Kitlo hematopoietic stem cells exhibit distinct lymphoid-primed chromatin landscapes that enhance thymic reconstitution

Abstract

Hematopoietic stem cells (HSC) with multilineage potential are critical for T cell reconstitution after allogeneic hematopoietic cell transplantation (allo-HCT). The Kit^lo HSC subset is enriched for multipotential precursors, but their T cell potential remains poorly characterized. Using a preclinical allo-HCT mouse model, we demonstrate that Kit^lo HSCs provide superior thymic recovery and T cell reconstitution, resulting in improved immune responses to post-transplant infection. Kit^lo HSCs with augmented bone marrow (BM) lymphopoiesis mitigate age-associated thymic alterations and enhance T cell recovery in middle-aged mice. Mechanistically, chromatin profiling reveals Kit^lo HSCs exhibiting higher activity of lymphoid-specifying transcription factors, such as, ZBTB1. Zbtb1 deletion diminishes HSC engraftment and T cell potential; by contrast, reinstating Zbtb1 in megakaryocytic-biased Kit^hi HSCs rescues hematopoietic engraftment and T cell potential in vitro and in vivo. Furthermore, age-associated decline in Kit^lo HSCs is associated with diminished T lymphopoietic potential in aged BM precursors; meanwhile, Kit^lo HSCs in aged mice maintain enhanced lymphoid potential, but their per-cell capacity is diminished. Lastly, we observe an analogous human BM KIT^lo HSC subset with enhanced lymphoid potential. Our results thus uncover an age-related epigenetic regulation of lymphoid-competent Kit^lo HSCs for T cell reconstitution.

Fig. 1. Age-related decline in multilineage HSCs marked by low Kit expression.

A–H Multiome single-cell RNA and ATAC sequencing was performed on HSC (Lineage-Sca-1+cKit+ CD34-CD48-Flt3-CD150+) cells isolated from 2-mo (young) or 24-mo (old) female C57BL/6 mice. nTOTAL = 12,350 cells. A Volcano plot of DGE analysis between MLin-HSC vs. q-HSC subsets in young HSCs, highlighting bona fide HSC markers. B Violin plot for imputed Kit gene expression by annotated HSC subsets (as described in Fig. S2C). C Frequency of computationally defined HSC subtypes in Kit^hi and Kit^lo subsets in young HSCs. D, E Combined Uniform manifold approximation and projection (UMAP) of young and old HSCs after Harmony batch correction, annotated by age (D) and annotated HSC subsets (q-HSC: Quiescent HSCs; Mgk-HSCs: Platelet-biased HSCs; Mlin-HSC: Multilineage HSCs; p-HSC: Proliferative HSCs; Int-HSC: Intermediate HSCs) (E). F Frequency of HSC subtypes in young and old HSCs. G Heatmap showing MAGIC-imputed gene expression values of bona fide HSC markers ordered by increasing Kit expression. H Violin plot for imputed Kit gene expression by age. Statistical analysis was performed using the Wilcoxon test. I Scaled change in frequency for each Kit HSC subset with age. J FACS analysis showing the frequency of Kit^hi and Kit^lo HSCs by age. All data are from n = 6 mice/group (young = 6; old = 6). Error bars represent mean ± SEM. **P < 0.01, ***P < 0.001. P-values calculated by two-way ANOVA. Source data are provided as a Source Data file, Source Data Fig. 1.

Fig. 2. Young Kit^lo HSCs exhibit enhanced T cell potential.

