Depleting myeloid-biased haematopoietic stem cells rejuvenates aged immunity

Abstract

Ageing of the immune system is characterized by decreased lymphopoiesis and adaptive immunity, and increased inflammation and myeloid pathologies^1,2. Age-related changes in populations of self-renewing haematopoietic stem cells (HSCs) are thought to underlie these phenomena³. During youth, HSCs with balanced output of lymphoid and myeloid cells (bal-HSCs) predominate over HSCs with myeloid-biased output (my-HSCs), thereby promoting the lymphopoiesis required for initiating adaptive immune responses, while limiting the production of myeloid cells, which can be pro-inflammatory⁴. Ageing is associated with increased proportions of my-HSCs, resulting in decreased lymphopoiesis and increased myelopoiesis^3,5,6. Transfer of bal-HSCs results in abundant lymphoid and myeloid cells, a stable phenotype that is retained after secondary transfer; my-HSCs also retain their patterns of production after secondary transfer⁵. The origin and potential interconversion of these two subsets is still unclear. If they are separate subsets postnatally, it might be possible to reverse the ageing phenotype by eliminating my-HSCs in aged mice. Here we demonstrate that antibody-mediated depletion of my-HSCs in aged mice restores characteristic features of a more youthful immune system, including increasing common lymphocyte progenitors, naive T cells and B cells, while decreasing age-related markers of immune decline. Depletion of my-HSCs in aged mice improves primary and secondary adaptive immune responses to viral infection. These findings may have relevance to the understanding and intervention of diseases exacerbated or caused by dominance of the haematopoietic system by my-HSCs.

Extended Data Figure 1 ∣. Expression of my-HSC markers in HSPCs, mature cells, and tissues

a-l, Expression of my-HSC candidate markers, Slamf1 (CD150) (a), Neo1 (NEO1) (b), Itga2b (CD41) (c), Selp (CD62p) (d), Cd38 (CD38) (e), Itgb3 (CD61) (f), Itgav (CD51) (g), Procr (CD201) (h), Tie2 (i), Esam (j), Eng (CD105) (k), Cd9 (CD9) (l), in HSC and HSPCs in normal mouse BM (top panels), and in young versus old bone marrow (bottom panels). Data from a–l obtained directly from Gene Expression Commons; scale bars represent log2 Signal Intensity (top) and Gene Expression Activity (bottom) as defined by Gene Expression Commons. m, Heatmap of relative RNA expression for CD150 (Slamf1), NEO1 (Neo1), CD62p (Selp), CD41 (Itga2b), CD38 (Cd38), CD51 (Itgav), and CD61 (Itgb3) in HSCs, MPPs, Progenitors, Myeloid, and Lymphoid cells. Processed data for 23 cell types were obtained directly from Gulati Supplementary Table 1. Fold-enrichment = [(average percentile of HSCs)/(average percentile of all other cell types)+100], as described in this publication. n–o, RNA expression of CD150 (Slamf1), NEO1 (Neo1), CD62p (Selp), and CD41 (Itga2b) in bulk mouse tissues from two independent datasets: Tabula Muris (n) or Kadoki (o). For n–o, Values are z-score normalized for each gene across all tissues.

Extended Data Figure 2 ∣. Gating strategy for total HSCs, my-HSCs, bal-HSCs, and HPCs

a, Schematic to identify and validate my-HSC cell-surface antigens. b, Representative flow-cytometry gating of mouse BM to identify total HSC (Lin⁻cKIT⁺Sca1⁺FLT3⁻CD34⁻CD150⁺), my-HSC (Lin⁻cKIT⁺Sca1⁺FLT3⁻CD34⁻CD150^High), bal-HSC (Lin⁻cKIT⁺Sca1⁺FLT3⁻CD34⁻CD150^Low), MPPs [MPPa (Lin⁻cKIT⁺Sca1⁺FLT3⁻CD34⁺CD150⁺), MPPb (Lin⁻cKIT⁺Sca1⁺FLT3⁻CD34⁺CD150⁻), MPPc (Lin⁻cKIT⁺Sca1⁺FLT3⁺CD34⁺CD150⁻)], OPP (Lin⁻cKIT⁺Sca1⁻), CMP&GMP (Lin⁻cKIT⁺Sca1⁻CD34⁺CD41⁻), MkP (Lin⁻cKIT⁺Sca1⁻CD34⁺CD41⁺), MEP (Lin⁻cKIT⁺Sca1⁻CD34⁻CD41⁺), CLP (Lin⁻cKIT^LoSca1^LoIL7Ra⁺FLT3⁺). Panels are after excluding dead cells, doublets, and lineage-positive (CD3⁺, or Ly-6G⁺/C⁺, or CD11b⁺, or CD45R⁺, or Ter-119⁺) cells. Used for Fig. 1b-k, Fig. 2a-f, Fig. 3b-d, Fig. 4a, Ex. Data Fig. 2c-k, Ex. Data Fig. 3a-l, Ex. Data Fig. 4a-s, Ex. Data Fig. 5c-s, Ex. Data Fig. 6a Ex. Data Fig. 8c-h. Illustration of Hematopoietic Stem and Progenitor Cell (HSPC) Tree Analysis. CMP is combined CMP&GMP. Gate to define my-HSC vs. bal-HSC was set as described previously. c–f, Relative expression of CD41 (c), CD38 (d), CD51 (e), CD61 (f), on HSC and HSPCs. MFI values for each marker were obtained for each population and normalized from 0–1 based on the lowest to highest expression. g–h, Relative cell-surface levels (g) and percent-positive cells (h) for CD150, NEO1, CD62p, CD41, CD38, CD51, and CD61, on lineage-positive high and low cells, total HSCs, and HPCs in the BM. For cell-surface levels (g), MFI values for each marker were obtained for each population and normalized from 0–100 based on the lowest to highest expression. i, Percentage of total HSCs that are CD41⁺ (y-axis) vs. mouse age in weeks (x-axis); n=21 mice. j, Mouse age (x-axis) vs. the frequency of total HSCs (my-HSC+bal-HSC) as a percentage of live cells in the (i) total BM (left y-axis, red) or (ii) cKIT-enriched BM (right y-axis, blue) in untreated mice; n=13 mice. k, Percent-positive of my-HSCs vs. bal-HSCs for CD47 (k, top) using independent anti-CD47 clones (MIAP301, left; MIAP410, right), and for cKIT (k, bottom) using independent anti-cKIT clones (ACK2, left; 2B8, right). Mouse ages: 4-6 months (b–i, k), 3-23 months (j). For a–k, BM was cKIT-enriched prior to analysis. For j, total BM (non cKIT-enriched) was also examined. p-values and R values calculated with one-tailed Pearson correlation coefficient (i–j). n represents independent mice.

