Achieving Selective Graft-Versus-Leukemia without Graft-Versus-Host Effects by WT1 Peptide Vaccination of Donors and Stringent Engineering of the Allo-Graft
Authors: Mueller Antonia MS , Shizuru Judith A. , Kohrt Holbrook E 
Published: October 2019

Abstract
Abstract 4108
A major goal for allogeneic hematopoietic cell transplantation (HCT) is to engineer grafts to contain only cell types required to rescue hematopoiesis, recover anti-infectious immune function, and provide curative graft-versus leukemia (GVL) effects. Such grafts will avoid the life-threatening complication of graft-versus-host disease (GVHD). Before manipulative approaches can be translated into clinical practice, the dynamics of engraftment, expansion, and interactions of isolated donor cell populations must be comprehensively understood. Here, in preclinical mouse models, we dissect the effects and potency of distinct purified donor cell populations to exert GVL. To enhance GVL we vaccinated donors with a tumor-antigen, WT1. AKR/b or C3H/SW mice were prepared with 4 weekly WT1-peptide/incomplete Freund's adjuvant vaccinations. Grafts consisted of FACS-purified hematopoietic stem cells (HSC [cKit+Thy1.1loLin−Sca1+]), augmented with CD4+, CD8+, naïve or memory CD4+ and CD8+T cells (TC), or WT1-tetramer selected (WT1)CD8+TC. HSC +/− TC subsets were infused into lethally irradiated, minor-antigen mismatched C57BL/6 mice. A luciferase-labeled, myeloid lineage C57BL/6 leukemia, FBL3, was inoculated on d0 or 14 post-HCT, modeling tumor persistence or early relapse after HCT, respectively. Graft compositions were tested for their strongest GVL potency measured by bioluminescence imaging and survival. When recipients were given tumor cells at the time of HCT (i) HSC+CD4++CD8+ TC grafts from vaccinated donors rescued most recipients, while those from unvaccinated mice resulted in rapid death from leukemia; (ii) recipients of HSC+CD4++CD8+ from unvaccinated mice died faster than recipients of HSC alone, suggesting GVH-related damage to lymphoid organs diminishes the host's endogenous tumor resistance; (iii) HSC + either CD8+, memory (mem)CD8+, or (WT1)CD8+TC from vaccinated donors provided improved but non-curative protection from leukemia compared to HSC alone; (iv) HSC + CD4+TC grafts (vaccinated or unvaccinated) were associated with rapid death; however, (v) CD4+TC were required to fortify tumor eradication by CD8+TC. To model the clinically relevant scenario of early relapse, we next infused tumor cells on d14 post-HCT. At 1 wk prior to tumor inoculation an in vivo boost WT1-peptide vaccination was applied. (i) Again, HSC+CD4++CD8+ from vaccinated donors achieved better tumor control as compared to HSC +/− unvaccinated CD4++CD8+ TC, but a high proportion of recipients died due to progressive leukemia. We speculate the reason for diminished GVL is a lack of tumor antigens present at the time of TC infusion which resulted in a different donor TC repertoire expanding during lymphopenia. (ii) All subgroups given combinations of naïve/memory CD8+ TC and naïve/memory CD4+ TC had prolonged survival compared with HSC recipients, but no long-term cure. (iii) The only group with improved overall survival received HSC + WT1CD8++ CD4+ TC grafts and a booster vaccine prior to tumor injection. Our findings suggest the benefit was due to an in vivo expansion of tumor-specific TC early post-HCT, driven by the tumor antigen + adjuvant re-exposure. We observed with peptide vaccination that: 1.) ∼2/3 of WT1CD8+TC retained a naïve phenotype, and CD4+TC were required to provide help and promote, suggesting that CD4+TC are required to convert naïve into effector memory CD8+TC. 2.) viral immunizations resulted in robust antigen specific responses compared to more modest development of WT1CD8+TC after the peptide-vaccine. However, the minimal numbers of WT1-specific CD8+TC transferred were highly efficient, whereas recipients of HSC+WT1-depleted TC died rapidly of leukemia, which supports the specificity of the response rather than general alloreactivity. Thus, dynamics of engraftment of donor effector populations are complex, modalities to promote tumor specific CD8+TC expansion early post-HCT are critical to maximize GVL and rely upon the interactions of co-transplanted cell subsets and timing of infusions. Immunogenic maneuvers such as vaccinations are powerful tools to enhance the potency of specific effector populations. Transplant approaches using pure HSC to restore hematopoiesis and manipulated TC populations without deleterious GVHD-inducing effects are the ultimate goal of allogeneic HCT and require informed translation of preclinical models.

Disclosures:
No relevant conflicts of interest to declare.