A Experimental schema to evaluate in vitro lymphoid progenitor and T cell differentiation potential of Kit^hi (red) and Kit^lo HSCs (blue) from 2-mo (young) C57BL/6 mice using the S17 lymphoid assay and murine artificial thymic organoid (M-ATO), respectively. Enumeration of absolute number of lymphoid progenitor cells following 14 days in S17 lymphoid assay (B) and absolute number of T cell subsets following 8 weeks of culture in M-ATOs (C). Refer to Supplementary Fig. 13A, B for gating strategies to define the above populations. Aggregated data across 3 independent experiments, each performed in triplicate (Kit^lo = 3; Kit^hi = 3). D Experimental schema for competitive allogeneic HCT (allo-HCT) using Kit^hi (red) and Kit^lo HSCs (blue) from 2-mo (young) C57BL/6 mice with competitor bone marrow (BM) cells from B6.SJL-PtprcaPepcb/BoyJ mice were transplanted into lethally irradiated 7-week-old (young) BALB/cJ recipients. E–J Eight weeks after competitive HCT, (E) frequency of donor-derived chimerism (CD45.2/ H-2Kb) of mature lineages in the peripheral blood (PB, CD45+, Myeloid cell: Gr-1+CD11b+; B cell: B220+; T cell: CD3 + ), (F) Enumeration of absolute number of donor-derived cells in the BM for LSK cells (LT HSC: Lineage⁻Sca-1⁺cKit⁺ CD34⁻CD48⁻Flt3⁻CD150⁺; ST HSC: Lineage⁻Sca-1⁺cKit⁺ CD48⁻Flt3⁻CD150⁻; MPP2: Lineage⁻Sca-1⁺cKit⁺ CD48⁺Flt3⁻CD150⁻; MPP3: Lineage⁻Sca-1⁺cKit⁺ CD48⁺Flt3⁻CD150⁺; MPP4/ LMPP: Lineage⁻Sca-1⁺cKit⁺ Flt3⁺CD150⁻) and Common Lymphoid Progenitors cells (CLP: Lineage⁻IL7Ra⁺Flt3⁺Sca^mid/lo Kit^lo). G–J Post-HCT thymi analysis for total thymic cellularity (G), H, I enumeration of absolute number of donor-derived cells for T cell precursors (ETP: Lineage⁻ CD4⁻ CD8⁻CD44⁺ CD25⁻Kit⁺; DN2: Lineage⁻ CD4⁻ CD8⁻CD44⁺ CD25⁺; DN3: Lineage⁻ CD4⁻ CD8⁻CD44⁻ CD25⁺) (H), Mature T cells (DP: Lineage⁻ CD4⁺ CD8⁺; SP4: Lineage⁻ CD4⁺ CD8⁻; SP8: Lineage⁻ CD4⁻ CD8⁺) (I), and analysis of thymic CD45- compartment for Thymic Epithelial Cells (TEC: CD45⁻ EpCAM⁺), Cortical TEC (cTEC: CD45⁻ EpCAM⁺UEA-1^lo 6C3^hi MHCII^hi/lo), Medullary TEC (mTEC: CD45⁻ EpCAM⁺UEA-1^hi 6C3^lo MHCII^hi/lo−), endothelial cells: CD45⁻ EpCAM⁻Ter119 PDGFRα⁻ CD31⁺ and fibroblasts: CD45⁻ EpCAM⁻Ter119⁻CD31⁻ PDGFRα⁺) (J). Data for (E–I) are from n = 10–11 mice/group (Kit^hi = 10; Kit^lo = 11) and (J) from 9 mice/group (Kit^hi = 9; Kit^lo = 9), across two independent experiments. K Experimental schema to evaluate thymic function following competitive allo-HCT using young HSCs. L Frequency of donor-derived Recent Thymic Emigrants (RTE: CD3 + GFP + ) in the PB at 8 weeks post-HCT. Refer to Supplementary Fig. 13C for the gating strategy to define the above population. All data are from n = 5 mice/group (Kit^lo = 5; Kit^hi = 5). M Experimental schema to investigate the functional response of differential Kit-expressing HSC-derived T cells in secondary recipients to L. monocytogenes expressing chicken ovalbumin (LM-OVA) infection. N Spleen analysis of secondary recipients for frequency of OT1-derived chimerism of CD8+ T cells (OT1+CD8 + : CD45.1⁺H2-K^bCD8⁺) 7 days post-infection. Refer to Supplementary Fig. 13D for the gating strategy to define the above population. All data are from n = 10 mice/group, (Kit^lo = 10; Kit^hi = 10), across two independent experiments. O Experimental schema for competitive allogeneic HCT (allo-HCT) using Kit^hi (red) and Kit^lo HSCs (blue) from 2-mo (young) C57BL/6 mice with competitor bone marrow (BM) cells from B6.SJL-PtprcaPepcb/BoyJ mice transplanted into lethally irradiated 14-mo (Middle-aged) BALB/cJ recipients. P Frequency of donor-derived T cell chimerism at the indicated timepoints. Q, R Sixteen weeks after competitive HCT, enumeration of donor-derived CLPs (Q) and total thymic cellularity (R). Data for (P–R) are from n = 6–7 mice/group, (Kit^lo = 7; Kit^hi = 6), across two independent experiments. Refer to Supplementary Fig. 12 for gating strategies to define the above populations. Error bars represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. P-values calculated by a nonparametric unpaired two-tailed Mann–Whitney U test. Panels A, D, K, M, and O were created in BioRender. Lab, K. (2025) https://BioRender.com/fl4hwgnand https://BioRender.com/oeh4i7x. Source data are provided as a Source Data file, Source Data Fig. 2.

