Allogeneic hematopoietic stem cell transplant is performed for high risk acute lymphoblastic leukemia (ALL). Complex allogeneic immune responses can produce graft versus host disease which is associated with antileukemia effects. The prevailing paradigm is that these antileukemia effects are mediated by allogeneic T cells. Our laboratory has been studying factors associated with long term disease free survival in murine models of allogeneic transplantation for ALL.
We employed a well characterized murine model of MHC-matched, multiple minor histocompatibility mismatched transplantation in which C3.SW mice are donors and C57BL/6 mice are recipients. The ALL model is a recently derived C57BL/6 murine pre-B ALL driven by a bcr/abl p210 oncogene and an Ink/Arf region deletion; such lesions frequently present in human ALL. Recipients underwent myeloablation with 5-FU and total body irradiation and received hematopoietic grafts comprised of donor marrow and spleen cells. Recipients were challenged with ALL cells three weeks after transplant and followed for survival up to 3 months. Some groups of mice were treated with vaccines comprised of irradiated leukemia cells, Freund's adjuvant and GM-CSF. Serum samples were collected prior to leukemia challenge, three weeks after leukemia challenge and at the conclusion of the experiments. T cell responses were measured with ELISPOT assays specific for minor histocompatibility antigens and leukemia antigen specific responses. Antibody responses were measured by flow cytometry and ELISA. Leukemia was measured by flow cytometry.
Across all experiments approximately 35% of animals challenged with ALL were long term, leukemia free survivors. Interferon-gamma ELISPOT T cell responses specific for minor histocompatibilty antigens were found in none of the survivors and ELISPOT responses to leukemia antigens were seen in only 20% of long term survivors and these were very modest in magnitude. In contrast, IgM and IgG antibodies that bound leukemia cells were found in the vast majority of survivors. Antibody responses were not specific for the ALL with which the animals were challenged. Cross reactivity with other C57BL/6 ALL and AML leukemia lines were observed. In addition, lower levels of antibody cross reactivity were observed against normal C57BL/6 and C3.SW hematopoietic cells. Titers of the antibodies ranged from 1:40 to > 1:100. Antibody responses were observed in mice that had not received post-transplant vaccination. However, post-transplant vaccination significantly increased the titers of leukemia reactive antibodies.
Statistical analysis was performed to determine if antibody levels at any point in the animal's course had any relationship with the risk of later death from leukemia or GVHD. Outcomes for each animal were categorized as alive, death from leukemia, death from GVHD or death of unspecified cause. Antibody levels for each animal were categorized as above or below the median for the entire experimental population. Chi square analysis demonstrated a statistically significant relationship between outcome and antibody level at the time of leukemia challenge. Few animals with high antibody levels died of leukemia (p = 0.0197). In comparison of death from leukemia with death from GVHD, higher antibody levels were associated with death from GVHD (p = 0.0099). A statistically significant difference was not observed between survivors and death from GVHD; however, the death from GVHD group was small, limiting the power of the analysis.
Antibody responses to ALL cells were consistently observed in survivors. These studies do not prove the antibodies play a role in control of leukemia, but suggest the hypothesis that antibody responses may contribute to GVL effects. Current experiments in B cell deficient mice are underway to test the mechanistic role of antibody responses in the GVL effect.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.