Fig 2: Il10 deficiency leads to increased infarct sizes, enhanced brain atrophy, and poorer long-term outcome following tMCAO. MRI was used to quantify infarct volume at day 3 (A) and cortical atrophy volume at day 14 (C) after tMCAO in WT control and Il10-/- mice (representative T2 image). B Triphenyltetrazolium chloride (TTC) staining was used for the evaluation of infarct volume at day 7 (representative TTC brain slices). D Composite neurological score was performed on day 14 after tMCAO. E Il10/GFP-positive cells were visualized in the penumbra area of the ischemic hemisphere by GFP counterstaining in FIR-tiger mice 14 days after tMCAO (green, Il10/GFP-positive cells; red, CD45-positive cells; blue, 40,6-diamidino-2-phenylindole nuclear staining; scale bar 20 µm). Infarct data are presented as mean ± SEM of 8–10 WT and 6–10 Il10-/- mice (A, B), atrophy data as mean ± SEM of 11 WT and 13 Il10-/- mice (C), and neurological score of 12 WT and 11 Il10-/- mice (D). Statistical significances were analyzed by Student t test (A–C) and Mann–Whitney U test (D). *P < 0.05 and ***P < 0.001

Fig 3: Flow cytometric analysis of antigen-matured engineered B cells.Host mice receiving LPS activated only (Mock), H + ?, or H -targeted B cells were analyzed 14 d after the first boost (w8). n = 4 mice in each group. a–c, Germinal center (GC) B cells. a Representative flow cytometry and statistical analysis of total GC B cells pre-gated as live, CD19+ singlets. b CD45.1+ (donor) GC B cells. c Fraction of cells gated in (b) which were class-switched IgG1+ and GT8++KO11- (engineered VRC01-expressing). d–e Plasma cells (PCs). Frequency of class-switched and antigen-specific PCs in the spleen (d) and bone marrow (e) as a fraction of total donor PCs. GT8++KO11–CD45.1+CD45.2-IgG+ PCs were analyzed by intracellular staining after the permeabilization of surfaced-stained cells. Plasma cells are gated as F4/80-IgD-TCRß-CD138+Sca-1+. f Memory cells (MCs). Enumeration of IgG1+GT8++KO11-, engineered MCs (sIgD-CD45.1+CD45.2-CD38+GL7-). g Vaccine-induced expansion of engineered cells. GT8++KO11-CD45.1+CD45.2- cell expansion at w8 compared with w0 as a percentage of the total donor cells. h Engineered memory cells have become germinal center dependent (CD73+) after vaccination. GT8+ or GT8- donor memory cells were compared with host naïve and memory B cell controls for expression of the indicated memory cell surface markers. i Quantification of B cells memory subtypes gated as in (h). a–i Bars represent mean ± SD for all data points in each group. *P < 0.05; **P < 0.01; ns, not significant; unpaired 2-tailed T test.

Fig 4: Cellular enlargement contributes to DNA damage–induced fitness decline in HSCs.(A) Mean volume (fl) of HSCs obtained from vehicle (n = 6), sublethally irradiated (3 Gy, n = 4), G0/1 2.8 to 3 Gy (n = 11), rapamycin-treated (RAP, n = 4), and RAP + 3 Gy–treated (n = 7) mice 2 weeks after irradiation (? = difference). (B) Measurement of DNA damage using CometChip: percentage tail DNA of HSCs (%) isolated from mice 2 weeks after treatment with vehicle, 3 Gy, RAP, or 3 Gy + RAP (n = 166). (C) Reconstitution assay: Donor (CD45.2) mice were pretreated with RAP or vehicle for 2 weeks, sublethally irradiated (3 Gy), and treated with RAP (n; donors = 12, recipients = 8) or vehicle (n; donors = 12, recipients = 15) for another 2 weeks before 1000 CD45.2 HSCs were isolated and transplanted into lethally irradiated recipient mice. Control donor HSCs were not treated (control, n; donors = 6, recipients = 9) or treated with RAP without irradiation (control, n; donors = 3, recipients = 5). Recipient mice were not treated with RAP after reconstitution. Percentage of donor-derived white blood cells in recipients and slope of reconstitution kinetics over time were determined.

Fig 5: CD45.2 antibody labeling and saturation. (a) Schematic of experimental timeline. (b) Schematic of antibody labeling. (c) Flow cytometry gating strategy for 0.4 mg/kg i.v. antibody dose. Leukocytes were identified and cell debris excluded using the P1 gate. P2 denotes single cells. Finally, the P3 gate was used to identify leukocytes that were CD45.2-APC+ and/or CD45.2-FITC+. CD45.2 negative cells were omitted from analysis. (d)–(f) Examples of flow cytometry results, each plot shows representative data from one mouse. The x-axis is FITC antibody fluorescence intensity, and the y axis is APC antibody fluorescence intensity. Quadrants I, II, IV quantify double positive, CD45.2-APC+, and CD45.2-FITC+ leukocyte fractions, respectively. (d) Quantification of CD45.2 antibody labeling for 0 mg/kg i.v. antibody dose. (e) Quantification of CD45.2 antibody labeling after 1 h for 0.04, 0.4, and 2 mg/kg i.v. antibody doses. (f) Quantification of CD45.2 antibody labeling after 24 h for 0.04, 0.4, and 2 mg/kg i.v. antibody doses. (g) Quantification of leukocytes labeled with i.v. CD45.2-APC antibody after 1 h (comparisons not shown: 0 versus 0.04****, 0 versus 0.4****, 0 versus 2****) and 24 h (not shown: 0 versus 0.04ns, 0 versus 0.4**, 0 versus 2****). (h) Quantification of cells where all CD45.2 sites are saturated with i.v. CD45.2-APC antibody after 1 h (comparisons not shown: 0 versus 0.04ns, 0 versus 0.4ns, 0 versus 2**) and 24 h (not shown: 0 versus 0.04ns, 0 versus 0.4ns, 0 versus 2ns, 0.04 versus 0.4ns, 0.04 versus 2ns, 0.4 versus 2ns). (g), (h) Each point represents data from one mouse (0 mg/kg n=2, 0.04 mg/kg n=4, 0.4 mg/kg n=3, 2 mg/kg n=4), bars show the mean and SEM, statistics by one-way ANOVA with Tukey correction for multiple comparisons between groups, ** P<0.01, *** P<0.001, **** P<0.0001, and ns denotes P=0.05.