Fig. 3. Kit^lo HSCs exhibit distinct epigenetic features that facilitate lymphoid potential.

A UMAP of 6 unsupervised HSC clusters identified by tile-based scATAC-seq analysis after Harmony batch correction. B–D Annotated by age (B), HSC subsets (C), and Kit subsets (D) on the scATAC-seq UMAP. E scATAC-seq cluster composition by HSC subsets (left) and Kit subsets (right). Color scale denotes correlation values between the ATAC clusters and cell annotations, generated using ArchR. F, G Matrix plots showing motif enrichment identified by ChromVAR (F) and gene accessibility (G) for lymphoid-specifying transcription factors for Kit subsets in young and old HSCs. Z-scores denote chromVAR motif enrichment in (F) and gene accessibility scores (peak matrix values) that have been row-scaled in (G), generated using ArchR.

Fig. 4. ZBTB1 regulates HSC multipotency and drives thymic reconstitution potential.

A Experimental schema for T cell differentiation assay and competitive allogeneic HCT with Zbtb1-deficient Kit^lo HSCs generated with 2-mo young Rosa26Cas9-eGFP KI mice. B Frequency of T cell subsets following 6 weeks of culture in M-ATOs. Refer to Supplementary Fig. 13B for gating strategies to define the above populations. Aggregated data across three independent experiments, each performed in duplicates (Control = 3; Zbtb1-KO = 3). C–E Competitive allogeneic HCT with Zbtb1-deficient Kit^lo HSCs generated with 2-mo young Rosa26Cas9-eGFP KI mice. Sixteen weeks after competitive HCT, frequency of donor-derived chimerism of mature lineages in the peripheral blood as described in Fig. 2E (C), donor-derived chimerism of LSK and MP cell subsets in the BM (D), and donor-derived chimerism of T cell precursor thymocyte and mature T cell subsets in the thymus (E). All data are from n = 6 mice/group, (Control = 6; Zbtb1-KO = 6), across two independent experiments. F Experimental schema for T cell differentiation assay and competitive allogeneic HCT with Zbtb1-OE Kit^hi HSCs from young mice. G Frequency of T cell subsets following 6 weeks of culture in M-ATO of young Kit^hi HSCs. Refer to Supplementary Fig. 13B for gating strategies to define the above populations. Aggregated data across three independent experiments, each performed in duplicates (Control = 3; Zbtb1-OE = 3). H–J Competitive allogeneic HCT with Zbtb1-OE Kit^hi HSCs generated with young mice. Sixteen weeks after competitive HCT, frequency of donor-derived chimerism of mature lineages in the peripheral blood as described in Fig. 2E (H), donor-derived chimerism of LSK and MP cell subsets in the BM (I), and donor-derived chimerism of T cell precursor thymocyte and mature T cell subsets in the thymus (J). All data are from n = 6 mice/group, (Control = 6; Zbtb1-OE = 6), across two independent experiments. Refer to Supplementary Fig. 12 for gating strategies to define the above populations. K Previously identified Zbtb1 targets and Notch1-interacting proteins enriched in Kit^lo HSCs. L Pathway enrichment analysis for non-Notch1 Zbtb1 targets enriched in Kit^lo HSCs by gseapy() using GO_Biological_Process_2023 libraries. Bubble plot showing representative pathways. Error bars represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. P-values calculated by nonparametric unpaired two-tailed Mann–Whitney U test. Panels (A) and (F) were created in BioRender. Lab, K. (2025) https://BioRender.com/zy2p156. Source data are provided as a Source Data file, Source Data Fig. 4.