Extended Data Figure 3 ∣. Anti-CD150 non-masking antibodies and FACS gating to isolate HSCs

a, Schematic to identify anti-CD150 antibodies not masked by anti-CD150 antibody clone 1 (TC15), used in panels b–d; BM cells were incubated with saturating concentrations of unlabeled anti-CD150 antibody clone 1 (TC15) and then stained with PE anti-CD150 clones 2, 3, 4 (Q38, 9D1, mShad150). b–d, Saturating concentrations of unlabeled anti-CD150 antibody clone TC15 blocks staining with PE anti-CD150 clone 4 (mShad150) (d), but does not block staining of PE anti-CD150 clones 2, 3 (Q38, 9D1) (b–c). e, Schematic to determine if anti-CD150 clones 2, 3, 4 (Q38, 9D1, mShad150) identify the same population as anti-CD150 clone 1 (TC15); used in panels f–h; BM cells were incubated with PECy-7 anti-CD150 antibody clone 1 (TC15) and with PE anti-CD150 clones 2, 3, 4 (Q38, 9D1, mShad150). f–h, Co-staining with anti-CD150 clones 2, 3 (Q38, 9D1) identifies the same population as anti-CD150 antibody clone TC15 (f–g). Co-staining with anti-CD150 clone 4 (mShad150) and anti-CD150 antibody clone 1 (TC15) is mutually blocked (h). i, Schematic to identify anti-CD150 antibodies that are not blocked by anti-CD150 clone mShad150, used in panel j; BM cells are incubated with saturating concentrations of unlabeled anti-CD150 antibody clone mShad150 and then stained with PE anti-CD150 clone 2 (Q38). j, Saturating concentrations of unlabeled anti-CD150 antibody clone mShad150 does not block staining of PE anti-CD150 clone 2 (Q38) (j). k, Schematic to determine if co-staining with anti-CD150 clone 2 (Q38) identifies the same population as anti-CD150 antibody clone mShad150; used in panel l; BM cells are incubated with PECy-7 anti-CD150 antibody clone mShad150 and with PE anti-CD150 clone 2 (Q38). l, Co-staining with anti-CD150 clones 2 (Q38) identifies the same population as anti-CD150 antibody clone mShad150 (l). m–n, Representative FACS gating of mouse BM to sort total HSC (Lin⁻cKIT⁺Sca1⁺FLT3⁻CD34⁻CD150⁺) from aged (11 months) control mice (Aged, A) or aged (11 months) mice with my-HSC depletion (Aged+Conditioning, A+C), used to sort HSCs for RNA-sequencing experiment presented in Fig. 2g-h, Ex. Data Fig. 4x, and used to sort HSCs for transplant experiments presented in Fig. 2i-k, Ex. Data Fig. 4t-v. My-HSC depletion was performed with anti-NEO1+anti-CD62p+anti-cKIT+CD47 and cells were collected at day 9 post-treatment; n=3 mice (A), n=3 mice (A+C). Mouse ages approximately: 3 months (a–h), 5-8 months (i–l), 11 months (m–n). For (a–n), BM was cKIT-enriched prior to FACS analysis or sorting. n represents independent mice.

