Fig 2: Aberrant colony formation and transplantability of Tet-deficient HSPCs.(A) CFU capacity of HSPCs isolated from E9.5 YS, E11.5 AGM, and E11.5 FL of indicated genotypes. E, erythroid (burst-forming unit); GM, granulocyte and macrophage (CFU); GEMM, granulocyte, erythroid, macrophage, and megakaryocyte (CFU). (B) Profiles of colonies formed by HSPCs isolated from E9.5 YS, E11.5 AGM, and E11.5 FL. (C) Schematic of transplantation assay. (D) Donor (CD45.2+) chimerism in peripheral blood (PB) of recipient (CD45.1+) mice after transplantation. Blood were collected every 4 weeks up to 12 weeks after transplantation. Each dot represents a recipient mouse. (E) Lineage distribution of engrafted mice showing CD3 ?+ T cell (T), B220+ B cell (B), and CD11b+ myeloid cell percentage in PB within donor-derived (CD45.2+) cells every 4 weeks up to 12 weeks after transplantation. (F) Kaplan-Meier curve representing percent survival of transplanted NSG mice. (G) May-Grünwald-Giemsa–stained PB smears of recipient mice 11 weeks after AGM transplantation shown at ×100 magnification (left) and hematoxylin and eosin staining of livers of recipient mice 12 weeks after AGM transplantation shown at ×40 magnification (right). (H) Gross images of the spleen of recipient mice transplanted with AGMs of indicated genotypes at 11 weeks after transplantation. (I) Gross images of femurs of recipient mice transplanted with AGMs of indicated genotypes at 11 weeks after transplantation. For all panels, data are presented as means ± SEM. Statistically significant (*P < 0.05, **P < 0.01, and ***P < 0.001).

Fig 3: B cells from aged donor mice have enhanced extrafollicular response and reduced GC formation early post-vaccination. (a) Representative flow cytometric plots of CD138+ IRF4+ plasma cells gated on donor cells from 10- or 93-week old donor mice in recipient spleens 6 days post-transfer and immunisation. Numbers adjacent to gates indicate percentage of donor HEL+ B220+ cells. Percentage and number of donor-derived CD138+ IRF4+ plasma cells are plotted on the graphs on the right. (b) Representative flow cytometric plots showing gating strategies for donor-derived GC B cells (CD38- GL7+), activated B cells (CD38+ GL7+) and memory B cells (CD38+ GL7- IgD-) from 10 weeks or 93 week old donor mice. Numbers adjacent to gates indicate percentage of donor HEL+ B220+ cells. (c) Percentage and number of HEL+ donor-derived GC B cells in recipient spleen on day 6 post-transfer and immunisation. (d) Graph showing the ratio of GC B cells: Plasma cells numbers derived from donor cells from 10 weeks or 93 weeks old donor mice. (e, f) Percentage and number of (e) CD38+ GL7+ B cells and (f) CD38+ GL7- IgD- memory B cells on day 6 post-transfer and immunisation. Bar graphs show the results of one of two independent experiments (n = 5–7 per group/experiment). Bar height corresponds to the mean, error bars indicate standard deviation, and each symbol represents one biological replicate. Statistics were calculated using the unpaired Mann–Whitney U test.

Fig 4: TET2-Loss-of-Function-Driven CH Aggravates Insulin Resistance and Hyperglycemia in Obese MiceBMTs following whole-body irradiation were used to generate mice carrying 10% Tet2−/− (10% KO BMT) or +/+ (10% WT BMT) hematopoietic cells, which were then fed HF/HS diet for 14 weeks. n = 15 mice per BM genotype unless otherwise noted.(A) Summary of the competitive BM transplantation approach and the timeline of these studies.(B) Percentage of CD45.2+ cells in the WBC population in blood, evaluated by flow cytometry. A CD45.1/CD45.2 dot plot representative of 10% KO BMT mice is shown.(C) Percentage of CD45.2+ cells within main WBC lineages after 12 weeks on HF/HS diet (17 weeks post-BMT), measured by flow cytometry.(D) Percentage of CD45.2+ cells within F4/80+ macrophage and CD3+ T cell populations in eWAT after 14 weeks on HF/HS diet (19 weeks post-BMT), measured by flow cytometry (n = 7 for 10% WT BMT, n = 6 for 10% KO BMT mice). Representative dot plots of macrophage populations are shown.(E) Body weight.(F) Percentage of body fat and lean mass, assessed by magnetic resonance imaging (n = 11 mice per BM genotype).(G) Insulin tolerance tests.(H) Fasting blood glucose levels.(I) Western blot analysis of insulin-induced Akt phosphorylation in eWAT (n = 6 mice per BM genotype).Data are represented as mean ± SEM.See also Figure S2.

Fig 5: Improved macrophage differentiation of GCase-targeted HSPCs in NSG-SGM3 mice.a Human cell engraftment 16-weeks post-transplantation in the bone marrow (BM), spleen (SP), and peripheral blood (PB) in transplants using CD68S-GCase-P2A-Citrine-targeted cells (n = 5 mice). b Modified allele frequency from engrafted CD68S-GCase-P2A-Citrine-targeted cells in the same tissues (n = 5 mice). c Percent human B-cell (CD19+), myeloid (CD33+), and monocyte (CD14+) populations in BM, SP, and PB shown in white. Citrine-positive cells in each population are shown in green (n = 5 mice). d Representative FACS plots showing gating strategy for mouse and human CD45+, CD45+/CD11b+, and CD45+/CD11b+/Citrine populations in macrophage preparations from lung, peritoneum, and liver. e Percent human CD45+ and human CD45+/Citrine cells in the same preparations (n = 5 mice). f Percent human CD45+/CD11b+ and human CD45+/CD11b+/Citrine cells in the same preparations (n = 5 mice). g Fold GCase activity in human Citrine+ cells compared to human Citrine- cells in BM, SP, and lung (n = 3 mice). h Modified allele frequency in human Citrine+ cells (green) compared to human Citrine- cells (white) in BM, SP, and lung in the same cells. a, b Median shown. c, e–g Data shown as mean ± SD. Source data are provided as a Source Data file.