Fig. 5. Old Kit^lo HSCs exhibit enhanced T cell potential.

A, B Evaluation of in vitro lymphoid progenitor and T cell differentiation potential of Kit^hi (red) and Kit^lo HSCs (blue) from or 24-mo (old) C57BL/6 mice using the S17 lymphoid assay and murine artificial thymic organoid (M-ATO), respectively, as shown in Fig. 2A. Enumeration of absolute number of lymphoid progenitor cells following 14 days in S17 lymphoid assay (A) and absolute number of T cell subsets following 8 weeks of culture in M-ATOs (B). Refer to Supplementary Fig. 13A, B for gating strategies to define the above populations. Aggregated data across 3 independent experiments, each performed in triplicate (Kit^lo = 3; Kit^hi = 3). C Experimental schema for competitive allogeneic HCT (allo-HCT) using Kit^hi (red) and Kit^lo (blue) HSCs from 22–24-mo (old) C57BL/6 mice with competitor bone marrow (BM) cells from B6.SJL-PtprcaPepcb/BoyJ mice transplanted into lethally irradiated young BALB/cJ recipients. D–H Eight weeks after competitive HCT, enumeration of absolute number of donor-derived T cells in the PB (Kit^hi = 9; Kit^lo = 9) (D), LSK cell subsets and CLP cells (Kit^hi = 10; Kit^lo = 10) (E), total thymic cellularity (Kit^hi = 9; Kit^lo = 9). F Donor-derived T cell precursor thymocyte subsets (Kit^hi = 9; Kit^lo = 9) (G), stromal subsets within thymic CD45-compartment (Kit^hi = 9; Kit^lo = 9) (H). Aggregated data across two independent experiments. I Experimental schema to evaluate thymic function following competitive allo-HCT using old HSCs. J Frequency of donor-derived Recent Thymic Emigrants (RTE: CD3⁺GFP⁺) in the PB at 8 weeks post-HCT. Refer to Supplementary Fig. 13C for the gating strategy to define the above population. All data are from n = 5 mice/group (Kit^lo = 5; Kit^hi = 5). K Experimental schema for competitive allogeneic HCT (allo-HCT) using Kit^hi (red) and Kit^lo HSCs (blue) from 22–24-mo (old) C57BL/6 mice with competitor bone marrow (BM) cells from B6.SJL-PtprcaPepcb/BoyJ mice transplanted into lethally irradiated middle-aged BALB/cJ recipients. L–N Eight weeks after competitive HCT, enumeration of the absolute number of donor-derived T cells in PB (L), donor-derived CLPs in the BM as defined in Fig. 3D (M), and thymic cellularity (N). All data are from n = 5 mice/group (Kit^lo = 5; Kit^hi = 5). Refer to Supplementary Fig. 12 for gating strategies to define the above populations. O Frequency of T cell subsets following 6 weeks of culture in M-ATO of old Kit^hi HSCs following Zbtb1 OE. Refer to Supplementary Fig. 13B for gating strategies to define the above populations. Aggregated data across three independent experiments, each performed in duplicates (Control = 3; Zbtb1-OE = 3). Error bars represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. P-values calculated by a nonparametric unpaired two-tailed Mann–Whitney U test. Panels (C), (I), and (K) were created in BioRender. Lab, K. (2025) https://BioRender.com/oeh4i7x. Source data are provided as a Source Data file, Source Data Fig. 5.

