Fig 2: TGF-ß1 at Concentrations of 10 and 100 pg/mL Reduces the Total and Myeloid Reconstitution Levels in a Single-Cell Culture(A) Single HSC1 cells, sorted from the BM cells of the B6-Ly5.1 mice, were transplanted into 30 lethally irradiated B6-Ly5.2 mice with 5 × 105 competitor cells from the B6-Ly5.2 mice. The single cells were also cultured in serum-free medium with SCF + TPO or SCF + TPO + TGF-ß1 (10 or 100 pg/mL) for 5 days and were then transplanted into 30 lethally irradiated B6-Ly5.2 mice with 5 × 105 competitor cells from the B6-Ly5.2 mice.(B) Clonal cell transplantation. The percentage of chimerism in the granulocytes/monocytes, B cells, and T cells of the peripheral blood was analyzed 1, 3, 6, 10, and 12 days after transplantation. Two separate cohorts of single-cell transplantation experiments were performed, but the data from one experiment are shown. Individual mice were identified by numbering.

Fig 3: Conditioned Medium from TGFB1-Selected MSCs Provided Superior Support of Human ChimersimMSCs were selected in MSC10 (MEM a + 10% human serum) or MSC10 with, in addition, 10 ng/mL TGFB1, washed with PBS, and cultured in fresh MSC10 for 24 h to produce conditioned medium. The supernatants and a control medium (StemSpan + KITLG 100 ng/mL) were used to cultivate human CD34+ cord blood cells overnight and to transplant the HSCs via tail vein injection into NBSGW mice. Mice were sacrificed and analyzed 12 weeks after transplantation.(A) Proliferation of hematopoietic cells after 24 h of cultivation in the different media. Cell numbers are shown relative to condition MSC10.(B) CD34+ cells were incubated in conditioned or control medium for 18 h and injected in the same medium used for cultivation. The figure shows percentage of human chimerism (CD45+ cells in all CD45+ cells) in the peripheral blood (PB) and bone marrow (BM) of transplanted NBSGW mice. Mouse cohorts 1, 2, and 3 are represented by different shapes.(C) Human cord blood CD34+ cells were thawed and transplanted in 150 µL conditioned medium. Depicted is the human chimerism (CD45+ cells in all CD45+ cells) in the BM of transplanted NBSGW mice.(D) One hundred CD34+ cells were seeded in MethoCult 4034 Optimum after cultivation for 18 h in conditioned medium. Colonies and cells were counted 16 days after seeding.MSC10c, conditioned medium by MSC10-selected MSCs; TSCc, conditioned medium by MSC10 + TGFB1-selected MSCs; GEMM, granulocyte, erythrocyte, monocyte, megakaryocyte; GM, granulocyte, macrophage; BFU-E, Burst-forming unit erythrocyte; ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001 calculated with ANOVA and Tukey post hoc test. The data represent means and the error bars show the SD.

Fig 4: Elevated autophagy in autoantigen-specific memory B cells. Splenocytes from MRL or MRL-lpr mice (4-month-old) were stained with PE-conjugated antibodies to CD11b, Gr-1, IgM, IgD and CD138, PE-Cy7-anti-CD19, Pacific Blue anti-CD38, APC-anti-IgG, and Alexa Fluor 488-conjugated dsDNA or RNP. DUMP– (IgM–IgD–CD11b–Gr-1–CD138–) CD19+ B cells were gated. (A) dsDNA-specific memory B cells (DUMP–CD19+CD38+dsDNA+IgG+) or (B) RNP-specific memory B cells (DUMP–CD19+CD38+RNP+IgG+) were analyzed by flow cytometry. (C) The frequency of dsDNA-specific memory B cells and RNP-specific memory B cells, as well as dsDNA-specific germinal center (GC) B cells (DUMP–CD19+CD38–dsDNA+IgG+) and RNP-specific GC B cells (DUMP–CD19+CD38–RNP+IgG+), in the spleen of each mouse was plotted. Data are presented as mean ± s.e.m. **P < 0.01, ***P < 0.001, ****P < 0.0001, (n=8 for MRL and 11 for MRL-lpr). (D) RNP-specific memory B cells (DUMP–CD19+CD38+RNP+IgG+) and RNP-negative memory B cells (DUMP–CD19+CD38+RNP–IgG+) were sorted from MRL-lpr mice and stimulated with LPS in vitro for 3 days, the number of ASCs producing RNP-specific antibodies were measured by ELISPOT. Data are presented as mean ± s.e.m. Experiments were performed twice in triplicates using cells from a pool of 3-4 mice. ****P < 0.0001, determined by two-tailed Student’s t-test. (E) Splenocytes from pooled MRL or MRL-lpr mice were stained as in (A) except that both Alexa Fluor 488-conjugated dsDNA and RNP were included for staining. Sorted dsDNA/RNP/Sm-specific memory B cells, and B220+IgMlowIgD+CD23+IgG- naïve B cells were used for real-time RT-PCR analysis of indicated autophagy-related genes. Data are presented as mean ± s.e.m. Experiments were performed three times using cells from a pool of 10-15 mice. *P < 0.05, **P < 0.01, determined by two-tailed Student’s t-test. (F) Western blot analysis of LC3 processing in Memory (DUMP-CD19+IgG+CD38+) and naïve B cells isolated from pooled MRL and MRL-lpr mice. Data are representative of two independent experiments.

Fig 5: H2afy-/- mice have defective B cell developmentA competitive repopulation assay was performed by transplanting bone marrow cells from H2afy+/+ or H2afy-/- littermates (CD45.2+) with the same number of WT congenic competitor marrow cells (CD45.1+/CD45.2+) into lethally irradiated congenic recipients (CD45.1+).(A) Overview of the competitive repopulation assay.(B) Peripheral blood chimerism of CD45.2+ cells at 1, 2, and 4 month(s) post-transplant (n = 16). Differences (two-way ANOVA) are indicated at each time point.(C) CD45.2+ bone marrow lineage cell frequencies at 4 months post-transplant (n = 7).(D) Donor-derived CD45.2+ B cell progenitors (ALP and BLP) and Hardy fractions (n = 7).Statistical analysis by two-tailed Student’s t test, except for (B). Error bars represent mean ± SD. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001.