 × 106) were adoptively transferred into either WT (B6) or IL-15KO recipient mice. Representative BLI images and the measurements of TBL (shown as mean ± SEM) of the indicated recipients collected 1 month after transfer are shown (n = 3). (E,F) The impact of IL-15 supplement on the homeostasis and homing of CD4 and CD8 TMs studied using BLI. MFG-labeled WT (B6) CD4 or CD8 TEs (5 × 106) were transferred into IL-15KO recipient mice. One month later, the recipients were supplemented with IL-15 (IP 10μg daily for 5 sequential days). (E) Time-course tracking of the fold change of TBL of the indicated recipients. Data are presented as mean ± SEM. (F) Representative BLI images of the indicated recipients on day 1 (right before the 1st IL-15 administration), day 6, and day 9. N = 4. Schematics showing the individual tissue localization in mice (ventral view and left side view) are provided for reference.

Homeostatic cytokines regulate the differentiated expression of gut-homing markers on CD4 and CD8 TMs in vitro and in vivo. (A) IL-7 and IL-15 regulation of homing marker expression on MP CD4 and CD8 TMs in vitro. MP CD4 and CD8 TMs (gated as CD4+CD25−CD44hi or CD8+CD44hi, respectively) purified from the spleen of 1-year-old B6 mice (pool of 8 mice) were cultured in triplicates in vitro for 3 days in the presence or absence of 100 ng/mL of IL-7 or IL-15. Their expression of homing markers, as well as IL-2/15Rβ (CD122), was measured by flow cytometry. Data are shown as histogram plots and measurements of mean fluorescence intensity (MFI, presented as mean of triplicates ± SEM), and are representative of at least 3 independent experiments. Red or blue *P < .05 (CD4 or CD8 TMs cultured with cytokine compared with CD4 or CD8 TMs cultured without cytokine). (B-C) Quantification of MP CD4 and CD8 TMs (gated as CD4+CD25−CD44hi and CD8+TCRβ+CD44hi, respectively) in 9-month-old IL-15KO mice with or without supplementation of IL-15 (IP 10μg daily for 5 sequential days). (B) Tissue distribution of CD4 and CD8 TMs. (C) The fold changes of CD4 and CD8 TM numbers in response to IL-15 treatment in indicated tissues. Data are shown as mean ± SEM (n = 3). (D) Comparison of CD4 and CD8 TM formation in WT or IL-15KO mice using BLI. MFG-labeled B6 CD4 or CD8 TEs (1 × 106) were adoptively transferred into either WT (B6) or IL-15KO recipient mice. Representative BLI images and the measurements of TBL (shown as mean ± SEM) of the indicated recipients collected 1 month after transfer are shown (n = 3). (E,F) The impact of IL-15 supplement on the homeostasis and homing of CD4 and CD8 TMs studied using BLI. MFG-labeled WT (B6) CD4 or CD8 TEs (5 × 106) were transferred into IL-15KO recipient mice. One month later, the recipients were supplemented with IL-15 (IP 10μg daily for 5 sequential days). (E) Time-course tracking of the fold change of TBL of the indicated recipients. Data are presented as mean ± SEM. (F) Representative BLI images of the indicated recipients on day 1 (right before the 1st IL-15 administration), day 6, and day 9. N = 4. Schematics showing the individual tissue localization in mice (ventral view and left side view) are provided for reference.

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