T cell and B cell reconstitution after allogeneic BMT. (a and b) Lethally irradiated (1,300 cGy) CBA/J recipients were transplanted with B10.BR TCD-BM (5 × 10⁶) with or without B10.BR T cells (0.5 × 10⁶) to induce GVHD. At different timepoints after BMT, animals were sacrificed and absolute numbers of donor-derived cell populations in the spleen were calculated from total cell counts and multicolor flow cytometric analyses of T cells with anti-CD3, -CD4, and -CD8 antibodies (a), and B cells with anti-B220 antibody (b). Donor or host origin was determined with anti-Ly9.1, which is present on host leukocytes. Allo, allogeneic. (c) The dot plots demonstrate how CD44^high and CD44^low T cells can be distinguished by flow cytometric analysis in the spleen of a normal mouse and that CD44^low populations represent naive CD4⁺ T cells, which express CD62L, or naive CD8⁺ T cells, which do not express CD122. CBA/J mice were transplanted as described in Figure 1a without the addition of T cells, and splenocytes were analyzed on day 28. Naive CD4 cells were defined as CD4⁺CD44^low, memory/activated (MEM/ACT) CD4 cells were defined as CD4⁺CD44^high, naive CD8 cells were defined as CD8⁺CD44^low, and memory/activated CD8 cells were defined as CD8⁺CD44^high. (d) CBA/J mice were transplanted as described in Figure 1a without the addition of T cells and received 10 μg/day IL-7 or PBS (control) on days 21–27 by intraperitoneal injection. Splenocytes were analyzed at different timepoints after transplant. Values represent the mean cell number ± SEM (n = 6–20). *P < 0.05.

IL-7Rα expression is downregulated on alloreactive T cells and IL-7 does not exacerbate the development of GVHD. (a) Lethally irradiated (1,300 cGy) CBA recipients were transplanted with B10.BR TCD-BM. IL-7 (10 μg/day) or PBS (control) was administered by intraperitoneal injection on days 21–27 after allogeneic BMT. Animals were sacrificed on day 28 and IL-7Rα expression was determined on donor-derived splenic CD4⁺ and CD8⁺ cells in IL-7–treated, PBS (control), and age-matched nontransplanted mice by flow cytometric analysis. Naive and memory/activated cells and donor/host origin were defined as in Figure 1. Iso-Ig, isotype control for the anti–IL-7Rα antibody. (b and c) BMT was performed as described in Figure 2a. Spleens were harvested from mice at 16 hours and on day 7, and stained with anti-CD8 and –IL-7Rα antibodies. (d) Sublethally irradiated (750 cGy) C3FeB6F1 recipients were infused with CFSE-labeled B6 purified T cells (2 × 10⁷). Spleen and superficial lymph nodes (axillary and inguinal) were harvested at day 3 and cells underwent flow cytometric analysis with anti-CD8 and –IL-7Rα antibodies to determine the number of cell divisions and IL-7Rα expression of CD8⁺ T cells. (e) Lethally irradiated (1,300 cGy) CBA mice were transplanted with B10.BR TCD-BM (5 × 10⁶) with T cells (0.5 × 10⁶). Mice were sacrificed at day 7 or day 14 after BMT. Spleen, gut, liver, and lymph nodes were harvested and mononuclear cells were obtained. Cells were stained with anti-CD4, -CD8, and –IL-7Rα antibodies for flow cytometric analysis. Representative histograms of four mice are shown. Dotted lines indicate isotype control for IL-7Rα. (f) Lethally irradiated (1,300 cGy) CBA mice were transplanted with B10.BR TCD-BM (5 × 10⁶) and varying T cell doses: 0.5 × 10⁶ (left graph), 0.2 × 10⁶ (right graph, circles), and 0.1 × 10⁶ (right graph, diamonds). IL-7 (10 μg/day) or PBS (control) was administered by intraperitoneal injection from day 0 to day 7 after allogeneic BMT. Open symbols represent the PBS (control) groups, and filled symbols represent the IL-7–treated groups. Kaplan-Meier survival curves are shown (n = 8–24).

De novo–generated T cells undergo homeostatic expansion in the recipient of an allogeneic BMT. (a and b) CFSE-labeled thymocytes (Thy) (a) and splenocytes (Spl) (b) (10⁷ each) from syngeneic donors (Syn) were transferred into lethally irradiated CBA/J recipients. On day 5, splenocytes from the recipients were harvested and stained with anti-CD4 and -CD8 antibodies for flow cytometric analysis. (c–e) Lethally irradiated CBA/J mice were transplanted with B10.BR TCD-BM and sacrificed on day 28 after allogeneic BMT. Thymocytes (c) and splenic T cells (d) were harvested, pooled, and labeled with CFSE, and 10⁷ cells were infused into irradiated CBA/J mice. On day 5, splenocytes from the recipients were harvested and stained with anti-CD4 and -CD8 antibodies for flow cytometric analysis. Infusion with CFSE-labeled B10.BR splenic T cells was performed as the allogeneic control (e). CBA, CBA/J.

Posttransplant IL-7 administration decreases apoptotic T cells in 9-month-old recipients of allogeneic BMT. (a–c) CBA/J mice (3 months or 9 months old) were transplanted as described in Figure 1 without the addition of T cells. All recipients received 10 μg/day IL-7 or PBS (control) by intraperitoneal injection from day 21 to day 27. Animals were sacrificed on day 28, and the percentage of donor-derived apoptotic T cells was determined by flow cytometric analysis using annexin V and anti-Ly9.1, -CD4, and -CD8 antibodies. Representative FACS profiles from control and IL-7–treated middle-aged mice are shown in a. The numbers of annexin V–positive donor-derived CD4⁺ or CD8⁺ T cells from normal B10.BR mice and young and middle-aged allogeneic BMT recipients are shown in b and c, respectively. Values represent percentage of apoptotic cells from ten animals per group, expressed as mean ± SEM. P values represent comparisons between IL-7–treated and PBS-treated groups (*P < 0.05).