Fig. 6. KIT^lo human BM HSCs exhibit enhanced lymphoid potential.

A–E Young and old human BM CITE-seq dataset generated and published by Sommarin et al. A–C UMAP of CD34+ young and old human BM annotated with HSC subsets (A), and age (B, C) HSC cluster composition by age. D Kit^lo gene signature (top 50 marker genes from 239 mouse genes identified with human orthologues) was overlaid on human HSC UMAP. E Violin plot for Kit^lo gene score by age. Statistical analysis was performed using the Wilcoxon test. F, G FACS analysis of human BM samples. F Violin plot of KIT protein expression on phenotypic HSCs (p-HSC: CD34 + CD38-CD10-CD45RA-CD90 + ) by age (young: 20–40 yrs; MA/old: >40 yrs). G Scatterplot showing distribution of KIT^hi and KIT^lo HSC subsets within p-HSCs in human BM samples with varying age. The two-tailed P-values generated for (G) were based on a single multivariable generalized estimating equation (GEE) logistic model. The model was adjusted for age, KIT^lo and KIT^hi groups, and their interaction, with an exchangeable working correlation structure to account for the matched data structure. H Experimental schema to evaluate in vitro T cell and lympho-myeloid potential of human BM HSC subsets based on differential KIT expression using human artificial thymic organoids (H-ATO) and MS5 assay, respectively. I Following 8–10-weeks of culture, enumeration of CD19+ B cells and T cell subsets within CD34+ T cell precursors (Early Thymic Progenitors, ETP: CD34⁺CD1a⁻CD7⁻; CD1a^neg- ProT: CD34⁺CD1a⁻CD7⁺; CD1a^pos-ProT: CD34⁺CD1a⁺CD7⁺), and mature T cells (DP: CD34⁻CD5⁺CD7⁺CD4⁺CD8⁺; SP4: CD34⁻CD5⁺CD7⁺CD4⁺CD8⁻; SP8: CD34⁻CD5⁺CD7⁺CD4⁻CD8⁺). Refer to Supplementary Fig. 10E for gating strategies to define the above populations. Aggregated data from 8 independent BM donors, each performed in duplicates, across two independent experiments (KIT^lo = 8; KIT^hi = 8). J Following 4 weeks of culture, enumeration of Lineage output (Neutrophil-Granulocytes: hCD45+CD15+; monocyte-macrophages: hCD45+CD14+; B cells: hCD45+CD19+; NK cells: hCD45+CD56+) after culturing 200 KIT^lo and KIT^hi HSCs.⁷⁹Aggregated data from 5 independent BM donors, each performed in triplicate or triplicates, across two independent experiments (KIT^lo = 5; KIT^hi = 5). Refer to Supplementary Fig. 10F for gating strategies to define the above populations. K Our model illustrates that hematopoietic stem cells (HSCs) with distinct lymphoid-primed chromatin states and differential ZBTB1 activity, which determines their lymphoid potential, can be distinguished by their CD117/KIT expression. In young bone marrow, Kit^lo HSCs (blue) with high ZBTB1 activity exhibit enhanced lymphoid potential, thymic recovery, and T-cell reconstitution, in contrast to Kit^hi HSCs (red) with reduced lymphoid potential. The age-related decline in lymphoid potential results from both a proportional shift toward Kit^hi HSCs and a global decrease in ZBTB1 activity across all HSC populations. Error bars represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. P-values calculated either by nonparametric paired two-tailed Mann–Whitney U test (I and J), unpaired Mann–Whitney U test (F). Panels (H) and (K) were created in BioRender. Lab, K. (2025) https://BioRender.com/fl4hwgn. Source data are provided as a Source Data file, Source Data Fig. 6.