Ex Vivo Expanded HOXB4-Transduced Bone Marrow Cells Enhance Allogeneic Repopulation in a Sublethal Irradiation Model of BMT.

Abstract

The successful development of molecular strategies to improve the competitive capacity of donor hematopoietic cells would have major implications for bone marrow transplantation (BMT). The forced expression of HOXB4 has been previously shown to increase the competitive capacity of donor marrow in congenic competitive repopulation assays. However, a major clinical objective is to improve allogeneic engraftment after BMT utilizing reduced intensity conditioning regimens. We hypothesized that expansion of HSCs resulting from forced expression of HOXB4 would enhance stable allogenic engraftment after sublethal myeloablative conditioning.BM mononuclear cells (BMMNC) isolated from B6(H2Kb) donor mice treated with 5-FU (150mg/Kg body weight) were prestimulated with cytokines (IL3, IL6 and SCF) and transduced with an MSCV based retroviral vector expressing either HOXB4-ires-GFP or only GFP. Following incubation with viral supernatants, BMMNC were expanded in the absence of virus, examined for transduction efficiency, and phenotyped by flow cytometry prior to administration of 2.5 x 106 cells to Balb/c (H2Kd) sublethally irradiated (450cGy) mice. Data is expressed as mean ± SEM. Transduction efficiency with both vectors was 45–50% as measured by GFP expression. Phenotypic analysis of transduced cultures showed no significant difference in surface phenotype between the HOXB4 or GFP transduced BM cells. The majority of the cells were CD45R/B220 positive (70%) and/or CD11b positive (55%). As early as 4 weeks post-transplantation, GFP and H2Kb positive donor derived cells were present in the peripheral blood of recipients. At 4 weeks, HOXB4 transduced donor cells engrafted at a higher rate 55% (5 out of 9 animals; donor cells in PB = 81.92 ± 10.25%; n = 5) compared to 33% engraftment (3 out of 9 animals) with GFP transduced cells (donor cells in PB = 39.11 ± 20.66; n = 3). Long term donor cell chimerism (16 weeks) could be measured in 3 of 9 HOXB4 transduced recipients while GFP engrafted recipients failed to survive beyond 6 weeks. Multilineage analysis of donor derived phenotypes in long term HOXB4 engrafted mice in PB, BM and spleen are shown in the table. Analysis of thymus in one of the HOXB4 engrafted recipients showed CD4 +/GFP+ H2Kb (76.49%) or CD8+/GFP+ H2Kb (76.95%) cells, indicating the development of T-cell lineages from donor derived HOXB4 transduced HSCs.Our results indicate engraftment and long term donor chimerism throughout the hematopoietic compartment in a murine allogenic model. However, forced HOXB4 expression in HSCs also resulted in increased myeloid lineage expression in chimeric animals, consistent with earlier observations in congenic transplants (Antonchuk et al, 2002). Though the mechanisms of HOXB4 induced HSC self renewal and multilineage hematopoiesis are still not clear, its capability to contribute to enhanced engraftment across major histocompatibility complex barriers supports its potential utility for allogeneic BMT.Lineage Engraftment (%) of Donor Cells (GFP+/H2Kb+) in Hematopoietic TissuesTissueCD3CD11bCD19CD45PB4.14 ± 0.3983.77 ± 10.1411.25 ± 11.7389.47 ± 8.17BM1.56 ± 1.165.81 ± 9.85.76 ± 2.980.54 ± 6.2Spleen12.15 ± 10.145.92 ±25.8124.15 ± 21.585.87 ± 5.3