Extended Data Figure 4 ∣. Antibody-mediated depletion of my-HSCs in vivo

a–d, Frequency (% live) (a, c) or absolute number (b, d) of my-HSCs and bal-HSCs (a–b) or of NEO1⁺ HSCs and NEO1⁻ HSCs (c–d) after anti-CD150 conditioning (anti-CD150, anti-CD150+anti-CD47, or anti-CD150+anti-CD47+anti-cKIT); n=3 mice per group. e, Total HSCs (my-HSCs+bal-HSCs) as percent-live in mice receiving anti-CD47; n=5 mice per group; P=0.3637. f, Percentage of total HSCs that are my-HSCs in mice receiving anti-CD47; n=5 mice per group; P=0.6628. g, Total HSCs (my-HSCs+bal-HSCs) as a percent-live, in mice receiving anti-CD47+anti-cKIT; n=4 mice per group; P=0.0973. h, Percentage of total HSCs that are my-HSCs in mice receiving anti-CD47+anti-cKIT; n=4 mice per group; P=0.2805. i, Percentage of total HSCs that are NEO1⁺ HSCs in mice receiving anti-CD150 (IgG2a)+anti-CD47+anti-cKIT (e.g., protocol CD150^v2); n=4 mice per group; *P=0.0353. j–n, Frequency (% live) of CLPs (j), IL7Ra⁺ cells (k), CMPs&GMPs (l), MkPs (m), and MEPs (n), after anti-CD150, anti-CD62p, or anti-NEO1 conditioning protocols. Values relative to mean of untreated control mice and log2-transformed; n=3 mice per group (NEO1^v1, NEO1^v2, CD150^v1); n=4 mice per group (CD62p, CD150^v2). o, Ratio of frequency (% live) for Lymphoid to Myeloid Progenitors (CLP)/(CMP&GMP), after anti-CD150, anti-CD62p, or anti-NEO1 antibody protocols. Values relative to mean of untreated control mice and log2-transformed; n=3 mice per group (NEO1^v1, NEO1^v2, CD150^v1); n=4 mice per group (CD62p, CD150^v2). p, Frequency (% live) of my-HSCs, bal-HSCs, NEO1⁺ HSCs, and NEO1⁻ HSCs after treatment with anti-CD150, anti-CD62p, or anti-NEO1 protocols; n=3 mice per group (anti-CD150^v1); n=4 mice per group (anti-CD62p); n=3 control mice and n=6 treated mice (anti-NEO1, combined 90ug&200ug protocols). Values relative to mean of control mice and log2-transformed. For j–p, values for treated mice (filled circles); values for control mice (unfilled circles). q, Correlation of fraction of live cells for my-HSCs vs. NEO1⁺ HSCs (left), and bal-HSCs vs. NEO1⁻ HSCs (right), of control mice and mice receiving anti-CD150, anti-CD62p, or anti-NEO1 protocols, in cKIT-enriched BM; n=3 mice (anti-CD150^v1); n=4 mice (anti-CD62p); n=3 control mice and n=6 treated mice (anti-NEO1, combined 90ug&200ug protocols). r, Comparison of the absolute number of cells in total (non-cKIT-enriched) BM (y-axis) vs. frequency of cells as a percent-live (in cKIT-enriched) BM (x-axis), for my-HSCs, bal-HSCs, CLP, CMP&GMP, and MkP in control mice and mice receiving anti-CD150, anti-CD150+anti-CD47, or anti-CD150+anti-CD47+anti-cKIT; n=3 mice per condition. s, Comparison of the frequency of cells as a fraction of total (non-cKIT-enriched) BM (x-axis) vs. the frequency of cells as a fraction of cKIT-enriched BM (y-axis), for cell populations in control mice and mice receiving anti-CD150, anti-CD150+anti-CD47, or anti-CD150+anti-CD47+anti-cKIT; n=3 mice per condition. Data for q–s log10-transformed. t–v, HSC lineage potential 8-weeks after transplantation of 100 total HSCs FACS-sorted from CD45.2 aged mice without (A) or with my-HSC depletion (A+C) into CD45.1 recipients. Donor Myeloid to Lymphoid Ratio (t, *P=0.0275) and Percent-Chimerism Ratio (u, *P=0.0340). For each recipient (y-axis), % donor cells (bottom x-axis) that are Myeloid (red bars) or Lymphoid (blue bars) and the Myeloid/Lymphoid log2-ratio (red circles) (top x-axis) (v); n=13 recipient mice (A), n=14 recipient mice (A+C). w, Donor Myeloid/Lymphoid Ratio 16-weeks after transplantation of 100 total HSCs FACS-sorted from CD45.2 aged mice without (A) or with my-HSC depletion (A+C) into CD45.1 recipients; n=12 recipient mice (A), n=14 recipient mice (A+C); *P=0.0232. x, Top 200 differentially expressed genes ranked by p-value for RNA-seq comparison between (A) vs. (A+C), based on Fragments Per Kilobase of transcript per Million mapped reads (FPKM); log2-transformed. Heatmap generated using Phantasus (v1.21.5) with FPKM values as input and Limma to define differentially expressed genes; n=3 mice (A), n=3 mice (A+C). Mouse ages approximately: 6-7 months (a–d, r–s), 7-9 months (g–i), 5-9 months (j–q), 11 months (donors, t–w), 2 months (recipients, v). For (a, c, g–s), BM was cKIT-enriched prior to analysis. For (b, d, e–f, r–s), total BM (non cKIT-enriched) was examined. P-values obtained by ordinary one-way ANOVA followed by one-tailed Dunnett’s multiple comparisons test with non-treated as control (a–d), unpaired parametric one-tailed t-test (i-k, p, t–u), unpaired non-parametric one-tailed t-test (w), or unpaired parametric two-tailed t-test (e–h, l–o). p-values and R values calculated with one-tailed Pearson correlation coefficient (q–s). CD150^v1 is rat IgG2b anti-CD150 protocol; CD150^v2 is rat IgG2a anti-CD150 protocol; NEO1^v2 includes mouse IgG2a anti-goat; α, anti-; ns, not significant. Bars indicate mean +/− s.e.m. n represents independent mice; *P<0.05, **P<0.005, ***P<0.0005; Exact P-values provided in Source Data.

