The Reduced Lymphopoietic Potential of a Novel Subclass of Hematopoietic Stem Cells Is Mediated by Specific Defects In the Production and Activity of Their Common Lymphoid Progenitor (CLP) Progeny

Abstract 576

Hematopoietic stem cells (HSCs) represent a rare subset of bone marrow (BM) cells in adult mice that are ultimately responsible for the replenishment of all different mature blood cell types through a hierarchically organized cascade of differentiation steps. Although HSCs were previously considered a functionally and phenotypically homogeneous population, recent studies tracking the 4 to 6 month progeny of a large number of highly purified, individually transplanted, single mouse HSCs and their serially generated derivatives in sublethally irradiated hosts clearly reveal a functional heterogeneity of HSCs. The results have revealed 2 distinct and stably propagated clonal subtypes of HSCs with unrestricted self renewal activity that we have termed α- and β-HSCs. β-HSCs are those that show a relatively balanced output of mature myeloid and lymphoid cells. α-HSCs have an equivalent self-renewal and myelopoietic activity to β-HSCs but, in contrast, are characterized by a variable and often extreme failure to produce mature lymphoid cells. Since it is well established that the reduced activity of the immune system with aging corresponds with a decrease in the frequency and activity of both B- and T lymphocytes and their respective progenitors, it is of interest to determine whether this simply represents a generalized aging of the hematopoietic system or rather an age-related change in the composition of the HSC compartment. The most rigorous approach to determine the time of appearance and kinetic changes in the distribution of different types of HSCs during development and aging is to study single-cell transplants and their clonal progeny using an ontogeny-independent HSC purification scheme. We recently showed that the E-SLAM (CD45⁺ Endothelial protein receptor EPCR⁺ CD150⁺ CD48⁻) phenotype could be used to achieve high HSC purity across development (E14.5 fetal liver, 3-week BM, 4-week BM, 10–12 week BM and aged BM) despite known ontological differences in HSC proliferative activity between these populations. These highly-purified populations of HSCs can thus be utilized to prospectively isolate and singly transplant these HSCs to enable study of their clonally progeny. Clonal analysis of the E-SLAM HSC compartment of aged mice showed an increase in frequency of α-HSCs. To investigate the mechanism underlying the lymphopoietic deficiency characteristic of α-HSCs, we undertook a set of experiments to compare the in vivo generated progeny of α- vs β-HSCs. We particularly focused on a quantitative and qualitative analysis of the common lymphoid progenitor (CLP) compartment (Lin⁻ CD127⁺ CD117^low Sca1^low). In contrast to previous reports of a decrease in CLP numbers in old mice, we simply found a broader distribution of CLPs in old mice. We did however find a decreased number of CLPs generated per HSC measured as the CLP/LSK ratio (Lin⁻ CD127⁻ CD117⁺ Sca1⁺) with age suggesting that – if the aged BM is dominated by α-HSCs – that α-HSCs possess a quantitative defect in CLP production. Intriguingly, the number of CLPs derived from mice reconstituted with an α stem cell were significantly reduced in comparison to CLPs derived from β-HSCs. To measure the B-cell production activity of CLPs produced from α- vs β-HSCs we utilized the OP9 co-culture system and plated limiting numbers of CLPs derived from reconstituted mice which were then compared to the CLP productivity of young and old CLPs from non-reconstitued mice. Functional analysis of CLPs from individual young mice showed a consistent B-cell frequency of 1/3.6 (n=13) in the OP9 co-culture system whereas CLPs from old mice showed either good B-cell potential comparable to young CLPs or very reduced B–cell potential. CLPs derived from β-HSCs show a consistent B-cell potential (1/11.3, n=9) even after reaching a physiological age of >100wks whereas when CLPs could be detected from α-HSCs they showed a reduced B-cell activity in vitro. The results of our study demonstrate that the decrease in lymphoid activity during aging is likely caused by the relative increase in α-HSCs which have both a quantitative and qualitative CLP defect.