Treatment with GM-CSF secreting myeloid leukemia cell vaccine prior to autologous-BMT improves the survival of leukemia-challenged mice

Abstract

Vaccination with irradiated autologous tumor cells, engineered to secrete granulocyte macrophage-colony stimulating factor (GM-CSF) (GM tumor), can generate potent antitumor effects when combined with autologous bone marrow transplantation (BMT). That notwithstanding, the post-BMT milieu, characterized by marked cytopenia, can pose a challenge to the implementation of vaccine immunotherapies. To bypass this problem, partial post-BMT immune reconstitution has been allowed to develop prior to vaccination. However, delaying vaccination can also potentially allow the expansion of residual tumor cells. Other approaches have used reinfusion of "primed" autologous lymphocytes and multiple administrations of GM tumor cells, which required the processing of large amounts of tumor. Utilizing the MMB3.19 murine myeloid leukemia model, we tested whether a single dose of GM tumor cells, 7 days prior to syngeneic BMT, could be a curative treatment in MMB3.19-challenged recipient mice. This vaccination protocol significantly improved survival of mice by eliciting long-lasting host immune responses that survived lethal irradiation, and were even protective against post-BMT tumor rechallenge. Furthermore, we demonstrated that mature donor lymphocytes can also play a limited role in mounting the antitumor response, but our pre-BMT vaccination strategy obviated the need for either established de novo immune reconstitution or the use of multiple post-BMT immunizations.

Figure 1

Effect of pre-BMT vaccination with GM-MMB3.19 or non-GM-MMB3.19 cells on GVL responses to MMB3.19 leukemia challenge. B6 mice were vaccinated with a single s.c. dose of 2×10⁵ GM-MMB3.19 or non-GM-MMB3.19 tumor cells. One week later, mice were exposed to 8.5 Gy and 4 h later transplanted with either B6 syngeneic 2×10⁶ ATBM cells alone (ATBM and MMB3.19 groups) or in combination with 4-5×10⁶ lymphocytes (DL, GM-MMB3.19 post and pre-BMT + DL and non-GM-MMB3.19 pre-BMT + DL groups). On day 1 post-BMT, all groups except the ATBM control were challenged with an i.p. injection of 1×10⁵ MMB3.19 cells. The GM-MMB3.19 post-BMT group was vaccinated with a single dose of 2×10⁵ GM-MMB3.19 cells on day 1 post-BMT. Data were pooled from 2-3 separate experiments consisting of 5-10 mice per group.

Figure 2

Role of syngeneic DL in the GVL response to MMB3.19 leukemia challenge associated with GM-MMB3.19 and non-GM-MMB3.19 pre-BMT vaccination. B6 mice were vaccinated with a single dose of 2×10⁵ GM-MMB3.19 or non-GM-MMB3.19 cells. One week later, mice were exposed to 8.5 Gy and 4 h later transplanted with either B6 syngeneic 2×10⁶ ATBM cells alone [ATBM, MMB3.19, GM-MMB3.19 pre-BMT (no DL) and non-GM-MMB3.19 pre-BMT (no DL) groups] or in combination with 4-5×10⁶ lymphocytes (DL, GM-MMB3.19 pre-BMT + DL and non-GM-MMB3.19 + DL groups). On day 1 post-BMT, all groups except the ATBM control were challenged with an i.p. injection of 1×10⁵ MMB3.19 cells. Data were pooled from 2-3 separate experiments consisting of 5-10 mice per group.

Figure 3

Role of host T cells in the GVL effect following pre-BMT vaccination with GM-MMB3.19 cells. B6 mice were treated with 0.2 ml of GK1.5 (1:6) and 2.43 (1:50) mAb against CD4⁺ and CD8+ T cells, respectively, 4 days prior to vaccination with a single s.c. dose of 2x105 GM-MMB3.19 cells. One week later (11 days after T cell-depletion), the mice were exposed to 8.5 Gy and 4 h later transplanted with either B6 syngeneic 2×10⁶ ATBM cells alone (ATBM, MMB3.19 and GM-MMB3.19 pre-BMT no DL (T depleted host) groups] or in combination with 4-5×10⁶ lymphocytes [DL, GM-MMB3.19 pre-BMT + DL, and GM-MMB3.19 pre-BMT + DL (T depleted host) groups]. On day 1 post-BMT, all groups except the ATBM control were challenged with an i.p. injection of 1×10⁵ MMB3.19 cells. Data were pooled from 2-3 separate experiments consisting of 5-10 mice per group.

Figure 4

Percentage of CD11c⁺eGFP⁻ cells in the skin-draining LN of vaccinated mice. B6 mice were vaccinated with a single dose of 2×10⁵ GM-MMB3.19, non-GM-MMB3.19 cells or PBS alone. One week later, mice were exposed to 8.5 Gy and 4 h later transplanted with ATBM and lymphocytes from eGFP⁺B6 syngeneic mice in order to be able to differentiated between host (eGFP⁻) and donor cells (eGFP⁺). Three weeks post-BMT, cell suspensions from the skin-draining LN of individual mice were prepared for flow cytometric analysis to determine the percentage of CD11c⁺ expressing cells. Data were pooled from 2 separate experiments consisting of 3-6 mice per group. Statistical difference between groups was determined using Kruskal-Wallis one-way ANOVA analysis (p < 0.01) followed by Dunn’s multiple comparison test on individual pairs (*p < 0.05, ** p < 0.01).

Figure 5

Effect of vaccination site on the survival rate of MMB3.19 myeloid leukemia-challenged mice. B6 mice were vaccinated with a single s.c. or i.p. dose of 2×10⁵ GM-MMB3.19 cells. One week later, mice were exposed to 8.5 Gy and 4 h later transplanted with either B6 syngenic 2×10⁶ ATBM cells alone (ATBM and MMB3.19 groups) or in combination with 4-5×10⁶ lymphocytes (DL, GM-MMB3.19 pre-BMT + DL s.c. and i.p. groups). On day 1 post-BMT, all groups except ATBM controls were challenged with an i.p. injection of 1×10⁵ MMB3.19 cells. Data were pooled from 2-3 separate experiments consisting of 5-10 mice per group.

Figure 6

Rechallenge of surviving GM-MMB3.19 vaccinated mice. Surviving GM-MMB3.19 vaccinated (i.p. or s.c.) mice were rechallenge with 5×10⁶ MMB3.19 cells (i.p.) at ≥ 12 weeks post-BMT, or were used as donors for adoptive transfer of 2×10⁶ ATBM cells along with 4-5×10⁶ DL. For control purposes, their ATBM counterparts were also challenged with an equal dosage of tumor cells or were used as donors in adoptive transfer experiments. Adoptive transfer data were pooled from 2 different experiments of 10 mice per group. Re-challenge data were pooled from 4-5 different experiments consisting of 3-5 mice per group.