Extended Data Figure 5 ∣. Optimization of NEO1 depletion protocol in vitro and in vivo

a–b, Strategy to restore balanced lineage output by depleting my-HSCs. c–h, Anti-NEO1 antibody saturation curve (c) determined from in vitro antibody dilution series (d–h). i, Schematic of in vivo saturation experiments with anti-NEO1 antibody; used in panels j–m. j–m, Dose-dependent relationship between anti-NEO1 antibody dose (0ug, 30ug, 90ug, 200ug), when combined with anti-CD47 and anti-cKIT, on the relative depletion of NEO1⁺ HSCs (j), and increase in CLPs (k). Optimal concentration in yellow; n=3 mice per group. l, The increase in NEO1⁻ HSCs after anti-NEO1 dose escalation (0ug, 30ug, 90ug, 200ug) is correlated with the increase in CLPs. m, Impact on the ratio of Bal-HSCs/My-HSCs (black) and NEO1⁻HSCs/NEO1⁺ HSCs (blue) as a percentage of live cells after anti-NEO1 antibody dose-escalation (0ug, 30ug, 90ug, 200ug), when combined with anti-CD47 and anti-cKIT; n=3 per group. Values relative to mean value of control (0ug) mice and log2-transformed. n, Schematic of double-antibody strategy to target NEO1, whereby mouse IgG2a or IgG2b monoclonal anti-goat antibodies are administered 24 hours after goat anti-NEO1^,. o–p, Schematic (o) to demonstrate that saturating concentrations of mouse IgG2a or IgG2b anti-goat do not reduce ability of donkey anti-goat AF488 detect goat anti-NEO1 antibody (p). q–s, Schematic (q) of experiment demonstrating that mouse anti-goat IgG2a A555 (r) and IgG2b PE (s) antibodies identify the same population as donkey anti-goat AF488 by flow-cytometry. Mouse ages approximately: 5-7 months (c–h, n–s), 6-9 months (i–m). For (c–s), BM was cKIT-enriched prior to analysis. For correlation p-values, one-tailed Pearson correlation coefficient (R_p), and one-tailed Spearman correlation coefficient (R_s) were calculated (l). p-values obtained by ordinary one-way ANOVA followed by one-tailed Dunnett’s multiple comparisons test with 0ug condition as control (m). MaG, mouse-anti goat; DaG, donkey anti-goat. Bars indicate mean +/− s.e.m. n represents independent mice; *P<0.05, **P<0.005, ***P<0.0005; Exact P-values provided in Source Data.

Extended Data Figure 6 ∣. My-HSC depletion restores features of a youthful immune system

a, Ratio of frequency of my-HSCs to bal-HSCs (% live) in aged (A), or aged+conditioning mice (A+C) after 8-weeks; n=4 (A), n=9 (A+C); P=0.0346. b–d, Statistical significance (y-axis, −log10p) vs. fold-change (x-axis, log2-FC) of plasma protein levels at week-8 for (A)/(Y) (b), (A)/(A+C) (c), or (A)/(Y & A+C) (d) comparisons. Gray bar (y=1–1.3); values above gray bar p<0.05; n=6 (Y, A), n=5 (A+C). e, Overlap of top 17% of plasma proteins, ranked by statistical significance, increased in (A)/(Y) and decreased in (A+C)/(A). f, Estimated plasma concentration of CXCL5 at week-8; n=6 (Y, A), n=5 (A+C). g, Relative plasma abundance of inflammatory proteins at week-8; n=6 (Y, A), n=5 (A+C). Values relative to the mean for (Y) and log2-transformed. h–i, Frequency of CD11b⁺Ly6G/C⁺ (h) and CD11b⁺SIRPa⁺ (i) mature myeloid cells in the blood approximately 1-week after antibody-conditioning; n=3 (A), n=4 (A, A+C). j–k, Frequency of mature B cells (B220⁺CD19⁺CD43⁻CD93⁻IgM⁺IgD⁺) (j), and progenitor B cells (B220⁺CD19⁻ B cells) (k), in the BM approximately 1-week after antibody-conditioning; n=5 (A), n=6 (A, A+C). Thymus weight (l) and frequency of thymic subsets 1–8 as defined by Akashi & Weissman, as % of total CD45⁺ cells in the thymus (n), 8-weeks after antibody-conditioning; representative FACS (m); n=3 (Y, A), n=9 (A+C). Populations enriched for transitional intermediate cells (areas 3&4) highlighted in box (n). Mouse ages: Y (3-6 months); A & A+C (18-24 months). For (a), BM was cKIT-enriched prior to analysis. p-values obtained by unpaired parametric one-tailed t-test (a), ordinary one-way ANOVA followed by one-tailed Dunnett’s multiple comparisons test using Aged as control (f), one-way ANOVA followed by Holm multiple comparisons test (b–d, g), ordinary one-way ANOVA followed by two-tailed Dunnett’s multiple comparisons test using Aged as control (h–i, l, n), or Brown-Forsythe and Welch ANOVA tests followed by Dunnett’s T3 multiple comparisons test using Aged as control (j–k). Bars indicate mean +/− s.e.m. n represents independent mice; *P<0.05, **P<0.005, ***P<0.0005; Exact P-values provided in Source Data.

