Acquired hematopoietic stem cell defects determine B-cell repertoire changes associated with aging

Abstract

Aging is associated with an inability to mount protective antibody responses to vaccines and infectious agents. This decline is associated with acquisition of defects in multiple cellular compartments, including B cells. While peripheral B-cell numbers do not decline with aging, the composition of the compartment appears to change, with loss of naïve follicular B cells, accumulation of antigen-experienced cells, and alteration of the antibody repertoire. The underlying cause of this change is unknown. We tested the hypothesis that aging-associated repertoire changes can be attributed directly to decreased B lymphopoiesis. Using an Ig transgenic model to report changes in the B-cell repertoire, we show that the reduced B-cell generative capacity of "aged" long-term reconstituting hematopoietic stem cells (LT-HSCs) alters the representation of antigen specificities in the peripheral B-cell repertoire. Further, we show that reconstitution using suboptimal numbers of fully functional LT-HSCs results in the generation of a similarly altered B-cell repertoire. This may be an important factor to consider when deciding the number of bone marrow cells to transplant in the clinical setting. In conclusion, when B lymphopoiesis is limited peripheral B-cell homeostasis is altered. This is reflected in reduced diversity of the B-cell repertoire, which likely reduces the protective quality of the immune response.

Fig. 1.

Aging-associated decline in B lymphopoiesis is B-cell/stem cell autonomous. Young or aged WT (B10.D2) mice were lethally irradiated, and lymphocyte-depleted bone marrow cells from young or prescreened immunologically aged 3-83μδ animals were transferred by i.v. injection. Donor BM was pooled from multiple animals before depletion, and BM equivalents were transferred at a ratio of one donor equivalent to four recipients. Reconstitution of the peripheral B-cell compartment was assessed 6 weeks posttransfer. Splenocytes were gated on live, CD19⁺ B cells, and 3-83μδ idiotype and total IgM expression was evaluated. Data are representative of analyses of a minimum of three individual animals in each category.

Fig. 2.

LT-HSCs from aged animals exhibit reduced B-cell generative capacity associated with repertoire alteration. Untreated whole BM was harvested from four young or four aged mice, RBCs were lysed, and LT-HSCs purified as described in experimental procedures. (A) Sorting profile of LT-HSCs from young and aged 3-83μδ lineage-depleted BM. (B) Reconstitution of young WT recipients 10 weeks posttransfer of 5000 LT-HSCs (Lin⁻Sca-1⁺cKit⁺Flk-2⁻CD34⁻) from young or aged 3-38μδ plus 3 × 10⁵ Rag2^−/− whole BM carrier cells. Total cells recovered were equivalent among recipients. Shown on the left are cytograms demonstrating IgM and 3-83μδ idiotype expression by CD19⁺ pregated splenocytes (B-cell plots). Shown on the right are cytograms illustrating T-cell antigen receptor (TCRβ) and CD4 expression by total splenocytes. Data are representative of analyses of a minimum of four individual animals in each category. (C) Summary of the percentage of idiotype-negative B cells present in the spleens of animals reconstituted with lineage-depleted (Fig. 1), 5-FU-treated, and flow-purified LT-HSC preparations (Fig. 2 A and B) derived from either young or aged donors. Means (horizontal bars) and statistically significant P < 0.0001* values are indicated.

Fig. 3.

Limitation of HSC function in young mice reduces total splenic B-cell numbers and alters the B-cell repertoire. (A) BM was harvested from 5-FU-treated young 3-83μδ mice and RBC lysed. Cells were then pooled, serially diluted (represented as BM equivalents) in PBS, and each dilution spiked with 3 × 10⁵ whole BM carriers from Rag2^−/− mice. Cell mixtures were transferred to lethally irradiated young WT recipients by i.v. injection. Six weeks posttransfer, live CD19⁺ splenic B cells were analyzed in terms of absolute number recovered and 3-83 μδ idiotype and IgM expression. BM equivalents transferred are indicated below each bar in the graphs and P-values provided. Statistics were generated from a minimum of three mice in each category, error bars represent SEM. P-values were determined by comparing the 1:10 dilution to each subsequent dilution in turn using the t-test: two samples assuming equal variances. (B) Effect of anti-IL-7 inhibition of B lymphopoiesis on repertoires development in young lethally irradiated WT mice that were reconstituted with stem cells from 5-FU-treated young 3-83 μδ mice. Control mice were sham treated by injection of PBS. Splenocytes were analyzed after the last antibody injection (6 weeks posttransfer) for 3-83 μδ idiotype and IgM expression. Splenocytes were pregated on live, CD19⁺ cells. Data are representative of analyses of a minimum of four individual animals in each category.

Fig. 4.

Reconstitution with increasing numbers of aged LT-HSCs increases B-cell genesis and partially restores a naive peripheral repertoire. Young WT (B10.D2) mice were lethally irradiated and reconstituted with 1000 or 10,000 LT-HSCs (Lin⁻Sca-1⁺cKit⁺Flk-2⁻CD34⁻) sorted from young or aged 3-83 μδ donors plus 3 × 10⁵ whole BM carriers from Rag2^−/− mice. Reconstitution was assessed at 10 weeks posttransfer. (A) Histograms show frequency of CD19⁺ B cells and TCRβ⁺ T cells relative to total splenocytes recovered from recipient mice. Histogram regions containing stained populations are marked with a bar and percentages within that region are indicated. (B) Cytograms showing 3-83 μδ idiotype and IgM expression (gated on CD19⁺ splenocytes). Percentages of B cells falling into each quadrant are indicated. Data are representative of analyses of a minimum of four individual animals in each category.