Effects of intrabone marrow-bone marrow transplantation plus adult thymus transplantation on survival of mice bearing leukemia

Abstract

We recently found that allogeneic intrabone marrow-bone marrow transplantation (IBM-BMT) plus adult thymus transplantation (ATT) from the same donor is effective in mice bearing solid tumors. In the current study, we examined the effects of this strategy on the survival of mice with leukemia. One week after intravenous injection of 1×10(6) leukemic cells (EL-4, H-2(b)) into 8-week-old B6 (H-2(b)) mice, the mice were 8 Gy irradiated and transplanted with 1×10(7) bone marrow cells (BMCs) from 8-week-old BALB/c mice (H-2(d)) by IBM-BMT with or without donor lymphocyte infusion (DLI) or ATT. All the mice without treatment died within 70 days after injection of EL-4. About 40% of those treated with IBM-BMT alone died within 100 days due to tumor relapse. In contrast, those treated with IBM-BMT+DLI or ATT showed the longest survival rate without relapse of leukemia. In addition, the former showed less graft versus host disease (GVHD) than the latter. The mice treated with IBM-BMT+ATT also showed an intermediate percentage of effector memory (EM) and central memory (CM) cells between those treated with BMT alone and those treated with IBM-BMT+DLI. The numbers and functions of T cells increased in those treated with IBM-BMT+ATT with interleukin-2 and interferon-γ production. These results suggest that IBM-BMT+ATT is effective in the treatment of leukemia with strong graft versus leukemia without increased risk of GVHD.

FIG. 1.

Survival rate and body weight in mice with leukemia treated with BMT+TT. Survival rate (A) and weight (B) of mice with advanced tumors are shown. *P< 0.0001 compared with those treated with BMT alone, BMT+ATT, or BMT+DLI. **P<0.05 compared with those treated with BMT+ATT or BMT+DLI. ^#P<0.05 compared with those treated with BMT alone, BMT+ATT, or BMT+DLI. ^##P<0.05 compared with those treated with BMT+ATT or BMT+DLI. Data are shown as means±SE. Untreated controls (n=12), those treated with BMT alone (n=12), BMT+ATT (n=12), or BMT+DLI (n=15). ATT, adult thymus transplantation; BMT, bone marrow transplantation; DLI, donor lymphocyte infusion; TT, thymus transplantation.

FIG. 2.

Chimerism, histology, transplanted thymus, and GVHD in mice with leukemia treated with BMT+TT. Chimerism in experimental mice (A), histology in the liver, lung, mesenterium, muscle, and BM from mice with leukemia (B), histology in transplanted thymus and the FACS profile in (C), histology for GVHD in chimeric mice (D) and the GVHD scores in the mice (E) are shown. Chimerism was analyzed in the spleens from mice transplanted with leukemia and those treated with BMT alone, BMT+ATT, or BMT+DLI (A). The numbers in the profiles show the percentage. Tumor cells (upper, left) infiltrated the liver, lung, mesenterium, muscle, and BM in the mice transplanted with leukemia (arrows) (B). Histological findings (left) and FACS profile (right) of thymocytes in the transplanted thymus from the mice treated with BMT and ATT (C). Plain arrow, cortex; dotted arrow, medulla. The numbers in the profiles show the percentage. Representative data from 4 experiments are shown. The histology of GVHD is shown in the liver (upper) and small intestine (lower) (D). Some lymphocytes infiltrated the liver and small intestine in BMT+ATT (arrows) and destroyed the tissue in BMT+DLI (dotted arrow). GVHD scores are shown in the liver (left) and small intestine (right) (E). The GVHD score was calculated as described in Materials and Methods. *P<0.05 compared with normal BALB/c mice and mice treated with BMT alone and BMT+ATT. **P<0.05 compared with normal BALB/c mice and the mice treated with BMT alone. ^#P<0.05 compared with normal BALB/c mice and mice treated with BMT alone and BMT+ATT. ^##P<0.05 compared with normal BALB/c mice and mice treated with BMT alone. The mice transplanted with leukemia cells (EL-4) were analyzed 5 weeks after transplantation and those treated with BMT alone, BMT+ATT, or BMT+DLI were analyzed 8 weeks after treatment. Data are shown as means±SE. Normal BALB/c (n=4), BMT (n=4), BMT+ATT (n=4), BMT+DLI (n=4). GVHD, graft versus host disease.

