![Fig. 4. IL-7 administration does not stimulate alloreactive donor T cells in recipients undergoing GVHD. / Lethally irradiated (B6 × C3H)F1 or CBA/J recipients (1300 cGy) received transplants with B6 or B10.BR TCD BM (5 × 106) and splenic T cells (0.5 × 106) on day 0 as described in Table 1. Osmotic minipumps were used for continuous subcutaneous administration of 1 μg/d IL-7 or PBS (control) from day −1 to day +13. All recipients were humanely killed on day 14 and spleens were harvested and processed for flow cytometric analysis and functional assays. (A) Numbers of splenic donor CD4+ and CD8+ T cells were calculated from total splenocyte counts and flow cytometric analyses with antibodies that recognize specifically host antigens (Ly9.1 and CD5.1) and T-cell antigens (CD4 and CD8). Values represent mean ± SE and each group contained 6 to 8 animals. (B) Intracellular cytokine expression of alloreactive T cells. Splenic B6 T cells were harvested on day +14 from IL-7– or PBS-treated recipients as described above and incubated with irradiated (20 Gy) (B6 × C3H)F1 splenic stimulator cells in 24-well plates for 5 days. Cells were harvested, and restimulated with TCD, irradiated (20 Gy) (B6 × C3H)F1 splenic stimulator cells for 16 hours. Brefeldin A (10 μg/mL) was added after the first hour of incubation. Intracellular cytokine expression in CD4 memory cells (CD4+, CD62L−, CD44+) was measured by flow cytometric analysis. Values represent mean ± SE and each group contained 3 animals. (C) Cytolytic activity of alloreactive T cells. Splenic B6 T cells were harvested on day +14 from IL-7– or PBS-treated recipients as described above and their cytotoxicity was determined against 32Dp210 and P815 (third party) cell lines in51Cr-release assays. (D) Alloreactive T-cell proliferation. Splenic T cells (4 × 105 cells/well) were incubated with irradiated (20 Gy) (B6 × C3H)F1 or CBA/J splenic stimulator cells (2 × 105 cells/well) in 96-well plates for 5 days and [3H]-thymidine was added during the final 20 hours of culture. The mean thymidine incorporation in unstimulated splenocytes ranged from 529 to 3336 cpm. Values represent means ± SE and each group contained 6 to 8 animals.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/98/7/10.1182_blood.v98.7.2256/5/h81911605004.jpeg?Expires=1724230466&Signature=uqcm3BwlShjae1KWjHmjgmpBUxjvA-6jUyOu9Jt41YTUhvz9nUzCp9RBEAwD9v-yy1p6TUcVHsrGP82DK07r7rFnumaDNqWWjCvjMJY77NLS7pRio6-kd7kUofP7ggayyrJwpu17iPzY-doQ50h6014FqC5899m5o8rNpcQX5x~X9-oMj~gfUeNMm1mAZ1jDqamb7JKwRHkIqiXAZ9Ua1e8AVdrpGlibo2vwPwq8iiL33jOENuCN2XAW-JjYh8~yrRjJPMB0p36AZCsctINGEK86naSwTHm93BONiSSlVweYM48IDtVH5BLih5pMWL0jfKUwWeuLWWiZdjww5A62-A__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
IL-7 administration does not stimulate alloreactive donor T cells in recipients undergoing GVHD.
Lethally irradiated (B6 × C3H)F1 or CBA/J recipients (1300 cGy) received transplants with B6 or B10.BR TCD BM (5 × 106) and splenic T cells (0.5 × 106) on day 0 as described in Table 1. Osmotic minipumps were used for continuous subcutaneous administration of 1 μg/d IL-7 or PBS (control) from day −1 to day +13. All recipients were humanely killed on day 14 and spleens were harvested and processed for flow cytometric analysis and functional assays. (A) Numbers of splenic donor CD4+ and CD8+ T cells were calculated from total splenocyte counts and flow cytometric analyses with antibodies that recognize specifically host antigens (Ly9.1 and CD5.1) and T-cell antigens (CD4 and CD8). Values represent mean ± SE and each group contained 6 to 8 animals. (B) Intracellular cytokine expression of alloreactive T cells. Splenic B6 T cells were harvested on day +14 from IL-7– or PBS-treated recipients as described above and incubated with irradiated (20 Gy) (B6 × C3H)F1 splenic stimulator cells in 24-well plates for 5 days. Cells were harvested, and restimulated with TCD, irradiated (20 Gy) (B6 × C3H)F1 splenic stimulator cells for 16 hours. Brefeldin A (10 μg/mL) was added after the first hour of incubation. Intracellular cytokine expression in CD4 memory cells (CD4+, CD62L−, CD44+) was measured by flow cytometric analysis. Values represent mean ± SE and each group contained 3 animals. (C) Cytolytic activity of alloreactive T cells. Splenic B6 T cells were harvested on day +14 from IL-7– or PBS-treated recipients as described above and their cytotoxicity was determined against 32Dp210 and P815 (third party) cell lines in51Cr-release assays. (D) Alloreactive T-cell proliferation. Splenic T cells (4 × 105 cells/well) were incubated with irradiated (20 Gy) (B6 × C3H)F1 or CBA/J splenic stimulator cells (2 × 105 cells/well) in 96-well plates for 5 days and [3H]-thymidine was added during the final 20 hours of culture. The mean thymidine incorporation in unstimulated splenocytes ranged from 529 to 3336 cpm. Values represent means ± SE and each group contained 6 to 8 animals.
