Lymphoid-Biased Hematopoietic Stem Cells Are Maintained with Age and Efficiently Generate Lymphoid Progeny

Abstract

Current models propose that reductions in the number of lymphoid-biased hematopoietic stem cells (Ly-HSCs) underlie age-related declines in lymphopoiesis. We show that Ly-HSCs do not decline in number with age. Old Ly-HSCs exhibit changes in gene expression and a myeloid-biased genetic profile, but we demonstrate that they retain normal lymphoid potential when removed from the old in vivo environment. Additional studies showing that interleukin-1 inhibits Ly-HSC lymphoid potential provide support for the hypothesis that increased production of inflammatory cytokines during aging underlies declines in lymphocyte production. These results indicate that current models proposing that lymphopoiesis declines with age due to loss of Ly-HSCs require revision and provide an additional perspective on why lymphocyte development in the elderly is attenuated.

Keywords: aging; hematopoietic stem cells; lymphopoiesis; myelopoiesis.

Graphical abstract

Figure 1

Quantification of Ly-HSCs and My-HSCs in the Marrow of Young and Old Mice (A) Fluorescence-activated cell sorting (FACS) plots showing the strategy used for the resolution of CD135⁻ CD150^low Ly-HSCs and CD135⁻ CD150^high My-HSCs within the lineage-negative, Sca-1⁺ CD117 (c-Kit)⁺ (LSK) CD48⁻ population. Figure S1A shows the gating strategy. (B) Relative frequency of Ly-HSCs and My-HSCs within the total HSCs in the bone marrow of young and old B6 mice. (C) Frequency and total number of HSCs in the bone marrow of young and old B6 mice. (D) Total number of Ly-HSCs and My-HSCs in the bone marrow of young and old B6 mice. (E) Number of total HSCs (left panel) and Ly-HSCs and My-HSCs (right panel) in the bone marrow of young and old BALB/c mice. (B–E) Each symbol represents a mouse (young, 8–12 weeks; old, 17–18 months); levels of significance for differences between populations are indicated. Error bars indicate means ± SEM.

Figure 2

Ly-HSCs and My-HSCs Exhibit Specific Changes in Gene Expression with Age (A) Venn diagram showing the number of genes whose expression changed significantly (adjusted p value <0.05) between young and old Ly-HSCs and young and old My-HSCs and which changed in common in both subsets. (B) Hierarchical clustering for the 44 genes classified as age regulated in both Ly-HSCs and My-HSCs for each individual biological replicate.

Figure 3

Ly-HSCs Acquire a Myeloid-Biased Pattern of Gene Expression with Age (A) Multidimensional scaling analysis of young and old Ly-HSCs and My-HSCs. Each symbol represents a distinct cohort of Ly-HSCs or My-HSCs isolated from 6 to 8 B6 mice. The scatterplot shows first multidimensional scales, obtained from all variable (age and lineage bias) genes (1,062 genes, y axis) or from those identified as regulated between young and old Ly-HSCs (337 genes, x axis). (B) Hierarchical clustering for genes classified as significant (adjusted p value <0.05) in a pairwise test between young Ly-HSCs and the rest of the samples. Genes are grouped by functional categories (only significant categories are shown, p < 0.01). Representative gene names are shown (blue, lower expression; red, higher expression in young Ly-HSCs). A list of the specific genes in each plot can be found in Table S2.

Figure 4

Young and Old Ly-HSCs Exhibit Similar Levels of Proliferation and Reconstitution Potential (A) Representative FACS plot showing Ki-67 staining of total young HSCs with Ki-67 antibody and isotype control. (B) Frequency of Ki-67 expressing young and old total HSCs. (C) Frequency of Ki-67 expressing young and old Ly-HSCs and young and old My-HSCs. Levels of significance are indicated. Each symbol in (B and C) represents an individual mouse. Error bars indicate means ± SEM. (D) Left panels: representative FACS plot showing resolution of CD45.1 recipient and CD45.2 donor cells in the bone marrow of Busulfan-conditioned CD45.1 B6 mice 5 and 16 weeks after transplantation. Right panels: percent donor chimerism in the marrow of Busulfan-conditioned CD45.1 mice at 5 and 16 weeks post-transplantation. No difference in total bone marrow cellularity was observed in any of the recipients at either time point. Error bars indicate means ± SD. Each symbol represents an individual recipient mouse.

Figure 5

The Developmental Potential of Young and Old Ly-HSCs Is Similar (A and B) Relative frequency of donor (A) HSCs and (B) MPPs in recipients of 200 young and 200 old Ly-HSCs at 5 and 16 weeks post-reconstitution. (C) Relative frequency of donor CLPs, Fraction A pre-pro-B, Fraction B early pro-B, Fraction C + C′ late pro-B, and Fraction D pre-B cells in recipients of 200 young and 200 old Ly-HSCs at 5 and 16 weeks post-reconstitution. (D) Relative frequency of donor CMPs, GMPs, and MEPs in recipients of 200 young and 200 old Ly-HSCs at 5 and 16 weeks post-reconstitution. (E) Donor cell engraftment and relative frequency of surface IgM⁺ B cells, combined CD4⁺ and CD8⁺ T cells, and CD11b⁺ myeloid cells in the peripheral blood of recipients of young or old Ly-HSCs 16 weeks post-reconstitution. The mice analyzed are the same as those described in Figure 4. Each symbol is an individual recipient mouse. Error bars indicate means ± SD.

Figure 6

Old Ly-HSCs Generate Lymphoid Progenitors In Vitro (A) Frequency and number of CD19⁺ B lineage cells produced in culture from 150 Ly-HSCs purified from young and old mice seeded on OP9 stroma in vitro. Representative FACS plots show CD19⁺ cell production at 3.5 weeks of culture. Data are representative of 3 experiments. (B) Diagram of the in vitro assay used to generate lymphoid progenitors from Ly-HSCs and My-HSCs. Representative FACS plots showing CD127⁺ CD135⁺ lymphoid progenitors generated from young Ly-HSCs and My-HSCs. MC, methylcellulose supplemented medium. (C) Total number of CD127⁺ CD135⁺ lymphoid progenitors generated by young and old Ly-HSCs and My-HSCs. (D) Total number of CD127⁺ CD135⁺ lymphoid progenitors generated by young and old Ly-HSCs and My-HSCs in the presence or absence of 1 ng/mL of IL-1. Each symbol in (C) and (D) represents production per culture dish. Cultures were initiated with Ly-HSCs and My-HSCs purified from 3 to 2 independent cohorts of 6 young (8–12 weeks) and 4 old (18 months) B6 mice, respectively. Levels of significance for the differences between populations are indicated.