Self Renewal and Proliferative Signals in the Bone Marrow Microenvironment Promote Homeostatic Peripheral Expansion of Donor-Derived T Cell Subsets Following Stem Cell Transplantation.
Authors: Chua Hui Lin , Ngyuen Ngoc-Thinh , Plett P. Artur , Orschell Christie M. 
Published: October 2019

Abstract
The bone marrow (BM), in addition to its role in providing hematopoietic stem cell support, is becoming increasingly recognized as a significant reservoir for T cells. Recent data have shown the BM to be the predominant site for homeostatic peripheral expansion (HPE). Constitutive HPE is a mechanism which allows maintenance of T cell numbers, while lymphopenia-induced HPE restores T cell numbers following T cell-depleting measures such as myelosuppression. While the role of BM in constitutive HPE in non-irradiated mice has been well documented, little is known of the role of BM in HPE following myeloablation. In the present study, we examined the contribution of BM to lymphopenia-induced HPE following myeloablative radiation conditioning and HSC transplantation, and documented the kinetics of expansion of donor-derived T cell subsets in the irradiated marrow and spleen. To this end, C57BL/6 and BoyJ congenic mice, differing in CD45 allelic expression, were used to track donor-derived T cell subsets using 8 color flow cytometry 5 days after transplantation of 10–20 × 10e6 low density BM cells (non-T cell depleted) into lethally-irradiated recipients (950cGy). At the time of analysis (d5 post transplant) the percentage of total donor cells in BM was approximately 20%, and 10% in the spleen. Of interest, an enhanced migration of CD8+ cells to BM was observed, increasing from 3±0.6% in steady state marrow to 8±3.3% of donor cells in transplanted mice. While only 10.1±1.0% of CD8+ cells in steady state BM exhibited a central memory phenotype (TCM, CD44hi, CD45RBhi, CD62Lhi), this percentage increased to 90±2.7% of donor CD8+ cells in day 5 transplanted BM (p<0.05). This preferential migration of TCM to irradiated BM is in contrast to the spleen, which contained approximately equal percentages of donor CD8+ naïve (CD44lo, CD62Lhi), effector (CD44hi, CD45RBlo, CD62Llo), and TCM cells. The reported increase in SDF-1 expression in irradiated BM relative to spleen may contribute to the enhanced TCM migration to BM, since TCM are more responsive to SDF-1 than naive cells. In experiments where donor cells were stained with CFSE prior to transplantation, proliferation of the different CD8+ subsets was followed. While the majority (∼97%) of BM TCM proliferated and lost CFSE fluorescence by day 5, a small percentage (2.8%) remained quiescent, exhibiting bright CFSE fluorescence (CFSEbright). In contrast, 10–20% of spleen TCM remained quiescent. These findings are in support of data documenting the BM as the predominant site for HPE, and illustrate the highly proliferative environment in the BM compared to spleen for expansion of TCM cells. Despite the higher degree of proliferation in the BM, 2-fold more BM CFSEbright TCM cells express IL7Ralpha, a molecule important in self renewal, compared to spleen CFSEbright cells. Given that TCM cells reportedly possess a similar self renewal genetic profile as hematopoietic stem cells, it is possible that TCM cells migrating through the BM following HSC transplantation are responsive to some of the same self renewal signals important for HSC self renewal, which would be presumably plentiful in the post-irradiation/transplantation BM microenvironment. In this regard, expression of IL7Ralpha by BM-homed TCM may allow for significant expansion while maintaining a self renewal phenotype, requirements which may be important for efficient HPE following myeloablation and stem cell transplantation.