CD8+ Effector T Cell Migration into the Bone Marrow of Transplanted Mice: Influence of Gvhd, Leukemia Cells and Variable Dependence on Chemokine Receptors
Authors: Zhao Kai , Zhu Jieqing , Rosenberger Sarah , Zhou Meng , Shlomchik Warren D 
Published: October 2019

Abstract
The graft-versus-leukemia (GVL) effect, mediated by alloreactive T cells, reduces the risk of leukemia relapse after allogeneic hematopoietic stem cell transplantation. While much work has focused on how T cells kill leukemia cells, less attention has been paid to the process whereby activated alloreactive T cells find leukemic targets. We initially focused on how chemokine receptor signaling impacts effector T cells egress from blood, entry into the marrow space and subsequent scanning of leukemia cells. We further tested how this process is influenced by transplantation and the consequences of alloreactive T cells. We tracked the migration of CD8+ RAG1-/- OT-1 TCR transgenic effectors (generated by in vivo vaccination of B6 mice seeded with naïve OT-1 cells) in recipients. OT-1 cells recognize the SIINFEKL peptide from ovalbumin and are not reactive in our models. OT-1 effectors were treated (or not) with pertussis toxin (PTX) to block chemokine receptor signaling, and labeled with fluorescent dyes so as they could be identified by flow cytometry and two photon intravital microscopy (2PIM). PTX+ and PTX- OT-1 cells were transferred together into the following recipients: unmanipulated B6 mice, irradiated (B6xBALB/c) CB6F1mice transplanted with B6 bone marrow (BM) cell, with or without B6 T cells. In some experiments on day 12 post-transplant, F1 mice also received WT or IFN-γR-/- B6 blast crisis CML cells (BC-CML) generated by transduction of BM with retrovirus encoding bcr-abl and the NUP98/HOXA9 fusion as we have described (J Clin Invest. 2017;127(7):2765-2776). OT-1 effectors were infused on day 14 and 3 hours later the calvarium was analyzed by 2PIM to enumerate PTX treated or untreated OT-1 effectors that were inside or outside the blood vessels. Blood vessels were identified using labeled dextran or Qdot 655.
PTX treatment was highly effective as PTX-treated OT-1 were nearly completely excluded from lymph node (LN). PTX treatment reduced OT1 migration into the BM of unmanipulated B6 mice and similarly in F1 recipients without T cells. However, the presence of a GVH response reduced the recruitment of PTX- OT-1 cells into BM. The ratio of OT-1 outside/inside vessels was still higher in the PTX- group; however, this was mostly driven by an increase of PTX+ OT-1 in vessels in GVH mice. These data suggest that GVH may reduce effector T cell recruitment to the BM, though some of this effect could be due to effectors trafficking instead to other locations, perhaps GVHD target tissues.
The presence of WT BC-CML leukemia cells in F1 GVH recipients increased recruitment of OT-1 PTX- effectors into the BM, both relative to PTX+ OT-1 and in total numbers compared to experiments wherein no BC-CML cells were infused. We considered the possibility that IFN-g-stimulation of BC-CML cells may have released chemokines that recruited PTX- OT-1. However, IFNγR-/- BC-CML also increased PTX- OT-1 recruitment in GVHD mice to a similar degree. Experiments are ongoing to better understand how BC-CML cells promote PTX-inhibitable effector T cell recruitment.

Disclosures
Shlomchik: NapaJen: Consultancy.