Different Subsets of Haematopoietic Cells and Immune Cells in Bone Marrow between Young and Old Donors

Background

Young donors are reported to be associated with better transplant outcomes than old donors in allo-HSCT, but the underlying mechanism is still uncertain. Successful allo-HSCT relies on the rapid reconstitution of donor-derived haematopoietic and immune systems in the recipient. Therefore, characterizing the differences in percentages of HSCs and progenitors and immune cell subtypes between young and old donors may help explain the disparities in transplant outcomes. In humans, HSCs give rise to multipotent progenitors (MPPs) that further segregate into either common myeloid progenitors (CMPs) or multipotent lymphoid progenitors (MLPs), which in turn segregate into either common lymphoid progenitors (CLPs) or granulocyte-macrophage progenitors (GMPs). CMPs further segregate into either megakaryocyte-erythroid progenitor (MEPs), or GMPs. However, differences in the frequencies of HSCs and their progenitors between young and old adults remain uncertain. In addition, aGVHD is generally considered to be associated with increased ratios of donor Th1/Th2, Tc1/Tc2 and M1/M2 macrophage. However, little is known about the cytokine-producing T cell subsets and macrophage subsets in BM between young and old donors.

Aims

To evaluate the different subsets of HSCs and their progenitors and immune cells among young (aged <30 years), middle-aged (aged 30-45 years), and old donors (aged >45 years). Moreover, to analyze the association between donor characteristics and HSC frequency.

Methods

In this prospective study, a total of 60 healthy adult donors, including 20 young donors, 20 middle-aged donors, and 20 old donors were enrolled. The frequencies and ROS levels of BM HSCs(CD34⁺CD38⁻CD90⁺CD45RA⁻) and progenitors including MPPs(CD34⁺CD38⁻CD90⁻CD45RA⁻), MLPs(CD34⁺CD38⁻CD45RA⁺), CLPs(CD34⁺CD38⁺CD7⁻CD10⁺CD45RA⁺), GMPs(CD34⁺CD38⁺CD7⁻CD10⁻CD45RA⁺), CMPs(CD34⁺CD38⁺CD7⁻CD10⁻CD135⁺CD45RA⁺), and MEPs(CD34⁺CD38⁺CD7⁻CD10⁻CD135⁻CD45RA⁻) were quantified by flow cytometry. Furthermore, T cell and macrophage subsets were analyzed in young, middle-aged and old donors by flow cytometry. Effector T cells, naïve T cells, effector memory T cells and central memory T cells were identified as CD45RA⁺CCR7⁻, CD45RA⁺CCR7⁺, CD45RA⁻CCR7⁻, and CD45RA⁻CCR7⁺. Th1, Th2, Tc1 and Tc2 were identified as CD3⁺CD8⁻IFN-γ⁺, CD3⁺CD8⁻IL-4⁺, CD3⁺CD8⁺IFN-γ⁺ and CD3⁺CD8⁺IL-4⁺, respectively. In addition, M1 and M2 were identified as CD14⁺CCR2⁺CD68⁺ and CD14⁺CX3CR1⁺CD163⁺. Moreover, the association of donor characteristics with HSC frequency was analysed by univariate and multivariate analysis.

Results

To determine the differences in HSCs and progenitors in different age donors, HSCs and six subpopulations were compared among young, middle-aged and old donors. The frequencies of HSCs and myeloid progenitors, including CMPs and MEPs in CD34⁺ cells were significantly lower and the frequencies of lymphoid progenitors including MLPs and CLPs in CD34⁺ cells were higher in the BM of young donors than in that of old donors. Significantly lower levels of ROS in HSCs and progenitors were observed in young donors than in the other donors. Furthermore, to investigate the differences in the differentiation potential from HSCs to immune cells in different age donors, T cell and macrophage subsets were compared among the three donor age groups. Young donors demonstrated a lower CD4⁺/CD8⁺ T cell ratio, lower memory T cell frequency and higher naïve T cell frequency in both CD4⁺ cells and CD8⁺ cells. Importantly, BM immune cells from young donors polarized towards less pro-inflammatory T cells characterized by Th1 and Tc1, and more immune suppressor cells, such as M2, than those from old donors. As a result, young donors had lower ratios of Th1/Th2, Tc1/Tc2 and M1/M2 in BM. In addition, multivariate analysis showed that age≥37 was independently correlated with a higher HSC frequency.

Conclusion

BM HSCs from young donors exhibited a lower frequency, balanced myeloid-lymphoid differentiation potential, lower ROS level and produced more immune suppressors and fewer immune effector cells than those from old donors. Donor age might be a good predictor of HSC frequency. Although further validation is required, the differences in the frequency and immune differentiation potential of HSCs in BM between young and old donors may partly explain the different outcomes of allo-HSCT.