Extended Data Figure 7 ∣. My-HSC depletion increases naïve T cells and B cells in aged mice

a–c, Absolute numbers of (a) naïve (CD44⁻CD62L⁺); *P=0.0430, (b) central memory (CM: CD44⁺CD62L⁺); *P=0.0177, or (c) effector memory (EM: CD44⁺CD62L⁻); P=0.576, T cells (CD4 & CD8), per mL of blood, approximately 8-weeks post-treatment. Values log2-transformed; n=9 (A), n=14 (A+C), mice pooled from 2 independent experiments. d, Absolute numbers of mature B cells (IgM⁺IgD⁺) per mL of blood, approximately 8-weeks post-treatment; *P=0.0498. Values log2-transformed; n=6 (A), n=5 (A+C). e, Absolute numbers of CD45+ cells per mL of blood, approximately 8-weeks post-treatment. Values log2-transformed; n=3 (A), n=9 (A+C); P=0.069. f–g, Percentage of CM (f) and EM (g) subsets per total T cells (CD4 & CD8), 8-weeks post-treatment; n=6 (Y, A), n=5 (A+C). h–j, Frequency relative to Aged mice of T cell (CD4 & CD8) subsets 8-weeks after antibody treatment (h). Naïve, CM, and EM subsets were defined by 12-marker cluster-based analysis (i–j); n=6 (Y, A), n=5 (A+C). Values relative to mean for Aged control mice and log2-transformed. Mouse ages: Y (3-6 months); A & A+C (18-24 months). p-values obtained by unpaired parametric one-tailed t-test (a, d), unpaired parametric two-tailed t-test (b–c, e), or ordinary one-way ANOVA followed by two-tailed (f–g) or one-tailed (h) Dunnett’s multiple comparisons test using Aged as control. Bars indicate mean +/− s.e.m. n represents independent mice; *P<0.05, **P<0.005, ***P<0.0005; Exact P-values provided in Source Data.

Extended Data Figure 8 ∣. Flow-cytometry gating strategy for T cells, B cells, and myeloid cells

a–c, Gating strategy to identify: (b) naïve (CD44⁻CD62L⁺), central memory (CD44⁺CD62L⁺), and effector memory (CD44⁺CD62L⁻) T cells (combined CD4 & CD8), or (c) CD4 T cells that are PD1⁺CD62L⁻ or PD1⁻CD62L⁺ in the blood, used for Fig. 3e, Fig. 3g, Extended Data Fig. 7a-c, Extended Data Fig. 7f-g. d–f, Gating strategy to identify: (e) mature B cells (CD19⁺B220⁺IgM⁺IgD⁺), or (f) Aged B Cells ABCs (CD19⁺IgM⁺CD93⁻CD43⁻CD21/CD35⁻CD23⁻) in the blood, used for Fig. 3f, Fig. 3h, Extended Data Fig. 7d. g–i, Gating strategy to identify (h) progenitor B cells (B220⁺CD19⁻), or (i) mature B cells (B220⁺CD19⁺CD43⁻CD93⁻IgM⁺IgD⁺) in the bone-marrow, used for Extended Data Fig. 6j-k. j–l, Gating strategy to identify (k) CD11b⁺Ly6G/C⁺ myeloid cells, or (l) CD11b⁺SIRPa⁺ myeloid cells in the blood, used for Extended Data Fig. 6h-i.

Extended Data Figure 9 ∣. Antibody-conditioning enhances functional immunity to infection

a, Schematic of model to determine the impact of antibody-conditioning on functional immunity. b, Gating to identify Ter119⁺ cells (Ter119⁺CD19⁻CD3⁻CD45^+/lo) and antigen-infected cells (Ag34⁺Ter119⁺) in mouse spleens, used for Extended Data Fig. 9i-j. c–h, My-HSC and NEO1⁺ HSC absolute numbers in total BM (c, e), or as percentage of total HSCs (d, f), with correlations (h), 10-weeks after anti-NEO1^v2 conditioning. Representative FACS on total HSCs (g). A and A+C mice received vaccination at Week 8; n=2 (Y), n=7 (A), n=10 (A+C). i, Total number of Ter119⁺ cells per mouse spleen in mice that were Naïve, Infected, or Vaccinated & Infected with FV. Representative FACS histograms for Ter119 expression, gated on all single cells. Each row represents an independent mouse. n=9 (Y, naïve), n=13 (Y, FV infected), n=13 (Y, vaccinated & FV infected), n=6 (A, naïve), n=10 (A, FV infected), n=8 (A, vaccinated & FV infected), n=5 (A+C, naïve), n=9 (A+C, vaccinated & FV infected). Data log10-transformed. j, Total number of antigen-infected (Ag34⁺Ter119⁺) cells per spleen in mice that were: Infected, or Vaccinated & Infected with FV. Representative FACS histograms for Ag34 expression, gated on Ter119+ cells. Each row represents an independent mouse; n=13 (Y, FV infected), n=13 (Y, vaccinated & FV infected), n=10 (A, FV infected), n=8 (A, vaccinated & FV infected), n=9 (A+C, vaccinated & FV infected). Data log10(x+1)-transformed. k, Correlation of Infectious virus levels (Infectious Centers; x-axis) vs. Dextramer⁺CD44⁺ cells (Percent of splenic CD8+ T cells; y-axis) in Vaccinated & FV-infected mice; n=8 (A, vaccinated & FV-infected), n=9 (A+C, vaccinated & FV-infected). Data log10-transformed. For i–k, data from experiments using the anti-NEO1^v1 protocol (open circles) or the anti-NEO1^v2 protocol (closed circles) were combined. Mouse ages: Y (3-6 months); A & A+C (21-22 months). For (c–f), BM was cKIT-enriched prior to analysis. For (c, e, h), total BM (non-cKIT-enriched) was also analyzed to calculate total numbers of cells. p-values obtained by one-tailed unpaired parametric t-test (c–f), two-tailed Pearson correlation coefficient (h, k), or ordinary one-way ANOVA followed by Tukey’s multiple comparisons test (i–j). NEO1^v2 protocol is NEO1^v1 protocol (anti-NEO1+ anti-CD47+anti-cKIT)+mouse (IgG2a) anti-goat. Inf., FV infected without vaccination; Vacc. & Inf., FV infected with vaccination, Vacc. & Inf., FV infected with vaccination; LOD, limit of detection; n.s., not significant. n represents independent mice. Bars indicate mean +/− s.e.m. (c–f) or bars depict median (i–j). *P<0.05, **P<0.005, ***P<0.0005; Exact P-values provided in Source Data.

