Fig 2: Anergic B cells lacking NDRG1 survive and compete normally in a polyclonal repertoire.a Experimental schematic; lethally irradiated WT or sHEL transgenic CD45.1+ recipients were reconstituted with 80:20 mixtures of WT or Ndrg1-/- IgHEL CD45.2+ BM and WT non-tg CD45.1+ BM. b Percentage of total HEL-binding B220+ CD45.1- cells in the BM and spleen of mice in (a). c Percentage of HEL-binding B220+CD45.1- cells labeled with BrdU after one week. In (b, c), dots are individual mice from one experiment with 5–6 mice per group, lines show means and 95% CI error bars. d. Representative flow cytometry plots of B220+CD43- B cells from BM of sHEL mice reconstituted with 50:50 mixtures of WT IgHEL CD45.1+ BM with either WT IgHEL or Ndrg1-/- IgHEL CD45.2+ BM. e Surface IgM expression on B cell populations gated as in (d). f Intracellular Bim quantification in B220+CD19+IgDa+splenocytes from (d). Each point represents individual mice and lines represent means with 95% CI error bars from one experiment (4–5 per group).

Fig 3: Loss of Kat2a diversifies cell fates and promotes RT1(9a) preleukemia progression.(A and B) Pseudo-time single-cell trajectory of (A) Kat2aWT cells (B) Kat2aNULL RT1(9a) cells 2 and 4 months after transplantation. Trajectories were inferred using Monocle3 (21); compartments were labeled as per hematopoietic markers in (fig. S5, C and D). (C) Proportion of candidate progenitor cell compartments in the RT1(9a) pseudo-time trajectory contributed by individual Kat2aWT or Kat2aNULL, 2-month or 4-month samples. (D) Representative flow cytometry histograms of B220 B cell marker detection in plate 2 CFC of RT1(9a)-transduced Kat2aWT and Kat2aNULL cells during in vitro transformation. (E) Aggregate results of B220 staining as in (D). Mean ± SD, n = 3. (F) Representative flow cytometry histograms of F4/80 monocyte marker detection in plate 2 CFC of RT1(9a)-transduced Kat2aWT and Kat2aNULL cells during in vitro transformation. (G) Aggregate results of F4/80 staining as in (F). Mean ± SD, n = 3.

Fig 4: L2pB1 cells actively accumulate in tumors. Tumors were dissected on day 18 post injection. Lymphocytes were subjected to immunophenotyping by flow cytometry with fluorescently-labeled antibodies for CD45, CD3e, CD19, B220, IgM, PD-L2, and ZsGreen. (A) Representative histograms of PD-L2+ZsGreen+ L2pB1 cells in tumor, lymph nodes (LN), and spleen (SP). L2pB1 cells were identified as CD3-CD19+B220+IgM+PD-L2+ZsGreen+. (B) Percentage of L2pB1 cells from total tumor-infiltrating B cells as isolated from tumors (TIL), lymph nodes (LN), or spleen (SP). (*P = 0.01, **P = 0.02). Data are representative of three independent experiments.

Fig 5: Depletion of L2pB1 cells reduces lipoptosis of both MC38 and B16F10 tumor cells. (A) MC38 colon cancer cells and (B) B16F10 melanoma cells were cultured alone or co-cultured with PCW cells from saline or DT treated mice for 72 hours, in the presence of LPS. Cells were stained with Hoechst 33342 (nuclei), CD45-AF488 (leukocytes) and Oil Red O at 72 hours. Representative images from triplicate wells were taken at 400x magnification. Scale bar (white) = 50µm. (C, D) Lipoptosis was quantified as average area of Oil Red O staining per tumor cell ( Supplemental Figure 3 ). MC38 and B16F10 tumor cells treated with L2pB1-depleted PCW cells showed significant reduction of lipoptosis as compared to wild type PCW cells, ****p < 0.0001 and *p = 0.0265.