Alloreactive and nonalloreactive donor T cells can be distinguished by their phenotype and proliferation kinetics. (a and b) Lethally irradiated (1,300 cGy) C3FeB6F1 (allogeneic, Allo) or B6 (syngeneic, Syn) recipients were transplanted with B6 BM (5 × 10⁶) and CFSE-labeled B6 (Ly5.1⁺) purified T cells (2 × 10⁷). At various timepoints after BMT, recipients were sacrificed and splenocytes were stained with anti-CD8, -CD25, -CD44, -CD62L, and -CD122 antibodies. Flow cytometric analysis is shown for expression of these antibodies on donor CD8⁺ T cells 64 hours after BMT (a).CD69 expression on donor CD4 and CD8 T cells in the spleen was determined 16 hours, 40 hours, and 64 hours after BMT (b). (c) Lethally irradiated (750 cGy) B6D2F1/J recipients were transplanted with CFSE-labeled B6 purified T cells (2 × 10⁷). After 64 hours, mice were sacrificed, splenocytes were stained with anti-CD4 and -CD8 antibodies, and FSC intensity was determined. A: nondividing cells; B: slow proliferating cells; C:fast proliferating cells. FSC, forward scatter; % of max, % of maximum number of the cells per group. (d) Lethally irradiated (750 cGy) C3FeB6F1 recipients were transplanted with CFSE-labeled B6 purified T cells (2 × 10⁷). After 64 hours, mice were sacrificed and splenocytes were sorted according to CFSE intensity, shown in a histogram plot as fast- and slow-proliferative cells. Cells were incubated with irradiated C3FeB6F1 splenocytes as stimulators for 5 days. IL-2 (20 IU/ml) was added after 72 hours, and proliferation was determined by ³H-thymidine incorporation.

Most de novo–generated T cells after allogeneic BMT with purified hematopoietic stem cells have a memory/activated phenotype. Lethally irradiated (1,300 cGy) CBA/J mice were transplanted with either B10.BR TCD-BM (allogeneic BMT) or lineage-negative SCA-1⁺c-kit⁺ B10.BR BM cells (2 × 10⁴). IL-7 (10 μg/day) or PBS (control) was administered by intraperitoneal injection from day 21 to day 28 after allogeneic BMT, and from day 28 to day 35 after allogeneic SCT. Animals were sacrificed and harvested on day 28 (BMT) and day 35 (SCT). Absolute numbers of specific donor-derived cell populations were calculated from total cell counts and percentages of T cells by flow cytometric analysis. Naive CD4⁺ T cells were defined as CD44^lowCD62L^high, activated CD4⁺ T cells as CD44^highCD62L^high, memory CD4⁺ T cells as CD44^highCD62L^low, naive CD8⁺ T cells as CD44^low, and memory/activated CD8⁺ T cells as CD44^high. Values represent the mean cell number ± SEM (SCT: n = 3; BMT: n = 16).

Posttransplant IL-7 administration increases the proliferation of nonalloreactive donor T cells in recipients of allogeneic BMT. (a) Lethally irradiated CBA/J recipients were transplanted with B10.BR TCD-BM (5 × 10⁶) and received 10 μg/day IL-7 or PBS (control) on days 21–27. Animals were sacrificed on day 28 and splenocytes were stained with cell surface antibodies (anti-CD4, -CD8, and -Ly9.1), followed by fixation, permeabilization, and intracellular staining with 7-AAD for cell cycle analysis. The values shown represent the mean percentage ± SEM of donor CD4⁺ or CD8⁺ T cells in S/G2/M phase with P values comparing IL-7–treated with PBS-treated groups (four mice per group). *P < 0.05. (b) Mice were transplanted as described in a, and BrdU was added to their drinking water at a concentration of 0.8 mg/ml on days 15–33 after allogeneic BMT. The mice also received 1 μg/day IL-7 or PBS (control) on days 15–33. Splenocytes were harvested at day 33 and stained with anti-CD4, CD8, -CD44, and -BrdU antibodies. The values shown represent the mean percentage ± SEM of donor naive or memory CD8⁺ T cells that are BrdU⁺, with P values comparing IL-7–treated with PBS-treated groups (four mice per group). (c and d) Sublethally irradiated B6 (Ly5.1⁺) (syngeneic) or C3FeB6F1 (allogeneic) recipients were infused with CFSE-labeled B6 purified T cells (2 × 10⁷). The recipients received 10 μg/day IL-7 or PBS (control) on days 0–3. On day 3, splenocytes were harvested and stained with anti-CD4 and anti-CD8 antibodies for flow cytometric analysis. Results shown are representative of three duplicate experiments. (e) Splenocytes were harvested from C3FeB6F1 (allogeneic) recipients as in d, and incubated with PMA (10 ng/ml) and ionomycin (2 μM) for 5 hours. Brefeldin A was added during the last 3 hours to inhibit IFN-γ secretion. Cells were harvested and stained with anti-CD4 and anti-CD8 antibodies, then washed, fixed, permeabilized, and stained intracellularly with anti–IFN-γ antibody.