Extended Data Figure 10 ∣. Mouse my-HSC markers are enriched in aged human HSCs

a, Relative mRNA expression of CD62p (Selp, P=0.0243), CD41 (Itga2b, P=0.0001), CD61 (Itgb3, P=0.0135), CD150 (Slamf1, P=0.179), and NEO1 (Neo1, P=0.433) in human HSCs isolated from young (age 20-26) and old (age >70) donors; data obtained from Nilsson, GSE69408. b, Relative mRNA expression of CD62p (Selp, P=0.0001), CD41 (Itga2b, P=0.0027), CD61 (Itgb3, P=0.0116), and NEO1 (Neo1, P=0.274) in human HSCs isolated from young (age 18-30) or old (age 65-75) donors; data obtained from Adelman (GSE104406). c–e, Correlation of relative mRNA expression of CD62p (c), CD41 (d), and CD61 (e) in human HSCs as compared to donor age; data obtained from Adelman (GSE104406). For a–e, values relative to mean of young samples. f, Heatmap depicting expression of candidate markers in independent datasets of human: HMGA2⁺ vs. HMGA2⁻ CD34⁺ cells (e), MPN (f) or MDS (g) vs. normal HSCs, and Pre-AML vs. normal HSCs (h). g–h, Representative FACS to identify human HSCs (Lin⁻CD34⁺CD38⁻CD45RA⁻CD90⁺), MPPs (Lin⁻CD34⁺CD38⁻CD45RA⁻CD90⁻), LMPPs (Lin⁻CD34⁺CD38⁻CD45RA⁺), CMPs & MEPs (Lin⁻CD34⁺CD38⁺CD45RA⁻), and GMPs (Lin⁻CD34⁺CD38⁺CD45RA⁺) in normal human BM, used for Fig. 5f-i, Extended Data Fig. 10i-s. Samples are post CD34⁺-enrichment. i–m, FACS of human HSCs depicting fluorescence-minus-one (FMO) control (i), anti-CD62p clone AK4 (j), anti-CD62p clone Psel.KO2.3 (k), anti-CD62p clone AC1.2 (l), and anti-CD150 (m); representative for n=3 donors (j); n=2 donors (k–m). n, Illustration of human Hematopoietic Stem and Progenitor Cell (HSPC) Tree Analysis (n). o–s, Relative expression of CD62p (o), CD150 (p), ESAM (q), and CD166 (r), on human HSPCs, with summary (s), showing percent positive HSCs, and normalized MFI for each marker on HSPCs. For o–s, FACS median fluorescent intensity (MFI) values for each marker were obtained for each population, divided by the MFI for the FMO control, and then normalized from 0–100 based on the lowest to highest expression. Red color scale corresponds to normalized MFI values. Blue, purple, maroon scale corresponds to bins for HSC positivity (20-50%, 51-70%, and 71-100). t, Model to rejuvenate aged immune systems by depleting myeloid-biased hematopoietic stem cells. p-values obtained by unpaired parametric one-tailed t-test (a–b), or p-values and R values calculated with one-tailed Pearson correlation coefficient (c–e). MDS, myelodysplastic syndrome; MPN, myeloproliferative neoplasms. Bars indicate mean +/− s.e.m. *P<0.05, **P<0.005, ***P<0.0005; Exact P-values provided in Source Data.

