Abstract
The use of cytotoxic T-lymphocytes (CTL) has been attempted experimentally with various tumors to achieve disease control. Factors that may influence GVT include CTL cytotoxicity, ability to home to disease sites, and survival of T cells in the host. The objective of our study is to evaluate the GVL effects of human alloreactive CTL against ALL in a chimeric NOD/scid mouse model. CTL were generated from random blood donor PBMCs stimulated with the 697 human ALL cell line and supplemented with IL-2, -7, or -15. CTL were analyzed for in vitro cytotoxicity against 697 cells, phenotype, and in vitro migration on day 14. NOD/scid mice were injected with 107 697 ALL cells followed by 5x106 CTL. Mice were sacrificed seven days following CTL injection and residual leukemia was measured in the bone marrow and spleen via flow cytometry. The ratios of CD8/CD4 positive T cells at the time of injection were 46/21% for IL-2, 52/31% for IL-7, and 45/14% for IL-15 cultured CTL (n=13). Control mice not receiving CTL had a baseline leukemia burden of 2.01% and 0.15% in the bone marrow and spleen, respectively (n=15). Mice treated with IL-15 cultured CTL had a reduction in tumor burden to 0.2% (n=13, p=0.01) and 0.05% (n=13, p=0.01) in bone marrow and spleen, respectively. Those treated with IL-2 or IL-7 cultured CTL showed no significant difference in leukemia burden in either the bone marrow (IL-2 1.28%, Il-7 5.97%) or spleen (IL-2 0.4%, IL-7 0.33%). No residual CTL could be identified in the bone marrow or spleen at the time of sacrifice in any CTL group. CTL grown in each cytokine resulted in similar in vitro cytotoxicity at an effector:target ratio of 10:1 (IL-2 41.3%, IL-7 37.7%, IL-15 45.3%, n=12–15, p>0.05 for all groups) and had statistically similar intracellular perforin and granzyme-B expression. In vitro CTL migration to a human mesenchymal stem cell line was greatest with IL-15 CTL (30.5%, n=4), followed by IL-7 CTL (18.9%, n=4), and least in IL-2 CTL (17.9%, n=4), though the differences were not significant. In vitro CTL migration was analyzed to an SDF-1α gradient as CXCR4/SDF-1α interactions are necessary for hematopoietic progenitor cell homing to the bone marrow. IL-15 cultured CTL showed the highest migration (48.8%, n=8) as compared to IL-2 (21.7%, n=6, p=0.048) or IL-7 CTL (35.9%, n=8, p>0.05). However, surface expression of CXCR4 measured by flow cytometry was significantly higher in IL-7 CTL (89.4%, n=9) compared to IL-2 CTL (52.2%, n=9, p<0.001) and IL-15 CTL (65.4%, n=10, p=0.002). Experiments are currently underway to further evaluate the role of CXCR4/SDF-1α in GVL. Preliminary in vivo experiments do not suggest any significant differences in CTL engraftment when evaluated at 24 hours post injection. Expression of the anti-apoptotic bcl-2 protein was greatest on IL-7 (MFI=5295, n=13) and IL-15 (MFI=4865, n=14) when compared to IL-2 CTL (MFI=3530, n=13, p=0.02 vs. IL-7, p=0.05 vs. IL-15), suggesting an increased in vivo survival ability. We hypothesize that IL-15 cultured CTL have greater GVL effects due to either higher in vivo survival, greater bone marrow homing efficiency, or both. Future experiments are planned to evaluate in vivo administration of IL-2 to enhance CTL survival in the host. In conclusion, IL-15 cultured CTL had significantly greater in vivo GVL effects compared to IL-2 and IL-7 CTL in the NOD/scid mouse model. This model can be utilized to evaluate the mechanism of T cell mediated GVL against ALL and potentially other human malignancies.
Generation of prolactin-inducible protein (Pip) knockout mice by CRISPR/Cas9-mediated gene engineering
