Anti-tumor Assessment Of Hoxb5-Converted T Cells And Functions Of Hoxb5-regulated Hematopoietic Multipotent Progenitors

The transcription factor Hoxb5 is highly expressed in hematopoietic stem cells(HSCs)and specifically marks the identity of HSCs.Hoxb5 is significantly downregulated in hematopoietic multipotent progenitor cells(MPPs)and completely silenced in lineage-committed progenitor cells.We recently reported that enforced expression of Hoxb5 converted B cell progenitors into functional T cells in vivo.However,the functions of Hoxb5 in the hematopoietic hierarchy,especially in MPPs,remains elusive.In this study,we first investigated the anti-tumor activity of Hoxb5-converted T cells,then explored the roles of Hoxb5 in regulating the functions of MPPs.The first chapter reported that Hoxb5 can convert the B cell progenitor(pro-pre-B)cells isolated from OT1(MHC-I restricted OVA antigen)transgenic mouse into the naive iT cells expressing TCRV?2 and TCRV?5(OTl-iT)in vivo,which exhibited strong anti-tumor ability.Briefly,we transduced the OT1 pro-pre-B cells with the retroviruses expressing Hoxb5(Hoxb5-retrovirus),and transplanted them into the Rag1-/-mouse.Four weeks post-transplantation,OT1-iT cells were generated in the recipients(OT1-iT-Rag1-/-),and BCR heavy chain(V(D)J)rearrangement analysis from the single OT1-iT cells shows that these iT cells carried the rearranged BCR heavy chain.Therefore,we proved that the OT1-iT cells were indeed regenerated from OT1 pro-pre-B cells.In order to evaluate the tumor-killing capacity of the regenerated OT1-iT cells,we co-cultured the splenic OT1-iT cells isolated from OT1-iT-Rag1-/-mouse with the melanoma cells expressing OVA(B16F10-OVA)and found that OT1-iT cells can effectively kill the tumor cells in vitro.Of note,the pre-activated OT1-iT cells showed stronger tumor-killing abilities.Next,we performed in vivo anti-tumor assays.We subcutaneously injected the B16F10-OVA cells into the OT1-iT-Rag1-/-mouse and found that the tumors didn't arise for a long time compared with the control group,which shows the preventive impact of OT1-iT cells in OT1-iT-Rag1-/-mouse.Moreover,we transferred the expanded OT1-iT cells into the tumor-bearing Rag1-/-mouse which were injected with B16F10-OVA cells.Our results showed that the OT1-iT cells significantly inhibited the growth of the tumors.In addition,retroviruses expressing with OT1-Hoxb5(OT1-Hoxb5-retrovirus)also converted the pro-pre-B cells isolated from the wide type mouse(C57BL/6),into OT1-iT cells in vivo,though with lower efficiency than transgenic OT1-pro-pre-B cells.Taken together,this study provides insights into the translational applications of blood lineage-transdifferentiated T cells in immunotherapy.In the second chapter,we explored the functions of the multipotent progenitor cells(MPPs)after the enforced expression of Hoxb5.We sorted the GFP-MPPs,wherein Hoxb5 is not yet turned on,from the bone marrow cells of the Hoxb5LSL/+Mx1-cre mouse.Then we transplanted them into the lethally irradiated recipients(CD45.1+)to enforce the Hoxb5 expression by polyinosinic-polycytidylic acid(pIpC).Interestingly,the Hoxb5-expressing MPPs exhibited long-term hematopoiesis in the primary recipients.Moreover,the Hoxb5-expressing MPPs generated a de novo Sca1+cKit+Mac1+CD48+(Mac1+CD48+SK)cell type,which was responsible for repopulating long-term multi-lineage hematopoiesis in serial transplant recipients,Furthermore,the Mac1+CD48+SK cells can not only self-renew but also give rise to the progenies of myeloid progenitors(MP)and common lymphoid progenitors(CLP),which suggest their multi-potent hematopoietic ability.Next,both bulk and single cell RNA-seq analyses revealed that Mac1+CD48+SK cells exhibited activated machineries of DNA replication and cell division whereas maintained their self-renewal potential.However,the HOXB5-expressing MPP cells isolated from human umbilical cord blood failed to produce long-term hematopoiesis in B-NDG recipients.One possible underlying reason is that HOXB5-overexpressing human MPPs require a humanized bone marrow micro-environment for HOXB5-reprogramming,which is not available in the current immunodeficient animal models.Collectively,our study uncovers that Hoxb5 can equip MPPs with self-renewal potential,thereby providing new strategies to convert blood progenitor cells into alternative self-renewable cell source for therapeutic purposes.