Figure 1 ∣. Identification of cell-surface antigens on myeloid-biased HSCs

a, Heatmap depicting candidate marker expression across independent datasets (left), with mean log2 fold-change (Old-HSC vs. Young-HSC; My-HSC vs. Bal-HSC) (right). Datasets: Old vs. Young HSCs (a, b, c, d, e, f, g, h) and My-HSCs vs. Bal-HSC (i, j, k). b, Percent-positive for each marker on my-HSCs vs. bal-HSCs (left); plot of my-HSC fold-enrichment for each marker (right). c, Plot of (my-HSC)/(bal-HSC) Fold-Change (log2) of RNA expression (y-axis) vs. Cell-surface Protein expression (x-axis) for candidate markers. d, Representative flow-cytometry to identify total HSCs (KLS FLT3⁻CD34⁻CD150⁺), my-HSCs (KLS FLT3⁻CD34⁻CD150^High), and bal-HSCs (KLS FLT3⁻CD34⁻CD150^Low) from mouse bone-marrow, after excluding dead cells, doublets, and gated on KLS (Lin⁻cKIT⁺Sca1⁺). e, Representative flow-cytometry of percent of bal-HSCs (left) or my-HSCs (right) that are NEO1⁺ or CD62p⁺. f–h, Relative cell-surface expression of CD150 (f), NEO1 (g), and CD62p (h), on total HSC (my-HSC+bal-HSC), my-HSC, bal-HSC, MPPa, MPPb, MPPc, CMP (CMP&GMP), MkP, and CLP. Flow-cytometry median fluorescent intensity (MFI) for antibodies to each marker were obtained for each population and normalized from 0–1 (from lowest to highest). i–k, Percentage of total HSCs (KLS FLT3⁻CD34⁻CD150⁺) that are my-HSCs (e.g., CD150^High) (i), NEO1⁺ (j), or CD62p⁺ (k) on the y-axis, vs. mouse age (weeks) on the x-axis; n=21 mice (i–j); n=10 mice (k). Mouse ages approximately: 4-6 months (b–h), 6-12 months (i–k). For (b–k), BM was cKIT-enriched prior to analysis. p-values and R values calculated with one-tailed Pearson correlation coefficient (i–k). n indicates independent mice. FC, fold-change.

Figure 2 ∣. Antibody-mediated depletion of my-HSCs in vivo

a–c, Percentage of Total HSCs that are my-HSCs after: anti-CD150 (a, **P=0.0045), anti-CD62p (b, *P=0.0374), or anti-NEO1 (c, *P=0.0465) optimized protocols (e.g., +anti-CD47+anti-cKIT), with representative flow-cytometry; n=3 mice per group (a, c); n=4 mice per group (b). d–f, Enrichment (blue) or depletion (red) as percent-live for Total HSCs (HSC), my-HSCs (My), bal-HSCs (Bal), MPPs, CLP, CMP&GMP, MkP after: anti-CD150 (d), anti-CD62p (e), or anti-NEO1 (f) protocols (+anti-CD47+anti-cKIT). Treated values relative to Control mean and log2-transformed; n=3 mice per group (d, f); n=4 mice per group (e). g–k, Total HSCs were FACS-sorted from aged mice without (Aged, A) or with my-HSC depletion (Aged+Conditioning, A+C) and underwent RNA-sequencing (g–h) or transplantation (i–k). Depletion was anti-NEO1+anti-CD62p+anti-CD47+anti-cKIT; n=3 mice (A), n=3 mice (A+C). g–h, GSEA comparison between (A) vs. (A+C) HSCs using gene-signatures from Young HSC vs. Old HSC (g^,,,-,) or bal-HSC vs. my-HSC (h^,,); n=3 mice (A), n=3 mice (A+C). i–k, HSC lineage potential 16-weeks after transplantation of 100 total HSCs FACS-sorted from CD45.2 aged mice without (A) or with my-HSC depletion (A+C) into CD45.1 recipients. Donor Lymphoid Percentage (i, *P=0.0126) and Myeloid/Lymphoid Percent-Chimerism Ratio (j, *P=0.0492). For each recipient (y-axis), % donor cells (bottom x-axis) that are Myeloid (red bars) or Lymphoid (blue bars) and the Myeloid/Lymphoid log2-ratio (red circles) (top x-axis) (k); n=12 recipient mice (A), n=14 recipient mice (A+C). Mouse ages 5-9 months (a–f), 11 months (donors, g–k), 2 months (recipients, i–k). For (a–k) BM was cKIT-enriched prior to analysis or sorting. P-values obtained by unpaired parametric one-tailed t-test (a–f, i–j) or GSEA with FDR adjustment (g–h). Bars indicate mean +/− s.e.m. n indicates independent mice; *P<0.05, **P<0.005, ***P<0.0005; Exact P-values provided in Source Data.

Figure 3 ∣. Depletion of my-HSCs in aged mice restores youthful immune features

a, Young-adult mice (Y) compared to aged mice without (A), or with anti-NEO1 conditioning (A+C), approximately 1-, 8-, or 16-weeks post-treatment. b, Frequency (% live) of my-HSCs and CLPs in (A+C) relative to (A); n=6 (A, A+C) at 1-week; n=4 (A), n=9 (A+C) at 8-weeks; n=4 (A, A+C) at 16-weeks. Treated (A+C) values relative to mean of aged control (A) and log2-transformed. c, Frequency of my-HSCs (%-live) in BM at week-1; n=5 (Y), n=6 (A, A+C). d, Frequency of CLPs (%-live) in BM at week-16; n=4 (Y, A, A+C). e–j, Naïve T cells (CD44⁻CD62L⁺) as percent total CD4 & CD8 T cells in blood (e), Mature B cells (IgM⁺IgD⁺) as percent total B cells (CD19⁺B220⁺) in blood (f), CD4 T cell ratio (% PD1⁺CD62L⁻)/(% PD1⁻CD62L⁺) (g), Aged B Cells (CD21/CD35⁻CD23⁻) as percent total mature B cells (CD19⁺IgM⁺CD93⁻CD43⁻) in blood (h), plasma concentration of IL-1α (i), plasma abundance of top 17% proteins increased in (A) vs. (A+C) (j); for (e–j), data at week-8 and n=6 (Y, A), n=5 (A+C). For j, values relative to Young (Y) mean and log2-transformed; gray squares are removed statistical outliers; significance −log₁₀p between (A) vs. (A+C). Mouse ages (b–j) approximately: young-adult (3-6 months), aged (18-24 months). For (b–d) BM was cKIT-enriched prior to analysis. p-values obtained by unpaired parametric one-tailed t-test (b), ordinary one-way ANOVA followed by one-tailed Dunnett’s multiple comparison test using Aged as control (c–d, f–i), Brown-Forsythe and Welch ANOVA followed by two-tailed Dunnett’s T3 multiple comparisons test using Aged as control (e), or one-way ANOVA followed by Holm multiple comparisons test (j). Bars indicate mean +/− s.e.m. n represents independent mice; *P<0.05, **P<0.005, ***P<0.0005; Exact P-values provided in Source Data.