FIG. 3.

Numbers of lymphocytes in the spleen from leukemia-bearing mice treated with BMT+ATT. Numbers of CD4⁺ T cells (A), percentage of FoxP3⁺ cells in CD4⁺ T cells (B), numbers of CD8⁺ T cells (C), and B220⁺ B cells (D) in the spleen were evaluated in normal BALB/c mice, leukemia-bearing mice treated with BMT alone, BMT+ATT, or BMT+DLI. The experiments were performed 5 weeks after BMT. *P<0.05 compared with BMT alone and BMT+DLI. **P<0.01 compared with BMT+DLI. ^#P<0.05 compared with BMT+ATT and BMT+DLI. ^##P<0.05 compared with BMT+DLI. ^§P<0.01 compared with BMT, BMT+ATT, and BMT+DLI. ^§§P<0.05 compared with BMT and BMT+DLI. ^§§§P<0.05 compared with BMT+DLI. ^‡P<0.01 compared with BMT+DLI. Data are shown as means±SD. Normal BALB/c (n=5), BMT (n=5), BMT+ATT (n=5), and BMT+DLI (n=5).

FIG. 4.

Proportions of EM, CM, and naïve T cells from leukemia-bearing mice treated with BMT+ATT. Percentages of CD62L^-CD44⁺ EM, CD62L⁺CD44⁺ CM, and CD62L^-CD44^- naïve cells in CD4⁺ (A) and CD8⁺ (B) T cell subsets were analyzed in the spleens from normal BALB/c mice and leukemia-bearing mice treated with BMT alone, BMT+ATT, and BMT+DLI. Analyses were performed at the same time as for the experiment in Fig. 3. *P<0.01 compared with BALB/c, BMT, and BMT+ATT. **P<0.01 compared with BALB/c and BMT. ^#P<0.05 compared with BMT+ATT and BMT+DLI. ^##P< 0.01 compared with BMT+DLI. ^§P<0.01 compared with BMT, BMT+ATT, and BMT+DLI. ^§§P<0.01 compared with BMT+ATT and BMT+DLI. ^‡P<0.01 compared with BALB/c, BMT, and BMT+ATT. ^‡‡P<0.01 compared with BALB/c and BMT. ^‡‡‡P<0.01 compared with BALB/c. ^¶P<0.05 compared with BMT, BMT+ATT, and BMT+DLI. ^¶¶P<0.05 compared with BMT+ATT and BMT+DLI. ^¶¶¶P<0.01 compared with BMT+DLI. ^fP<0.01 compared with BMT, BMT+ATT, and BMT+DLI. ^ffP<0.05 compared with BMT+ATT and BMT+DLI. Data are shown as means±SD. Normal BALB/c (n=5), BMT (n=5), BMT+ATT (n=5), and BMT+DLI (n=5). CM, central memory; EM, effector memory.

FIG. 5.

Mitogen responses and percentages of cytokine-producing cells in the spleens from leukemia-bearing mice treated with BMT and ATT Mitogen responses: Con A and LPS (A) and percentages of cytokine-producing cells (B) in the spleen were evaluated in the spleens from normal BALB/c mice and leukemia-bearing mice treated with BMT alone, BMT+ATT, and BMT+DLI. Analyses were performed at the same time as for the experiment in Fig. 3. *P<0.05 compared with BMT and BMT+DLI. **P<0.05 compared with BMT+DLI. ^#P<0.05 compared with BMT+DLI. ^§P<0.05 compared with BMT and BMT+DLI. ^‡P<0.05 compared with BMT, BMT+ATT, and BMT+DLI. ^‡‡P<0.05 compared with BMT and BMT+ATT. ^‡‡‡P<0.05 compared with BMT. Data are shown as means±SD. Normal BALB/c (n=4), BMT (n=4), BMT+ATT (n=4), and BMT+DLI (n=4). LPS, lipopolysaccharide.