Under the first aim of this project we have generated a precursor B cell leukemia line derived from mice with transgenic expression of E2aPBX1, a recurring translocation present in approximately 5% of pediatric ALL (Bijl et al, Genes and Development, 2005). Injection of these cells intravenously reproducibly results in leukemia development from as few as 10,000 cells with distribution to bone marrow, blood, lymph nodes, spleen, liver and central nervous system. Using congenic markers, leukemia cells can be identified in the bone marrow as early as 3 days after intravenous injection with a sensitivity of 0.01% of bone marrow cells. A subline has been transfected with luciferase and leukemia development can also be tracked in situ by luminescent imaging. The cell line was confirmed to be immunogeneic as vaccination with irradiated leukemia cells protects against subsequent challenge with E2aPBX1 but not other tumors. Using monoclonal antibodies to deplete cell subsets, we demonstrated that protection in immunized mice requires both CD4 and CD8 T cells and is impaired following NK cell depletion.
Under aim 2 we have performed syngeneic bone marrow transplantation experiments in which mice were injected with E2aPBX1 one week following transplant. This was followed by adoptive transfer of T cells from donors primed with an irradiated E2aPBX1 whole-cell vaccine. While leukemia development was delayed compared to BMT recipients receiving purified T cells from unprimed mice, all recipients eventually succumbed to leukemia. Together, this data indicates that whole-cell vaccination induces a tumor-specific, T cell mediated immune response that is unable to prevent the ultimate develop of leukemia following syngeneic BMT. Importantly, these experiments also demonstrate that ALL can be targeted by T cells but requires prior priming to leukemia antigens The use of allogeneic bone marrow rather than syngeneic bone marrow introduces minor histocomaptibility antigens as potential targets on leukemia cells. Indeed, allogeneic transplantation followed by E2aPBX1 leukemia challenge and subsequent transfer of primed allogeneic T cells results in cure of leukemia in all mice. However, the mice develop weight loss and histologic changes consistent with GVHD that results in late mortality. Interestingly, priming T cell donors with recipient (and leukemia) strain non-malignant B cells did not cure the mice indicating that both minor antigens and leukemia-associated antigens are responsible for cure in this model. We next sought to separate the anti-leukemic GVL effect from GVHD by selecting for T cells subsets. Neither CD4 nor CD8 T cells from primed donors alone were sufficient to cure all of the mice. Using flow sorting based on expression of CD44 and CD62L (L-selectin) we have demonstrated that central memory phenotype T cells (CD44+/CD62L+) can cure leukemia without the induction of GVHD whereas nave T cells (CD44-/CD62L+) induce rapidly lethal GVHD. In summary, we have demonstrated that ALL can be effectively targeted by a T cell response in vivo but that this response requires vaccination of the donor T Cell inocula. Second, allogeneic antigens contribute to the cure following T cell infusion but results in GVHD. Finally, sorted populations of T cells from primed donors can mediate selective graft versus leukemia responses. Using this model we have begun studying the early progression of the leukemia in bone marrow and the impact of this progression on T cells. We have identified that a surprising large percentage of T cells in leukemia-infiltrated compartments express high levels of the negative regulator of T cell function, programmed death 1 (PD-1) receptor. In addition, E2aPBX1 cells express the ligand for this receptor (PD-L1) in vitro and in vivo. Addition studies have shown that the percentage of PD-1+ T cells correlates with the extent of leukemic involvement and that PD-1+ T cells also express other markers of a senescent phenotype such as T cell immunoglobulin and mucin domain 3 (Tim-3). Ongoing studies are exploring the mechanism of PD-1 induction on T cells. Initial studies indicate that this does not happen during coculture in vitro suggesting that factors present in the bone marrow environment may be required Finally, preliminary studies have indicated that blockade of PD-1 can enhance the therapeutic efficacy of adoptive T cell therapy. Another area of investigation in the laboratory is focused on understanding the impact of the response to minor histocompatibility antigens expressed in normal tissues (graft versus host) on the anti-tumor immune response following allogeneic HSCT. Under project Project ZIA BC 011320, we have demonstrated and published that even mild GVHD can significantly impair responses to vaccines targeting tumors (Capitini et al, Blood, 2009). We have now shown that this attenuation of vaccine responses results from both diminished proliferation and increased apoptosis. The tumor antigenic complex used in these studies is the HY system in which a solid tumor that naturally expresses Y chromosome-derived antigens was injected into female mice receiving female allogeneic bone marrow and T cells (where the mouse strain-specific minor allogeneic antigens are distinct from the HY-derived tumor antigens). Ongoing studies are now assessing HY tumor responses in male mice of the same strain from which the tumor is derived. Importantly, in this model, the tumor antigens completely overlap with tumor antigens, a clinically relevant scenario in which tumor-specific antigens may be weak or absent. The use of CD4 and CD8 HY specific T cell receptor transgenic donors allows careful tracking of T cells targeting shared antigens. Preliminary studies indicated that, while HY-specific T cells mediate mild GVHD and expand to a much larger extent in males than in female hosts (despite HY vaccination), these T cells with specificity to both normal tissues and tumors are less potent at inducing tumor regression. Ongoing work is exploring the mechanism of this T cell dysfunction. We are currently breeding E2aPBX1 transgenic mice from which HY-expressing pre-B cell leukemia lines can be generated. Thus, we will be able to study whether the impact of normal tissue expression of tumor antigens will have a similar impact on immune responses to hematologic malignancies.
Aim 3 is ongoing and involves the extension of work conducted under aim 2 to clinically relevant leukemia targets. We have established that E2aPBX1 overexpresses the Wilm's Tumor 1 gene, also overexpressed on approximately 70-80% of human leukemias and validated as a target in patients. In order to obtain large numbers of WT-1 specific T cells, we have generated mice that express a T cell receptor specific for the dominant class I epitope derived from WT-1 (called Db126). T cells from these mice show robust expansion to Db126 peptide in vitro resulting T cells with the capability of targeting peptide pulsed targets in vitro. While E2aPBX1 cells can also be targeted, the results have been variable. We are in the process of optimizing the use of these T cells in vivo against E2aPBX1 and other hematologic malignancies that overexpress WT-1. An open protocol in the Pediatric Oncology Branch (Dr. Alan Wayne, Principal Investigator) is utilizing dendritic cell vaccination targeting WT-1 peptides as a therapeutic intervention in patients relapsing following allogeneic HSCT. Results obtained from our preclinical studies assessing the impact of alloreactivity on vaccine responses will be used to develop subsequent post-transplant vaccine intervention trials to reduce the risk of relapse in appropriately selected high-risk groups. We hope to conduct these trials in collaboration with the Pediatric Blood and Marrow Transplant Consortium.
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|Chung, Yang Jo; Fry, Terry J; Aplan, Peter D (2017) Myeloablative hematopoietic stem cell transplantation improves survival but is not curative in a pre-clinical model of myelodysplastic syndrome. PLoS One 12:e0185219|
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