Figure 4 ∣. Antibody-conditioning enhances functional immunity in aged mice

a, Frequency of my-HSCs in young-adult (Y) or aged mice without (A), or with aged+conditioning (A+C), 10-weeks after anti-NEO1^v2; (A) and (A+C) mice were vaccinated 2-weeks before analysis; n=2 (Y), n=7 (A), n=10 (A+C); (A) vs. (A+C) *P=0.0159. b, Percentage of Dextramer⁺CD44⁺ splenic CD8+ T cells 10-14 days after vaccination in aged-matched (20-26 months) mice without (A), or with antibody-conditioning (A+C). For (A+C), my-HSC-depletion was with anti-NEO1^v2 2-months before vaccination; n=13 (A, vaccinated), n=15 (A+C, vaccinated); *P=0.0412. c, Spleen weight (mg) for naïve, FV-infected, or Vaccinated & FV-infected mice; n=9 (Y, naïve), n=13 (Y, FV-infected), n=13 (Y, vaccinated & FV-infected), n=6 (A, naïve), n=10 (A, FV-infected), n=8 (A, vaccinated & FV-infected), n=5 (A+C, naïve), n=9 (A+C, vaccinated & FV-infected). Data log10-transformed. d, Infectious virus levels in FV-infected or Vaccinated & FV-infected mice; n=13 (Y, FV-infected), n=13 (Y, vaccinated & FV-infected), n=10 (A, FV-infected), n=8 (A, vaccinated & FV-infected), n=9 (A+C, vaccinated & FV-infected). Data log10-transformed. e, Percent of Dextramer⁺CD44⁺ splenic CD8+ T cells in Vaccinated & FV-infected mice; n=8 (A, vaccinated & FV-infected), n=9 (A+C, vaccinated & FV-infected); *P=0.0473. For a, c–e, mouse ages approximately: (Y) 3-6 months; (A) & (A+C) 21-22 months. For c–e, data from experiments with anti-NEO1^v1 (open circles) or anti-NEO1^v2 (closed circles) were combined. For (a) BM was cKIT-enriched prior to analysis. p-values obtained by one-tailed unpaired parametric t-test (a–b), ordinary one-way ANOVA followed by Tukey’s multiple comparisons test (c–d), or one-tailed unpaired parametric t-test with Welch’s correction (e). anti-NEO1^v2 is anti-NEO1^v1 (anti-NEO1+anti-CD47+anti-cKIT)+mouse (IgG2a) anti-goat. Bars depict mean +/− s.e.m. (a–b, e) or bars depict median (c–d). n represents independent mice; *P<0.05, **P<0.005, ***P<0.0005; Exact P-values provided in Source Data.

Figure 5 ∣. Mouse myeloid-biased HSC markers are enriched in aged human HSCs

a, Heatmap depicting RNA expression of candidate human my-HSC antigens in independent datasets of human Old vs. Young HSCs (a, b, c, d). b, Relative RNA expression of CD62p (Selp; ***P=0.000211), CD41 (Itga2b; *P=0.0325), CD150 (Slamf1; *P=0.0359), and NEO1 (Neo1; P=0.362) in human HSCs isolated from young (ages 20-31); n=14 donors, middle & old (ages 42-85); n=13 donors; data obtained from Pang (GSE32719). c–e, Correlation of relative RNA expression of CD62p (c), CD41 (d), and CD150 (e) in human HSCs compared to donor age; n=27 donors; data obtained from Pang (GSE32719). For b–e, values are relative to mean of young samples. f, Representative flow-cytometry of CD34⁺-enriched donor bone-marrow to identify human HSCs (Lin⁻CD34⁺CD38⁻CD45RA⁻CD90⁺). g, Representative flow-cytometry staining of HSCs with anti-CD62p antibody (red) compared to fluorescence-minus-one (FMO) control (black); n=3 independent donors. h–i, Histograms for flow-cytometry staining of HSCs with antibodies to CD304, CD150, TIE2, CD62p, ESAM, CD9, CD47, CD105, CD166; black line represents FMO control (h), with percent of HSCs positive for each marker (i). p-values obtained by unpaired parametric one-tailed t-test (b), or p-values and R values calculated with one-tailed Pearson correlation coefficient (c–e). FMO, fluorescence-minus-one. Bars indicate mean +/− s.e.m. *P<0.05, **P<0.005, ***P<0.0005; Exact P-values provided in Source Data.